Wedge-shaped sites don&rsquo;t usually inspire envy among architects, but for Soeda and Associates, the constraints of a sliver-thin plot in <a href=https://www.designboom.com/tag/architecture-in-japan/ >Japan</a>&lsquo;s Kawasaki City inspire a playfully precise solution. The team&rsquo;s latest <a href=https://www.designboom.com/tag/residential-architecture-interiors/ >residential</a> project
pinched between a standard road and a culvert
into a sharp-edged composition that cuts clean through conventional typologies
every opening calculated for rhythm and relief
The architecture responds directly to the site&rsquo;s tight
The building occupies a footprint formed by two intersecting roads &mdash; only one of which qualifies as a legal street under Japan&rsquo;s Building Standards Act
That technicality dictated setbacks on one side only
allowing the architects to stretch the form as close as possible to the boundary on the other
The result is a volume that looks sliced on the bias
a gesture that gives it a surprisingly sculptural street presence
images &copy; <a href=http://www.phota.jp/ target=_blank rel=noopener>Takumi Ota</a>
Rather than emphasize the structure&rsquo;s four-unit layout
Soeda and Associates visually unifies Sonata2&rsquo;s mass by tapering the form and playing with fenestration
varied openings cut into the walls with intentional asymmetry
dissolving the usual cues of residential repetition
The strategy disguises the building&rsquo;s function while animating its facades
a formal solution that&rsquo;s as design-minded as it is about obfuscation
With the site too compact for an elevator, the <a href=https://www.soeda-a.net/ target=_blank rel=noopener>architects</a> embrace verticality the old-fashioned way
The highest unit is accessed at the fourth floor
with the apartment split into a fourth&ndash;fifth floor duplex
The stairs thus become an architectural narrative device
connecting not just levels but experiences
Diagonal cuts in both plan and section introduce unusual sightlines and interior voids
creating an illusion of expanded space inside a decidedly compact volume
Sonata2 is designed for a triangular site in Kanagawa
Soeda and Associates&rsquo; Sonata2 residence is structured in reinforced concrete with walls carefully placed to handle seismic loads
Though the floor plan&rsquo;s acute angles might suggest structural imbalance
the architects used both material heft and spatial strategy to distribute weight
The external staircase contributes to this equilibrium
aligning the building&rsquo;s center of gravity with its center of rigidity
It&rsquo;s a thoughtful strategy that keeps the building stable while maximizing usable interior space
A standout structural element of the house is its top-floor roof
supported at a single point like a precariously balanced toy
T-shaped steel beams embedded in the slab mitigate this drama
offering stability without undermining the delicate aesthetic
a 100 millimeter- (3.9 inch)-square concrete column supports the external stairs
puncturing through the landings with minimalist confidence
While the project may read as compact in plan
it proves that welcoming interiors don&rsquo;t rely on scale
the building turns constraint into concept
and every inch has been earned by strategy
the four-unit layout is disguised with bold punched windows
Soeda and Associates uses setbacks and angular slices to shape the form
diagonal cuts generate unique spatial effects
the architects achieve structural balance with strategic stair placement
tight urban constraints become architectural opportunity
architect:&nbsp;<a href=https://www.soeda-a.net/ target=_blank rel=noopener>Soeda and Associates</a>
structure: Ryotaro Sakata Structural Engineers
area: 133 square meters photography: &copy; <a href=http://www.phota.jp/ target=_blank rel=noopener>Takumi Ota</a>
happening now! partnering with antonio citterio, AXOR presents three bathroom concepts that are not merely places of function, but destinations in themselves — sanctuaries of style, context, and personal expression.
Volume 13 - 2020 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fnmol.2020.590896">https://doi.org/10.3389/fnmol.2020.590896</a>
This article is part of the Research TopicTau Protein: Mechanisms from Health to Degeneration<a class="Link Link--linkType Link--maincolor Link--medium Link--icon Link--chevronRight Link--right" aria-label="View all 22 articles" href="https://www.frontiersin.org/research-topics/12120/tau-protein-mechanisms-from-health-to-degeneration/magazine" target="_self" data-event="customLink-linkType-a_viewAll22Articles">View all 22 articles</a>
Microtubule-associated protein tau is characterized by the fact that it is an intrinsically disordered protein due to its lack of a stable conformation and high flexibility
Intracellular inclusions of fibrillar forms of tau with a &#x003B2;-sheet structure accumulate in the brain of patients with Alzheimer&#x00027;s disease and other tauopathies
detachment of tau from microtubules and transition of tau from a disordered state to an abnormally aggregated state are essential events preceding the onset of tau-related diseases
Many reports have shown that this transition is caused by post-translational modifications
including hyperphosphorylation and acetylation
The misfolded tau is self-assembled and forms a tau oligomer before the appearance of tau inclusions
Animal and pathological studies using human samples have demonstrated that tau oligomer formation contributes to neuronal loss
tau seeds are released from cells and incorporated into other cells
leading to the propagation of pathological tau aggregation
Accumulating evidence suggests several potential approaches for blocking tau-mediated toxicity: (1) direct inhibition of pathological tau aggregation and (2) inhibition of tau post-translational modifications that occur prior to pathological tau aggregation
(3) inhibition of tau propagation and (4) stabilization of microtubules
In addition to traditional low-molecular-weight compounds
newer drug discovery approaches such as the development of medium-molecular-weight drugs (peptide- or oligonucleotide-based drugs) and high-molecular-weight drugs (antibody-based drugs) provide alternative pathways to preventing the formation of abnormal tau
Of particular interest are recent studies suggesting that tau droplet formation by liquid-liquid phase separation may be the initial step in aberrant tau aggregation
as well results that implicate roles for tau in dendritic and nuclear functions
we review the mechanisms through which drugs can target tau and consider recent clinical trials for the treatment of tauopathies
we discuss the utility of these newer strategies and propose future directions for research on tau-targeted therapeutics
We also discuss perspectives for drug development in this area
as well as oligonucleotides to reduce tau expression
Tauopathies are neurological disorders (<a href="#B20">Avila et al., 2004</a>; <a href="#B106">G&#x000F6;tz and G&#x000F6;tz, 2019</a>), characterized by aberrant tau aggregates (NFT and tau inclusions) in neurons and glial cells. These aggregates represent tau gene mutations or hyperphosphorylated tau (<a href="#B161">Kovacs, 2015</a>). The majority of tauopathic patients also show depositions of A&#x003B2;, &#x003B1;-synuclein, or huntingtin (<a href="#B112">Guo et al., 2017</a>)
These observations suggest that tau abnormalities have a common pathological role across neurodegenerative diseases
Tau-targeted drugs may be a promising disease-modifying therapy because previous studies focusing on the correlation of AD neuropathological changes (A&#x003B2; plaques and NFT) with cognitive impairment have shown that the severity of cognitive impairment correlated best with the burden of abnormal tau (<a href="#B213">Nelson et al., 2012</a>)
many clinical trials of drugs targeting tau have been conducted
Mechanism of tau post-translational modification inhibitors in clinical trials
regulate the binding of tau to microtubules
Microtubule instability and depolymerization are observed in tauopathies
suggesting a therapeutic role for microtubule stabilizers
O-GlcNAcylation at serine and threonine compete with phosphorylation of the same residues
Tau degradation is inhibited by acetylation
The post-translational modifications are tightly regulated by various enzymes that mediate the addition and removal of the modifying groups
tau kinase inhibitors or P300 acetyltransferase inhibitors have been investigated for their ability to inhibit tau phosphorylation or tau acetylation
The usefulness of O-GlcNAcase inhibitors to elevate tau O-GlcNAcylation has also been examined in clinical trials
these observations suggest that tau hyperphosphorylation is involved in the development and pathogenesis of tauopathies and that its inhibition may be a therapeutic strategy
suggesting that CDK5 inhibition enhances tau phosphorylation by activating GSK3&#x003B2;
As CDK5 can phosphorylate molecules other than tau
therapeutic agents targeting CDK5 should be developed with great caution
These findings suggest that the development of TAOK inhibitor should proceed with caution
ACI-35 has been used in a phase 1 trial (Main ID in the WHO International Clinical Trials Registry Platform: ISRCTN13033912) (see section on Tau Clearance and Immunotherapy)
These facts suggest that tau acetylation may be important for tau-induced toxicity
This may be explained by either the poor penetration of salsalate into the brain (&#x0003C;3%)
or by an increase in tau aggregation following reduced tau acetylation
These findings suggest that upregulation of tau O-GlcNAcylation may be a therapeutic strategy for tau-related neurodegeneration
The facts suggest that compounds that directly target tau aggregation may be more effective tauopathies than tau kinase inhibitors
The recombinant tau protein is polymerized in the presence of polyanion, including heparin (<a href="#B97">Goedert et al., 1996</a>) or RNA (<a href="#B139">Kampers et al., 1996</a>), and the aggregation level can be monitored by fluorescent dye Thioflavin-T (S). Using this experimental system, many researchers screened tau aggregation inhibitors (<a href="#B291">Taniguchi et al., 2005</a>; <a href="#B42">Bulic et al., 2009</a>; <a href="#B68">Crowe et al., 2009</a>)
Many of the aggregation inhibitors discovered share a common characteristic: a negative or positive charge in their structure
The results of clinical trials of these drugs are expected
suggesting it may be suitable for the treatment of tau-related dementia
Ginseng is the root of Panax ginseng Meyer and has been used as an herbal medicine for various diseases. Red ginseng is believed to be a processed form of ginseng with enhanced pharmacological efficacy (<a href="#B170">Lee et al., 2015</a>). Tau aggregation was inhibited by the red ginseng treatment in vitro (<a href="#B270">Shin et al., 2020b</a>). As ginseng includes saponin and flavonoids (<a href="#B53">Choi, 2008</a>)
the inhibitory effect may be involved in the surfactant action or antioxidation by ginseng
indicating that the nickel administered to humans requires careful observation
Results of clinical trials on drugs against tauopathies besides AD are expected
a phase-3 low-dose study of LMTM in AD (LUCIDITY; NCT03446001) was eventually launched
Hyperphosphorylated tau is detached from microtubules and mislocalized in the somatodendritic compartment of neurons
In vitro studies have shown that tau is self-assembled to form tau oligomers and granular tau oligomers before forming NFTs
Tau aggregation inhibitors that halt these processes may be useful in the treatment of tauopathies
Immunotherapy is one potential therapeutic approach for preventing tau aggregation, a line followed by a number of academic and industrial research groups. These efforts have resulted in the generation of the TOC1 (<a href="#B230">Patterson et al., 2011</a>), TOMA (<a href="#B45">Castillo-Carranza et al., 2014</a>), and T22 (<a href="#B163">Lasagna-Reeves et al., 2012</a>) antibodies that recognize intermediate tau aggregates
While ongoing tau immunotherapy-based clinical trials mainly targeted phosphorylated tau
and aberrant conformational changes in tau (details below)
it is likely that the next generation of antibodies will be directed at tau intermediate aggregates
The traxane-derivative TPI287 (abeo-taxane) which has high BBB permeability proved safe and well-tolerated in a phase 1 study (NCT02133846) in patients with CBD or PSP (n = 44) and a phase 1 study (NCT01966666) in patients with mild to moderate AD (n = 33). However, the drug caused adverse effects in the AD group and worsened dementia symptoms in the CBD and PSP patients. It appears that TPI287 is no longer being developed for clinical use (<a href="#B307">VandeVrede et al., 2020a</a>)
Recent reviews propose that tau is not a stabilizer of microtubules in the axon but rather confers flexibility to the labile domain of microtubules and leads to microtubule elongation (<a href="#B240">Qiang et al., 2018</a>; <a href="#B21">Baas and Qiang, 2019</a>). Further, an analysis of fast single-molecule tracking showed that microtubule assembly is regulated by more rapid tau dynamics, kiss-and-hop mechanism, than previously reported (<a href="#B132">Janning et al., 2014</a>)
These observations suggest that microtubule stabilizers may not be suitable as inhibitors of tau-related dysfunction
Active immunization is an attractive therapeutic approach because it can induce a sustained autoantibody response in small doses
the therapeutic effects should not be limited by the production of anti-drug antibodies
there are 10 tau antibodies that have entered clinical trials
BIIB092 is in a phase 2 clinical trial for AD (TANGO; NCT03352557)
trials with C2N-8E12 in AD patients were conducted (NCT02880956)
An extension study (NCT03712787) is being conducted for patients who have successfully completed the phase 2 trial to evaluate long-term safety and tolerability
The monoclonal antibody UCB0107 binds to the mid-region of tau (amino acids 235&#x02013;246). Its preceding mouse version (antibody D) reportedly inhibited transneuronal propagation of pathogenic and aggregated tau in vivo (<a href="#B7">Albert et al., 2019</a>), and seeding activity of human AD and PSP tau in vitro (<a href="#B65">Courade et al., 2018</a>)
Two phase 1 studies aimed to evaluate the safety
and pharmacokinetic properties of UCB0197 in healthy adult males were conducted (NCT03464227 and NCT03605082)
A phase 1 study is ongoing in patients with PSP (NCT04185415)
The results of the trials are not yet available
LY3303560 binds preferentially to tau aggregates rather than monomers
a phase 1 trial of LY3303560 has been conducted to evaluate safety in MCI and mild to moderate AD (NCT03019536) while an ongoing phase 2 trial (NCT03518073) is being undertaken in early symptomatic AD patients
The results of both studies are currently awaited
BIIB076 is a human recombinant monoclonal anti-tau antibody. Although the epitope to which this antibody is directed has not been revealed, it is known that it recognizes monomeric and fibrillar tau (Alzforum.org, Therapeutics; BIIB076. <a href="https://www.alzforum.org/therapeutics/biib076">https://www.alzforum.org/therapeutics/biib076</a>)
a phase 1 trial to examine the safety of BIIB076 has been conducted in healthy volunteers and AD patients (NCT03056729)
JNJ-63733657 is a monoclonal antibody that recognizes the central domain of tau (<a href="#B271">Sigurdsson, 2018</a>)
but the exact epitope is unknown and results of preclinical testing are not available
A phase 1 trial of JNJ-63733657 in healthy volunteers and patients with prodromal or mild AD has been conducted (NCT03375697 and NCT03689153)
The monoclonal antibody Lu AF87908 binds to the phospho-S396 region of tau (<a href="#B253">Sandusky-Beltran and Sigurdsson, 2020</a>). A preclinical study showed that the original mouse antibody inhibited tau propagation induced by exposure of tau transgenic brain lysates in vivo and in vitro (<a href="#B249">Rosenqvist et al., 2018</a>)
a phase 1 trial (NCT04149860) is assessing the tolerability of Lu AF87908 in healthy individuals and AD patients (NCT04149860)
Currently a phase 1 study is being undertaken in healthy volunteers to evaluate the safety
and pharmacokinetics of this antibody (NCT04096287)
The trial showed that no serious adverse events occurred
and RO7105705 plasma half-life was 32 days
Two phase 2 trials were conducted in AD patients to evaluate efficacy and safety (NCT03289143 and NCT03828747)
Genentech announced top-line results from a phase 2 trial
RO7105705 did not meet the primary efficacy end-point of reducing the decline of the cognitive score (Genentech press release)
such as JNJ-63733657 and UCB0107 may be more effective than N-terminal-directed antibodies at disrupting the seeding and propagation of aberrant tau
Genentech showed no efficacy of the N-terminal-directed antibody
close monitoring of adverse effects of oligonucleotide therapies is essential
tau dissociated from microtubules migrates into the cytoplasm and forms the first aggregates
the localized gathering of tau is a singularity of aggregation
identification of the droplets formed specifically in tauopathies and the search for inhibitors of the droplets will be a challenge for the future
Tau droplet formation by liquid-liquid phase separation (LLPS) is a key initial step in aberrant tau aggregation
Tau must be abundant before it begins to aggregate
LLPS-mediated formation of droplets supersaturated with tau may be a key step in the latter process
A droplet is a reversible structure formed by weak interactions
suggesting that tau droplets may be a better drug target than irreversible tau aggregation
it appears that properly-regulated inflammation may protect against tau pathology and tau-mediated toxicity
These findings indicate that tau has other physiological functions other than its better-known microtubule-stabilizing effects; therefore
treatments that restore tau function may be helpful the management of tauopathies
a re-focusing on the regulation of tau ubiquitination may help efforts to inhibit tau aggregation
in which cDNA expressing a tau intrabody is loaded into neurons
may also prove efficacious for treating tauopathies
We are currently investigating the mechanism
which may lead to discovering new drugs targeting tau loss-of-function
Clinical trials of tau-based drugs aimed at gain-of-toxic-tau function (e.g.
dysregulation of post-translational modifications and tau aggregation) or loss-of-function (microtubule instability) have been conducted in tauopathy patients
Once these clinical trials have been completed
the potential benefit of tau in the treatment of progressive neurodegenerative dementias may be revealed
they will serve as a foundation for the next generation of tau-based drugs
New tau abnormalities in the pathological and (previously unknown) physiological functions of tau have been reported in many papers
The research reviewed here amply shows that basic research related to tau drug discovery
and clinical studies on candidate therapeutics must continue in our effort to treat tauopathies
YS and AT conceived and designed this article and revised and approved the final version of the manuscript revision
All authors contributed to the article and approved the submitted version
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas &#x02018;Singularity Biology (No
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
The authors would like to thank Enago (<a href="http://www.enago.jp">www.enago.jp</a>) for the English language review
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<a name="B106" id="B106"></a> G&#x000F6;tz
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Citation: Soeda Y and Takashima A (2020) New Insights Into Drug Discovery Targeting Tau Protein
Received: 03 August 2020; Accepted: 10 November 2020; Published: 03 December 2020
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Diabetes is known to increase the risk of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC)
Here we treat male STAM (STelic Animal Model) mice
NASH and HCC associated with dysbiosis upon low-dose streptozotocin and high-fat diet (HFD)
Although both treatments ameliorate hyperglycemia and NASH
insulin treatment alone lead to suppression of HCC accompanied by improvement of dysbiosis and restoration of antimicrobial peptide production
There are some similarities in changes of microflora from insulin-treated patients comorbid with diabetes and NASH
fails to suppress HCC in the male STAM mice lacking insulin receptor specifically in intestinal epithelial cells (ieIRKO)
which show dysbiosis and impaired gut barrier function
male ieIRKO mice are prone to develop HCC merely on HFD
These data suggest that impaired gut insulin signaling increases the risk of HCC
which can be countered by restoration of insulin action in diabetes
it remains unclear whether any specific strategy in diabetes treatment may lead to the inhibition of HCC
mainly because the pathophysiological mechanism of NASH and HCC associated with diabetes is not fully understood
leading to a tumor-favorable microenvironment
how the systemic effect of insulin as it is mediated by multiple organs may have a role in this pathophysiology remains poorly understood
it is necessary from a systemic pathophysiological viewpoint to determine what kind of diabetes treatment is suitable for patients with diabetes and NASH to prevent the progression of NASH and the development of HCC
we demonstrate the protective role of insulin signaling in the gut against hepatocarcinogenesis associated with maintaining intestinal barrier function and suppressing dysbiosis using diabetic NASH-HCC model mice and samples from patients with diabetes and NASH
These data suggest that certain somatic mutations caused by STZ treatment on birth had been maintained by cell proliferation in STAM mice until HCC became evident
the characteristics of this animal model may prove helpful in investigating the pathogenesis of non-obese diabetes comorbid with NASH/HCC as well as in exploring treatment strategies suitable for patients with these diseases
Given the results from insulin-treated and PHZ-treated mice
it was suggested that insulin signaling has certain protective effects against the development of HCC as well as hepatic steatosis and fibrosis
was induced in the STAM model and suppressed by treatment of insulin or PHZ
suggesting its involvement in effects of both treatments
These data suggest that insulin signaling promoted tight suppressive regulation of collagen production in STAM mice
We also investigated the expression of inflammatory cytokines. Ccl2, Tnfa, and Il6 were significantly suppressed by insulin treatment (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-42334-y#Fig2">2i</a>)
suggesting a protective role of insulin signaling against chronic hepatic inflammation
While insulin suppressed the progression of NASH in the liver of STAM mice to a similar extent with PHZ
insulin alone suppressed the development of HCC
We therefore tried to explore how insulin suppressed hepatocarcinogenesis in STAM mice
suggesting that the Warburg effect was present in STAM mice but suppressed by insulin treatment
and malonyl CoA could act as a substrate of lipogenesis
The relevance of the data from PCoA and individual analyses suggest that these bacteria could be a good marker for dysbiosis in this model mice
The bacterium was reported to have bile salt hydrolase (BSH) responsible for the deconjugation of taurine- or glycin-conjugated primary bile acids
Bacteroides massiliensis B84634 = Timone 84634 = DSM 17679 = JCM 13223)
and thus could be involved in supplying a substrate for secondary bile acids production
although it has not been experimentally confirmed because of the difficulty in inoculation of the bacteria
These data suggested that insulin signaling upregulated some antimicrobial peptides (AMPs) in the ileum
which could affect the proportion of B.massiliensis
These data suggest that insulin promotes chemical barrier function
Host preference is also assumed between mice and humans
To evaluate the impact of dysbiosis on the development of HCC in STAM mice, STAM mice were orally administrated triple antibiotics, including aminobenzyl penicillin (ABPC), neomycin (NEO), and vancomycin (VCM), to establish intestinal sterilization (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-42334-y#Fig6">6a</a>).
supporting the contribution of the certain bacteria in the gut flora of STAM mice
promoted the development of tumor in the liver in STAM mice
the vehicle treatment might have a beneficial effect thereby diminishing the difference by the treatment
These data support the possibility that certain bacteria including B
massiliensis promoted liver tumor development in this model
although gnotobiotic experiments are needed to demonstrate specific mechanisms
In ileum of STAM mice, microscopic analysis revealed fewer eosinophilic granules and decreased alucian blue positive granules in crypt, which were ameliorated by insulin treatment in STAM mice, but not in ieIRKO-STAM mice (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-42334-y#Fig7">7b, c</a>)
These microscopic findings in crypt granules
which represented antimicrobial peptides (AMPs) in Paneth cells
suggested that ieIR signaling deficiency resulted in dysfunctional AMPs production in Paneth cells
suggesting that Akt-TSC2/mTORC1 signal facilitates AMP production in intestinal epithelium
these data suggest that insulin action in intestinal epithelial cells plays a protective role against the development of hepatocellular carcinoma associated with diabetes and NASH
while the number of patients with viral hepatitis has been continuously decreasing
Given that diabetes is a known strong risk factor for NAFLD/NASH
management of diabetes may prove vital to suppressing the occurrence of HCC
suggesting that inhibition of SGLT may be beneficial for the suppression of NASH/NCC associated with severe insulin resistance
Since diabetes is caused by decreased insulin action in various tissues
impaired insulin action in the liver or other tissues may lead to carcinogenesis supported by driver mutations in STAM mice and in patients with diabetes and NASH
insulin treatment suppresses HCC in STAM mice
insulin treatment ameliorates the Warburg effect with the associated reduction of HIF-1α and its downstream molecules
This may contribute to the suppression of tumor growth
but may not be sufficient for preventing tumor development
insulin action in intestinal epithelial cells may be impaired
and patients comorbid with NASH could be susceptible to HCC
the current study revealed that supplementation of insulin effect in the gut recovers expression of AMPs and ameliorates dysbiosis through insulin receptor in the gut
massiliensis plays a role in promoting HCC in this model
Although isolation and implantation to germ-free mice are needed to assert robust conclusions
it is extremely difficult to isolate this specific bacterium
The vulnerability of germ-free mice and hygienic control against polyurea and high fat diet feeding to generate STAM mice in germ-free isolators are other technical issues to conquer
We could not determine any species with statistical significance in non-biased differential analysis such as ANCOM
and many clinical parameters also remain unknown in the human study
further efforts using animal models are needed
and the collection of various specimens and clinical data from patients with NASH and diabetes are also required to clarify the direct relationship between the specific species and HCC development in the future
intestinal insulin signaling plays a protective role against the development of nonalcoholic steatohepatitis and hepatocellular carcinoma associated with diabetes in mice
and treatments leading to the preservation of insulin action in the gut of patients with diabetes could be useful for prevention of NASH and HCC
Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Center for Global Health and Medicine (approved protocol number; Med-P16-113
Med-P16-114) and the Animal Care Committee of the University of Tokyo (approved protocol number; 21077
All relevant ethical guidelines were followed
The experiment involving administration of insulin or phlorizin until the age of 20 weeks (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41467-023-42334-y#Fig2">2a</a>) was performed on request of the University of Tokyo at Stelic Institute &amp; Co.
and the other experiments were performed at the University of Tokyo and Research Institute of the National Center for Global Health and Medicine
The mice were euthanized when we found severe impairment of activity and decrease of more than 25% of body weight independent of tumor size to save animal welfare
All mice were housed under a 12-hour light/12-hour dark cycle at macroenvironmental temperature and humidity ranges of 20 to 22 °C and 40 to 60%
and had free access to sterile water and pellet food
Except for STAMTM mice and diet-induced-obese mice
Insulin or phlorizin (PHZ) treatment was implemented by subcutaneous injection twice daily (b.i.d.) from 6 weeks of age
Sanofi Aventis) was diluted in citrate buffer (100 mM
Novo Nordisc) was diluted in normal saline
The injection dose was determined by blood glucose level
and was gradually increased until 12 U/kg BW (insulin glargine for study at 20 weeks of age)
50U/kgBW (insulin glargine for study at 9 weeks of age)
12U/kgBW (insulin degludec for study at 20 weeks of age)
PHZ (Sigma) was dissolved in a solution containing 10% ethanol
and 75% saline and was injected subcutaneously (0.4 g/kg) twice daily
In the study involving insulin-treated ieIRKO-STAM mice
control mice were treated with normal saline
To elucidate how these drugs influence the prognosis of these model mice
we planned both long- and short-term studies
The long-term study was implemented until 20 weeks of age when the mice reached the phase where HCC became evident or the hepatic “burn-out” phase
The short-term study was performed until they reached 9 weeks of age
when the mice exhibited steatosis and fibrosis with increased expression level of relevant genes that allowed for appropriate analysis of treatment effects on changes in gene profiling
Antibiotic treatment was performed by adding 1 g/L of ampicillin (Nacalai)
0.5 g/L of vancomycin (WAKO) to drinking water
in C57B/6 J mice from 6 weeks of age to 20 weeks of age
Fecal microbiota transplantation (FMT) was conducted after 5-days treatment of triple antibiotics in C57B/6 J mice
Immediately after quitting antibiotics administration
fresh fecal suspension in normal saline (0.1 g/mL
supernatant obtained by centrifuge at 600 x g for 1 minute) was administrated by oral gavage once a day for the first 2 weeks
oral gavage was carried out three times a week
FMT was conducted from 6 weeks of age to 20 weeks of age
C57B/6 J mice at the age of 8 weeks were used
For inhibition of bile acid hydrolase (BSH)
Medchem express) was fed by oral gavage (20.8 mg/kgBW) with vehicle once a day
The vehicle consisted of 10% DMSO and 18% Sulfobutylether-β-Cyclodextrin (SBE-beta-CD
GR-7 was given mice from the age of 6 weeks to the age of 20 weeks
The mice in control group were given vehicle
the difference with or without treatment 0.154
Mice were euthanized by cervical dislocation
and tissues were quickly removed and frozen instantly in liquid nitrogen
RNA was extracted using a tissue total RNA mini kit (FAVORGEN). Real-time quantitative PCR was performed using a KAPA SYBR Fast qPCR kit (KAPA BIOSYSTEMS) or a Taqman probe (ABI). Expression levels were normalized to that of the 18 S rRNA gene. Primer sequences are shown in Supplementary Data <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-023-42334-y#MOESM4">1</a> in this article
The specific gene expression patterns in the liver of STAM mice were examined by Agilent Expression Array analysis on request of the researchers by Takara Bio Inc
Japan) from total liver RNA with SurePrint G3 Mouse GE microarray 8 x 60K
Total RNA was extracted from snap frozen liver samples from three STAM mice at 20 weeks of age in each group with Qiagen RNeasy Mini Kit
according to the manufacturer’s information
target samples were prepared with Low Input Quick Amp Labeling Kit
They were hybridized with Gene Expression Hybridization Kit (Agilent) and washed with Gene Expression Wash Buffers Pack (Agilent)
GO enrichments were performed using Metascape (version 3.0) and GO TRRUST (version 2)
To explore related signaling pathways in the liver of STAM mice
comprehensive hepatic gene expression analyses were performed by microarray between normal mice
and insulin-treated STAM mice at HCC phase (20 weeks of age)
Snap-frozen liver samples were prepared from three or four STAM mice in each group at 9 weeks and 20 weeks of age
approximately 40 mg of frozen liver was immersed into 750 μL of 50% acetonitrile/Milli-Q water containing internal standards at 0 °C to inactivate enzymes
The tissue was then homogenized and centrifuged (2300 × g
the upper aqueous layer was centrifugally filtered to remove proteins
The filtrate was vaporized and resuspended in water for capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis
approximately 45 mg of frozen liver was immersed into 1% formic acid in acetonitrile containing internal standards and homogenized
followed by addition of 167 μL Milli-Q water
The supernatants were added to the former one
and phospholipids removed with the solid phase extraction method
The filtrate was vaporized and resuspended in 100 μL 50% isopropanol (v/v) for LC-TOF/MS
Cationic compounds were measured in the positive mode of metabolome analysis using Agilent 1200 series RRLC system SL (Agilent Technologies)
and anionic compounds were measured in the negative mode of the same system
developed by Keio University) and SampleStat (version 3.14) respectively
16S ribosomal RNA was analyzed using fecal DNA extracted using QIAamp Fast DNA Stool Mini Kit (QIAGEN)
Human 16 S metagenomic study was performed in the University of Tokyo Hospital
Department of Diabetes and Metabolic Diseases
The 16 S rRNA sequencing library was constructed according to the Illumina 16 S Metagenomic Sequencing Library Preparation protocol (Illumina) targeting the V3 and V4 hypervariable regions of the 16 S rRNA genes using primers 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 805 R (5′-GACTACHVGGGTATCTAATCC-3′),
KAPA HiFi HotStart Ready Mix (KAPA) was used for the PCRs (95 C
The sequence of primers with illumina adaptors were TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG and GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG
Index PCR was performed with these amplicons with Nextera XT Index Kit v2 Set A (Illumina) to produce 16 S rRNA amplicon library
The proportion of spiked-in PhiX control was 20%
For 2 × 300 bp paired-end sequencing of the V3 and V4 hypervariable regions on the MiSeq platform
Illumina MiSeq v3 Reagent Kit was used in each experiment
DNA sequences were processed using Miseq Control Software (MCS)
and BaseSpace Onsite 16 S Metagenomics App version 1.1.0 (Illumina
and the bacterial abundance was expressed as read proportion to all read
Primary component analysis in human study was conducted by Graphpad Prism 9.5.0 software
Only in the validation of the experiment in which FMT from STAM was conducted
and sequenced in 2 x 150 bp paired-end with iSeq100 system
Akkermansia muciniphila(AY271254), Bacteroides massiliensis(AY126616), Prevotellamassilia timonensis(NR144750.1), Acetatifactor muris(HM989805), Bacteroides acidifaciens(AB021164), Muribaculum intestinale(NR_144616.1) was selected by following criteria in Supplementary Fig. <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-023-42334-y#MOESM1">15c</a>
ratio of Control-STAM-INS to control-STAM-nonINS was less than 1
and the maximum group mean was more than 5%
and species count in csv format generated from BaseSpace 16 S Metagenomics App is shown in Source Data
Secondary HRP-conjugated antibodies (SantaCruz
Quantitative densitometric analysis was exerted by gel analyzer in ImageJ software (version 1.52)
cholesterol and NEFA were measured using LabAssay™ Triglyceride and NEFA (FujiFilm
Insulin was measured using a high-sensitive insulin ELISA kit (Morinaga)
Plasma AFP was measured using Mouse alpha-Fetoprotein/AFP Quantikine ELISA Kit (R&amp;D SYSTEMS) Plasma LPS was measured using LPS ELISA Kit (CUSABIO)For FD-4 (Fluorescein isothiocyanate–dextran– 4 kDa) assay
mice were fasted 4 hours before oral feeding and for the duration of the experiment
4 kDa FITC-Dextran (dissolved at 40 mg/mL in PBS
administrated at 20uL/gBW) was fed by oral gavage
and fluorescence intensity at Excitation: 490 nm and Emission: 530 nm was read
Sirius red positive area proportion was measured by Image J (version 1.52) in 3 images per mouse on request of the researchers at Stelic Institute &amp; Co.
Intestinal samples were also stained with PAS-alucian blue stain
Referring to previous report<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 37" title="Kawashima, R. et al. Interleukin-13 damages intestinal mucosa via TWEAK and Fn14 in mice-a pathway associated with ulcerative colitis. Gastroenterology 141, 2119–2129.e2118 (2011)." href="/articles/s41467-023-42334-y#ref-CR37" id="ref-link-section-d17650552e3375">37</a>
the mouse at 8 weeks of age ileal tissues were washed with phosphate buffered saline and cut into 1-cm lengths
Tissues were then cultured with 100 nM of human recombinant insulin (Humarin R
Further information on research design is available in the <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41467-023-42334-y#MOESM7">Nature Portfolio Reporting Summary</a> linked to this article
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This work has been funded by a grant for the Research Program on Hepatitis from Japan Agency for Medical Research and Development (JP17fk0210304
JP21fk0210090 to K.U.); a Grant-in-Aid for Challenging Exploratory Research (21659227
Ronald Kan (Joslin Diabetes Center) and Dr
Morris Birnbaum (Pfizer) for sharing Akt1 floxed mice and Akt2 floxed mice
Yasuko Ota for their technical assistance with animal care
Minori Matsuda for the excellent secretarial work
National Center for Global Health and Medicine) for the assistance of morphological microscopic observation
Yuki Kawamura (Department of Gastroenterology
National Center for Global Health and Medicine) for her advice on ex vivo ielum experiments
National Center for Global Health and Medicine
Diabetes and Metabolism Information Center
RIKEN Center for Integrative Medical Sciences
Department of Prevention of Diabetes and Lifestyle-Related Diseases
designed the study and wrote the manuscript; K.S
performed and analyzed most of the experiments; K.E.
performed liver biopsies from NASH-suspected patients; M.K.
acquired and analyzed whole exome sequence data; K.H.
The authors declare no competing interests
Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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<a href="https://www.houyhnhnm.jp/en/news/797059/" rel="noopener" target="_blank">Last December, Lacoste opened a store in Harajuku, Tokyo.</a>In addition to selling new clothing and shoes
the store also offers a polo shirt customization service
and has a café where customers can enjoy drinks and snacks
all of which add to the brand's multifaceted appeal
One of the contents of the Harajuku store is the "My Lacoste Artist Program"
This is a project to release limited-edition collections with crocodile motifs by teaming up with artists who are active in a variety of fields
This third installment of the series features Nana Soeda
For those who are not familiar with her work
she creates two-dimensional works and animations based on the theme of cheapness
influenced by toys and counterfeit mascot characters found in Asian shopping malls
The dark characters depicted in her works are cute
<a href="https://www.houyhnhnm.jp/en/news/880182/?image_page=2" class="image-page-link"></a>
Each one has a unique graphic touch with the word "Harajuku" attached to it
<a href="https://www.houyhnhnm.jp/en/news/880182/?image_page=4" class="image-page-link"></a>
<a href="https://www.houyhnhnm.jp/en/news/880182/?image_page=5" class="image-page-link"></a>
<a href="https://www.houyhnhnm.jp/en/news/880182/?image_page=6" class="image-page-link"></a>
<a href="https://www.houyhnhnm.jp/en/news/880182/?image_page=7" class="image-page-link"></a>
Please note that they are only available at the Harajuku store
SIE Shanghai vice chairman explains the company&#39;s struggles
triumphs at gaining a foothold in the Chinese market
When it first launched the PlayStation 4 in China
Sony was effectively starting from nothing
The country's restrictions on consoles had only just been lifted when the system arrived there in 2015
console gaming effectively had zero market presence
That meant that Sony Interactive Entertainment Shanghai vice chairman and president Takehito Soeda had to find ways to build an entire console market
"This country has the single largest legal market in the world for software, mobile, computer and console gaming," Soeda said <a href="https://www.theceomagazine.com/executive-interviews/it-electronics/takehito-soeda/">in an interview with CEO Magazine</a> on the company's efforts in China
"Because console gaming was illegal for so long
we started operations with a market share that was practically zero
"...We had to develop entire networks from scratch
we went back to where we started and founded an entire business."
Soeda says some examples of what Sony did included storefront demos of games and various trade shows for both business partners and consumers that offered the opportunity for people to get a look at exactly what the PS4 was and what kinds of experiences they could play on it
Soeda goes on to say that despite the ban on consoles being lifted allowing the PS4 to have a presence in the country
it's nonetheless been challenging due to issues with censors
as well as a lack of any console business groundwork there to begin with
you can release a game with minimal interaction with censors," he said
"...We're only able to get 30 or 40 games out a year
We want more content available in the market
and that in turn means more opportunities for us."
One response Sony has had to this has been to work with Chinese developers to release games that implement elements of local culture and build relationships within the industry there
"China hasn't had decades of console gaming like everywhere else in the world
so we need to work together to cultivate and grow the console gaming segment," he concludes
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Although China has the biggest gaming market in the world
its audience for console gaming is one of the most underdeveloped due to a government ban implemented in 2000
Worried about the effects console gaming may have on children, industry stalwarts such as <a href="https://asia.playstation.com.cn/chs-cn/" rel="noopener" target="_blank">Sony Interactive Entertainment</a> (SIE) were for years barred from selling consoles to the Chinese mainland
with companies such as Nintendo entering into joint ventures to circumvent the rules
While regulation remains a challenge in the country
the lifting of the ban in 2015 showed an eagerness to embrace an industry that exceeded US$135 billion globally in revenue in 2018
“You don’t see a lot of industry-wide gaming names in China,” concedes Takehito Soeda
Shanghai-based regional Vice-Chairman of Sony Interactive Entertainment Shanghai
and that’s why I’ve been interested in bringing the PlayStation 4 (PS4) to the Chinese market
This country has the single largest legal market in the world for software
Because console gaming was illegal for so long
we started operations with a market share that was practically zero.”
Having originally worked with Sony between 1992 and 2006
with his current tenure coinciding with the easing of restrictions on console gaming in China
“The company knew my background was a good fit and that once they had done the research and met with prospective partners
Sony’s latest iteration of its home gaming console
to the Chinese market was a daunting challenge
he engaged in extensive negotiations with regulators to assuage concerns about games that would appear on the console
you can release a game with minimal interaction with censors,” he says
Even when software was approved by government regulators
SIE lacked partnerships and local infrastructure
Takehito says getting games on shelves became a difficult task
“We had to develop entire network s from scratch
we went back to where we started and founded an entire business.” He says that to build interest in the console
he thought of ways to have customers engage with the hardware firsthand
see the graphics the PS4 can process and engage with the game’s story and style of play
they begin to understand how it’s different from PC or mobile gaming,” Takehito says
“It’s the content of the games that’s attracting people
We knew that if we were going to engage customers
we had to let them experience the games before buying them
we opened storefront demonstrations where customers could come
pick up a controller and play a game for 10 minutes to get a feel for it
Then we had trade industry shows that engaged businesses and customers
and they were important for inviting new players to our consoles
We rolled that into product demonstrations in shopping malls on weekends
where we built small stages in stores and let people have a go with the games,” he says
we were accelerating the evolution of the market
There are a lot of customers who simply aren’t familiar with console gaming
and this was our way of letting them know the uniqueness and beauty behind it.”
Takehito’s efforts have yielded strong results for the company
helping drive cumulative worldwide sales of 91.6 million PS4s as of the end of 2018
with strict censoring reducing the number of games regionally available for the console
That’s why SIE has focused on partnering with local publishers to develop games that appeal specifically to the market
“We’re only able to get 30 or 40 games out a year
and that in turn means more opportunities for us
“When we work with local developers to make local games
It’s like the distinction between a locally made film and a Hollywood movie
but sometimes you want something with which you are familiar and where you can recognise a touch of your own culture.”
All of this is part of developing a workable strategy for console gaming in the region
we need to reach new customers,” Takehito says
“We need a sustainable and growing market and we want customers to have more choices
not only for mobile and PC games but also in the console space,” he explains
“China hasn’t had decades of console gaming like everywhere else in the world
so we need to work together to cultivate and grow the console gaming segment.”
Takehito sees no reason why the Chinese market can’t catch up with the West
“The PlayStation has only been around for a few years in China,” he says
with an international catalogue from which to draw on
selling to a country whose economy is opening and growing.”  
The CEO Magazine is more than a business title; it’s a source of information
inspiration and motivation for the world’s most successful leaders
Learn all about The CEO Magazine at <a style="color: #fff; text-decoration: underline !important;" href="https://theceo.com/" target="_blank" rel="noopener">TheCEO.com</a>
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Japan – Hiromi Soeda always had trouble hearing what people were saying
whether they were her teachers at school or – later – clients at the hair salon where she worked
she struggled to hear her children over domestic noises like a ventilator fan or running water.&nbsp;&nbsp;
Doctors could find nothing wrong with her ears
was diagnosed with Auditory Processing Disorder (APD)
a form of Listening Difficulty (LiD) where the brain can’t process the words one is hearing.&nbsp;
With low public awareness of the condition in Japan
those with APD say they can feel lonely or isolated
and have trouble keeping a job or simply taking part in daily interactions.&nbsp;
“I’m just nodding and pretending I understand
Sometimes I get the time wrong for meeting people
“They just drift away because they think I can’t keep a promise.”&nbsp;
While the YYProbe app is used by the wider community of people who are deaf or hard of hearing
a new generative AI-powered summarization feature provided by Microsoft Azure OpenAI Service is particularly helpful for those with APD.&nbsp;
Generative AI tools are built on large language models (LLMs) that synthesize troves of data to generate text
when her mother was hospitalized with Covid-19
Soeda used the app to understand what doctors were telling her
Doctors subsequently discovered her mother had other ailments
including Parkinson’s disease and water in her lungs and had suffered a cerebral infarction.&nbsp;&nbsp;
Soeda used YYProbe on a tablet to follow what doctors were saying
summarize the information and send the transcript to her younger sister.&nbsp;&nbsp;
“It’s much better to read [the text] to follow and help my understanding,” she said
is known primarily as a manufacturer of automotive components
initially developed YYProbe during the pandemic as a speech-to-text tool for all employees to create business records
Aisin employees who were deaf or hard of hearing found it particularly useful
The team went on to develop an audio recognition system called YYSystem
as a tool for wider society which could be used by people who are hard of hearing
as well as a free version which has more than 10,000 active monthly users
These include those with listening difficulties
though Nakamura says it’s hard to know the breakdown
brought summarization and translation abilities.&nbsp;&nbsp;&nbsp;
YYSystem is also deployed via counter-top screens at government departments and retail stores and will be used by spectators at the 2025 Deaflympics in Tokyo.&nbsp;
Globally, between two and 10 percent of children have APD, and it is more common in children with other learning or developmental disabilities, according to the <a href="https://www.who.int/publications/i/item/9789240020481" target="_blank" rel="noreferrer noopener">World Report on Hearing</a>
published by the World Health Organization in 2021
Japan has a fairly well-developed network of schools for people who are deaf or hard of hearing
and it also has legislation to protect those with disabilities from discrimination in the workplace
Both people who are deaf or hard of hearing and those with APD are often reluctant to admit they need help
“Japanese people do not like troubling other people,” said Kaori Nasu, president of <a href="https://4hearts.net/about-us/" target="_blank" rel="noreferrer noopener">4Hearts</a>
an advocacy organization that aims to break down communication barriers – including for people who are deaf or hard of hearing – in Japan
“Sometimes you just give up trying to get involved in the conversation or just keep smiling even though you don’t understand what is being said.” 
Those who wear hearing aids often hide them under their hair
“That person does not have the information to make a judgement
4Hearts runs awareness and empathy workshops in government departments
Participants are given ear plugs and headphones with loud static
so they can experience what it’s like to be deaf or hard of hearing
and then come together to think about what they can do.&nbsp;
The community is starting to step out of the shadows.&nbsp;&nbsp;
a group of about 300 members of the deaf community
organizes outings to watch a pro volleyball league where YYSystem is hooked up to the arena’s sound system and transcribes the sounds from the venue
“People who cannot hear or [find it] hard to hear can have a fuller experience of viewing sports,&#8221; said volunteer Taiyo Akashi.&nbsp;
A Japanese sign-language band named <a href="http://kokoro-oto.com/biography" target="_blank" rel="noreferrer noopener">Kokoro Oto</a> performs pop, hip hop and rock at live music venues, offering those who are deaf or hard of hearing a chance to experience live music. When she’s not performing, sign-language vocalist Kuniko Nishimaki, who is deaf, uses the YYProbe app to navigate convenience stores and has used the summarization function to keep up in parent-teacher meetings.&nbsp;
Growing up, Soeda did well in elementary school as she could read what the teacher wrote on the blackboard. Gym class was harder. “I couldn’t understand verbal instructions,” she said. “The teacher would think I was joking around and not being serious.”&nbsp;
In high school, when teaching moved to lecture mode, Soeda struggled. She ended up going to beauty school and began working as a hairdresser in a salon. But loud hair dryers and surrounding clatter made it hard for her to chat with customers, which was part of the job. “The owner of the salon told me it’s not working out,” she said.&nbsp;
Subsequent stints at a noisy manufacturing plant and at a chain restaurant, where she had to wear headphones to get instructions from a supervisor, didn’t last either. She now waits tables at a small restaurant.&nbsp;
Three years ago, Soeda came upon an APD activist on the internet who had been featured on national TV and who had a checklist for APD symptoms. “I did the checklist and thought – this really sounds like me.” That was how Soeda came to be diagnosed by Dr Koji Hirano, an APD expert who wrote a book titled, “I can hear it, but I can’t hear it.”
Today, Soeda runs an online LiD/APD parent support group with 123 members, including doctors and others who work in the field. Since there is no cure, they discuss ways to mitigate the effects, for example, advocating for kids to be able to bring devices into classrooms to help them learn.&nbsp;
They also work with app developers. In May this year, Soeda’s parent support group visited Aisin’s research and development office in Akihabara, the video gaming and anime hub in Tokyo, and met with Nakamura, the developer of YYSystem. Nakamura says he is in constant contact with users and regularly adds features based on their requests – “I don’t actually sleep! I am always writing code!”
Soeda’s group suggested wider line spacing and shorter sentences, as well as different colored text to denote different speakers – changes that have been adopted.&nbsp;
These days, Soeda uses YYProbe for seminars that she runs for her APD support group. And she uses it for fun – when out for drinks with friends at the local izakaya.&nbsp;
“It’s quite noisy inside,” she said. “When we have several people together, I’m in trouble.” The app helps her follow the conversation and translates music, laughter and clapping as simple emoticons on the screen.&nbsp;
In the future, said Nakamura, the app will go beyond text and speech, so users can input as well as generate pictures and videos and graphs to communicate. Generative AI is already making this possible.&nbsp;
Top image: Hiromi Soeda, who has Auditory Processing Disorder, chats using the YYProbe app with Minori Oba, who works for Aisin, maker of the app. Photo by Noriko Hayashi for Microsoft.
Text description provided by the architects. The site is located at 300 meters from Tokyo Bay. This apartment building stands on a wholesale block marked by the urban grid on the land that had been reclaimed 50 years ago. 
Located in close proximity to the sea, it is our top priority to protect the building from strong sea breezes and salt damage. Selected materials are concrete and glass only, without corrosive materials such as steel and stainless steel. 
©  Takumi Ota</a>The building consists of housing units sandwiched between four walls made of concrete and glass
Construction joints of concrete are erased so that expression of materials is emphasized more
and each unit is designed to be adaptable to different life styles of residents
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MARGE FUNASAKI general manager of Mid-Town Radio/Disco Mart
When Mid-Town Radio/ Disco Mart opened in 1947
Harry Truman was in the second year of his presidency
Jackie Robinson made history as the first African-American to play Major League Baseball and Miracle on 34th Street premiered in movie theaters
Mid-Town Radio/Disco Mart celebrated its 75th anniversary
and we extend our profound gratitude to the people of Hawai‘i for allowing us to serve you for three-quarters of a century
Our story is a familiar one in the islands
emigrated from Japan to work at the Waipahu Sugar Mill
and later opened what was then a small radio repair and electronics shop on Depot Road
They were the mom and pop of our family’s “mom and pop” shop
and began selling furniture as well as appliances and electronics
“Disco” is an abbreviation of the word “discount” but humorously caused some confusion during the 1970s for young folks thinking our big warehouse
Mid-Town Radio/Disco Mart celebrates its 75th anniversary
Ehukai Wong and owner/general manager Marge Funasaki
we have worked each day to continue our parents’ legacy and provide the friendly
local customer service that is so special in Hawai‘i
we understand the important role we play in supporting our community and have had the good fortune of caring for and providing quality furniture and appliances to multiple generations of island families at the best prices in town (or country)
Mahalo nui loa to our generations of loyal customers
The Yamamoto family is proud to have been a part of your lives for the past 75 years and honored to continue serving you and our incredible island home
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A Swedish agency for grants for faith institutions said Thursday it was cutting support to the Russian Orthodox Church
after Sweden's intelligence service warned the church was used for intelligence activities
The Swedish Agency for Support for Faith Communities said in a statement that it was also cutting financial support for the Church
for not living up to its "democracy criteria."
It said that Sweden's Security Service (Sapo) believed the Church was used by the Russian state "as a platform for gathering intelligence and other security-threatening activities."
"In the Swedish Security Service's remarks
it appears that representatives of the religious community have had contact with people who work for Russian security and intelligence services," the agency said in a statement
It added that the Church had received significant funding from the Russian state
and that representatives had acted in a manner that seemed to encourage "support for Russia's invasion of Ukraine."
The agency also noted that the Russian Church denied the allegations made against it
The Moscow Patriarchate was already among the smaller recipients of grants for faith-based institutions
and in 2022 the Church received just under 200,000 kronor ($19,300) from the Swedish state
In its annual assessment published last week
Sapo pointed to Russia as one of the main threats to Sweden
a former Swedish intelligence officer was handed a life sentence for spying for Russia
a Russian-Swedish national went on trial accused of passing Western technology to Russia's military
A Stockholm court found he had exported the material but ruled his actions did not amount to intelligence gathering
Sweden also dropped two centuries of military non-alignment and applied for NATO membership in the wake of Russia's invasion of Ukraine
and is expecting to become a full member within days after the last holdout Hungary ratified the country's membership on Monday
Russia's Prosecutor General's Office has designated The Moscow Times as an "undesirable" organization
criminalizing our work and putting our staff at risk of prosecution
This follows our earlier unjust labeling as a "foreign agent."
These actions are direct attempts to silence independent journalism in Russia
The authorities claim our work "discredits the decisions of the Russian leadership." We see things differently: we strive to provide accurate
We, the journalists of The Moscow Times, refuse to be silenced. But to continue our work, <a href="https://www.themoscowtimes.com/contribute?utm_source=contribute&utm_medium=article" title="we need your help">we need your help</a>
please support us monthly starting from just $2
and every contribution makes a significant impact
independent journalism in the face of repression
August 10, 2022 by <a href="https://theirturn.net/author/donny/" title="Posts by Donny Moss" rel="author">Donny Moss</a> &mdash; <a href="https://theirturn.net/2022/08/10/activists-protest-humane-society-animal-warehousing/#comments">Leave a Comment</a>
Animal rights activists staged a protest at the Humane Society of New York (HSNY) to sound the alarm about the plight of the animals who have been warehoused at the Manhattan shelter for 28 months
the activists spoke to dozens of area residents and clients of HSNY’s vet clinic
who are not allowed in the building with their companion animals for exams or euthanasia appointments
They also confronted several employees exiting the building
Many of the interactions were caught on camera
The protest comes 13 months after a whistleblower at the HSNY informed local animal rights activists that adoptions had come to a virtual standstill since the HSNY closed its building to the public in April 2020
The whistleblower also reported that the HSNY’s Executive Director
is keeping the building closed to the public under false pretenses
DeFeo claims that the building is closed due to COVID
according to the whistleblower and lawyers advising the activists
she cannot reopen the building because of violations of the Americans with Disabilities Act (ADA)
The HSNY already settled one ADA lawsuit and
unless it takes steps to make the building wheelchair accessible according to ADA guidelines
it cannot reopen to the public without subjecting itself to another
Animal rights activists are calling on the Humane Society of New York
which has been closed to the public for 28 months
to send the animals to adoption centers that are open to adopters
activists spoke to several area residents who said that they attempted to adopt from the HSNY in recent months but were either ignored or turned away
These and other neighbors said they were perplexed by the ongoing closure of the building and disturbed to learn that the building is filled with homeless animals who hadn’t been seen by adopters in over two years
Clients of the vet clinic who spoke to the activists expressed frustration about not being able to be with their companion animals during exams and about being forced to wait outside in the extreme heat instead of in the HSNY&#8217;s air-conditioned lobby
the Today Show and several other mainstream media outlets reported on an animal warehousing protest against the Humane Society of New York during Broadway Barks
a star-studded adoption event during which Bill Berloni
Activists attempted to speak to two of the HSNY’s staff veterinarians as they exited the building
Soeda used a hand gesture to dismiss the activists and their concerns about the animals
“Veterinarians take an oath to relieve animal suffering,” said Christina Fritz
a former client of the HSNY’s vet clinic who joined the activists
&#8220;Turning a blind eye to the cats and dogs who have been stuck in cages right under their noses makes them complicit in the cruelty.”
veterinarians at the Humane Society of New York&#8217;s vet clinic
ignore the activists attempting to talk to them about animal warehousing at organization&#8217;s adoption center
During a call in July 2022, DeFeo told Fritz that the HSNY had adopted out about <a href="https://www.facebook.com/groups/534733101036426/posts/630782484764820/">150 animals</a> since closing its building to the public 27 months earlier &#8212; in April 2020
then the HSNY has sent home an average of 1.4 animals per week
wealthy shelter in Manhattan that claims on its tax forms to have 125 &#8211; 175 animals
In an apparent effort to minimize the number of adoptions she facilitates while keeping the Humane Society of New York closed to the public
Executive Director Sandra DeFeo lists just 14 animals on its website and does virtually no adoption promotion on social media
“The Humane Society of New York is located in a bustling neighborhood at the crossroads of the Upper East Side and Midtown,” said Matthew Schwartz
a New Yorker whose adoption application was ignored by the HSNY
“A shelter in that location with ample resources should be doing adoptions every day.”
no adopters have entered the Humane Society of New York to meet the animals who need homes because the Executive Director
refuses to make the building wheelchair accessible
If she reopens the building without making the renovations
then she would be subjecting the organization to another ADA lawsuit
recorded a testimonial about the plight of the animals being warehoused
has since joined dozens of animal rights activists in NYC calling on the HSNY to send the animals to foster homes and/or adoption centers where adopters can meet them
“Twenty eight months is far too long for homeless animals to not be seen by adopters who would otherwise be rescuing them,” said Tischler
because the HSNY lists just a small fraction of its animals on its website and does little adoption promotion on social media
potential adopters don’t even know that the animals exist.”
During a phone call in August 2021, Donny Moss of TheirTurn asked DeFeo, the Executive Director, to send the adoptable animals to shelters that are open to the public or to foster homes. In response, she claimed that she has been “doing adoptions all along,” which Moss knew to be false based on his own <a href="https://theirturn.net/2021/09/25/humane-society-new-york-warehousing-animals/">two-month investigation</a>
DeFeo also said that the shelter is the animals’ foster home; that their cages are like “apartments;” and that she would barricade the building if anyone tried to take out the animals
“My heart sank when I heard the Director of an animal shelter describe cages as ‘apartments,’” said Moss
“Shelters are stressful for animals and are no substitute for loving homes where they can roam freely
Forcing cats and dogs to live in cages indefinitely is animal cruelty.&#8221;
Three members of the Board of Directors of the Humane Society of New York have resigned amid the warehousing scandal &#8211; photographer Alexandra Rowley and attorneys C
Since launching the campaign to help the animals being warehoused at the HSNY, activists have staged seven protests — five at the home of <a href="https://theirturn.net/2022/04/20/alexandra-rowley-humane-society-animal-warehousing/">Alexandra Rowley</a>
a board member who resigned amid the protests; one at Broadway Barks
an adoption event in the theater district; and one at the HSNY
Activists say they will continue to protest the HSNY and the individuals enabling the organization&#8217;s bad behavior until DeFeo and her board of directors send the adoptable animals to adoption centers that are open to adopters
 Filed under: <a href="https://theirturn.net/category/companion-animals/" rel="category tag">Companion Animals</a>
Sandra DeFeo will state that &#8220;we&#8217;ve been doing adoptions all along&#8221; and that she doesn&#8217;t know what &#8220;warehousing&#8221; even means and that they&#8217;re very particular about who they adopt to
but that&#8217;s not what is happening at all
They list just 14 of their animals; do hardly any adoption promotion and ignore the few adoption applications that do come in
She simply can&#8217;t be bothered with processing adoptions while the building is closed
and she won&#8217;t send the animals to adoption centers that are open because that would be acknowledging the problem
 Click link to article below for more info before calling
An organization that&#8217;s supposed to be taking care of animals is instead warehousing them in cages
How can people who can&#8217;t make it up to the protests contact Sandra DeFao and demand that the animals be sent to shelters that are adopting out the animals
Governor Kathy Hocul is refusing to sign a bill (by Linda Rosenthal) that would stop puppy mill and kitten mill animals from being sold in pet stores
This of course exacerbates the problem of there not being enough adoptive homes for shelter animals
I have left a message on Governor Hocul&#8217;s site saying that I will not vote for her in November if she doesn&#8217;t sign the bill
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PET radioligands with low molar activity (MA) may underestimate the quantity of the target of interest because of competitive binding of the target with unlabeled ligand
The aim of this study was to evaluate the change in the whole-body distribution of 18F-PSMA-1007 targeting prostate-specific membrane antigen (PSMA) when solutions with different peptide concentrations are used
Methods: Mouse xenograft models of LNCaP (PSMA-positive prostate cancer) (n = 18) were prepared and divided into 3 groups according to the peptide concentration injected: a high-MA group (1,013 ± 146 GBq/μmol; n = 6)
a medium-MA group (100.7 ± 23.1 GBq/μmol; n = 6)
and a low-MA group (10.80 ± 2.84 GBq/μmol; n = 6)
Static PET scans were performed 1 h after injection (scan duration
SUVmean in tumor and normal organs was compared by the multiple-comparison test
Immunohistochemical staining and Western blot analysis were performed to confirm expression of PSMA in tumor
1.12 ± 0.30) showed significantly lower uptake of 18F-PSMA-1007 in tumor than did the high-MA group (1.97 ± 0.77) and the medium-MA group (1.81 ± 0.57)
0.24 ± 0.04) and the medium-MA group (0.57 ± 0.08) showed significantly lower uptake than the high MA group (1.27 ± 0.28)
The tumor-to-salivary gland SUVmean ratio was 1.73 ± 0.55 in the high-MA group
The immunohistochemical staining and Western blot analysis revealed significant overexpression of PSMA in tumor and low expression in salivary gland and kidney
Conclusion: A decrease in the MA level of the injected 18F-PSMA-1007 solution resulted in decreased uptake in tumor and
there is a possibility of minimizing the adverse effects in salivary gland by setting an appropriate MA level in PSMA-targeting therapy
Different peptide concentrations of nonradiolabeled PSMA between diagnostic PET and radionuclide therapy might result in different degrees of radioligand accumulation in the target tissues and normal organs
we evaluated changes in whole-body distribution using 18F-PSMA-1007 solutions with different peptide concentrations to optimize the MA level for subsequent targeted radionuclide therapy
18F-PSMA-1007 was synthesized by a 1-step method optimized using a MPS-200 cassette-type synthesizer (Sumitomo Heavy Industries), based on a previous report (<a id="xref-ref-11-1" class="xref-bibr" href="#ref-11">11</a>)
18F-F− produced by a Cypris-HM-18 cyclotron (Sumitomo Heavy Industries) was trapped on a quaternary methyl amine cartridge (carbonate form) and eluted into a reaction vessel with 0.075 M tetrabutylammonium HCO3 solution (600 μL)
This solution was azeotropically dried with acetonitrile (1 mL)
a dimethylsulfoxide solution of the PSMA-1007 precursor
5-((S)-4-carboxy-1-((S)-4-carboxy-1-(4-((S)-1-((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentylamino)-3-(naphthalen-2-yl)-1-oxopropan-2-ylcarbamoyl)benzylamino)-1-oxobutan-2-ylamino)-1-oxobutan-2-ylcarbamoyl)-N,N,N-trimethylpyridin-2-aminium 2,2,2-acetate (2 mg/2 mL)
was fed into the reaction vessel and fluorination was performed at 95°C for 10 min
The reaction mixture was diluted with 5% ethanol solution (10 mL) and passed through PS-H+ and C18ec cartridges
these cartridges were washed with 5% ethanol solution (23 mL) and 30% ethanol solution (3 mL) for removal of any chemical or radiochemical impurities
18F-PSMA-1007 trapped on the cartridge was finally eluted with 30% ethanol solution (4 mL) into the product vial containing 0.9% NaCl solution (11 mL) plus 100 mg of sodium ascorbate
The radiochemical purity of 18F-PSMA-1007 was 96.9%–97.6%
and the MA was 1,260–1,476 GBq/μmol at the end of the synthesis as analyzed with radio–high-performance liquid chromatography
which was performed on a Chromolith Performance RP-18e (100 × 4.6 mm; Merck) using a Shimazu high-performance liquid chromatography system
95% to 5% from 13 to 19 min); and A + B = 100%
The 18F-PSMA-1007 solution without dilution was used for injection in the high-MA group
nonradiolabeled PSMA-1007 standard (ABX) was added to the 18F-PSMA-1007 solutions (1.5 μg and 11 μg of PSMA-1007 for 1 mL of each 18F-PSMA-1007 solution)
The final MA levels of the 18F-PSMA-1007 solutions for the PET/CT study were 1,013 ± 146 GBq/μmol (high MA)
the final MA levels were 863.6 ± 64.3 GBq/μmol (high MA)
All animal experiments were performed under the guidelines of the Institute of Experimental Animal Sciences
The protocol was approved by the Animal Care and Use Committee of the Osaka University Graduate School of Medicine (approval 25-097-015)
Male NOD/SCID mice (4 wk old) were purchased from Charles River Japan
a prostate cancer cell line derived from a lymph node metastasis in a human
LNCaP cells were cultured in RPMI-1640 medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum (Sigma-Aldrich) at 37°C in a humidified incubator containing 5% CO2
LNCaP cells were collected in a 1:1 mixture (v/v) of medium and Matrigel (Corning) and subcutaneously implanted into either the left or right shoulder of the mice (∼5 × 106 cells were inoculated at each site)
The tumor xenograft models (n = 18; body weight
18.0 ± 2.1 g) were evaluated by small-animal PET/CT 40 d after implantation (tumor volume
Male ICR mice (10 wk old; n = 9; body weight
39.6 ± 2.1 g) were purchased from Japan SLC
and used as a non–tumor-bearing cohort for biodistribution experiments
Data were acquired with a small-animal PET/CT system (Inveon) (<a id="xref-ref-12-1" class="xref-bibr" href="#ref-12">12</a>). The mice were anesthetized with an inhalational mixture of 2% isoflurane plus 100% oxygen, and 18F-PSMA-1007 (16.5 ± 2.7 MBq) was administered through the tail vein. One hour after the injection, and after CT, static PET scanning was performed for 10 min (<a id="xref-ref-13-1" class="xref-bibr" href="#ref-13">13</a>)
All PET data were reconstructed by 3-dimensional ordered-subset expectation maximization (16 subsets
with correction for attenuation and scatter
Kidney and salivary gland obtained from male NOD/SCID mice (6 wk) were also subjected to immunohistochemical staining and Western blot analysis
ICR mice were divided into the high-MA (n = 3)
One hour after injection of solution prepared as described above
and the radioactivities of the samples were measured using a γ-counter (BeWell; Molecular Imaging Laboratory)
The results were described as percentage injected dose
and muscle with reference to the fused CT images
Regional uptake of radioactivity was decay-corrected to the injection time and expressed as SUVmax and SUVmean
corrected for injected dose (MBq) and body weight (g)
SUVmean for tumor and salivary gland was plotted against MA level of the injected radiotracer solution using the Michaelis–Menten model
The Michaelis–Menten constant (KM) and maximum rate (Vmax) were calculated using JMP software (version 14.0.0; SAS Institute Inc.)
Scatchard analyses were performed to obtain maximal binding capacity (Bmax) and dissociation constant (Kd) for tumor and salivary gland
JMP software and SPSS (version 25) were used for the statistical analyses
and the 3 groups were compared using the independent t test with Bonferroni correction and the Tukey honestly-significant-difference test
A P value of less than 0.05 was considered to indicate a significant difference
(A–E) Immunohistochemical staining (high magnification) of tumor in high-MA group (A)
(F and G) Western blot analysis for evaluation of PSMA expression in high-
Urinary excretion of the radiotracer was significantly higher
and kidney and muscle uptake significantly lower
in the low-MA group than in the high- and medium-MA groups
no significant differences among the groups were observed for liver and gallbladder
although a trend toward decreased uptake in these organs was observed with a decrease in the MA level of the injected radiotracer solution
Maximum-intensity projections of 18F-PSMA-1007 PET in xenografts of mice of high-MA (A)
Tumor uptake was lower in low-MA group than in medium- and high-MA groups
(A and B) SUVmean of major organs (A) and SUVmax of tumor (B) on 18F-PSMA-1007 PET at 60 min after injection
(C) Ratios of SUVmean between tumor and salivary gland
The 18F-PSMA-1007 biodistribution in normal mice is shown in <a id="xref-table-wrap-2-1" class="xref-table" href="#T2">Table 2</a>
the medium- and low-MA groups showed a significant decrease in salivary gland and testis uptake
Biodistribution of 18F-PSMA-1007 Solutions with Different Peptide Concentrations 60 Minutes After Administration in Non–Tumor-Bearing Mice
<a id="xref-fig-4-1" class="xref-fig" href="#F4">Figure 4</a> shows the Michaelis–Menten curves for uptake in tumor and salivary gland. KM and Vmax were significantly lower for tumor (8.78 GBq/μmol and 2.07/h, respectively) than for salivary gland (126 GBq/μmol and 1.41/h, respectively). The results of the Scatchard plot analyses are shown in <a id="xref-fig-5-1" class="xref-fig" href="#F5">Figure 5</a> for tumor and salivary gland
Bmax and Kd were not estimated for salivary gland
Michaelis–Menten curves showing relationship between MA level of injected solution and SUV in tumor (A) and salivary gland (B)
Scatchard plot analyses in tumor (A) and salivary gland (B)
analysis failed to obtain Bmax in salivary gland
This study used PET and tissue sampling to evaluate the influence that different peptide concentrations in 18F-PSMA-1007 injection solution have on tissue uptake
Uptake in major organs and tumor was significantly lower
in the low-MA group than in the medium- and high-MA groups
Wurzer et al. recently reported that the MA levels of 68Ga-labeled PSMA inhibitor conjugates had a pronounced influence on the results of PSMA PET imaging (<a id="xref-ref-9-2" class="xref-bibr" href="#ref-9">9</a>,<a id="xref-ref-10-2" class="xref-bibr" href="#ref-10">10</a>)
addition of unlabeled compounds to the injection solution
which changed the MA levels from 1,200 to 8 MBq/nmol
resulted in differential accumulation between tumor and normal organs such as kidney and salivary gland
They concluded that there is a need for careful evaluation of the influence of the nonradiolabeled mass or MA on the tumor-to-organ uptake ratios of PSMA-targeted radiopharmaceuticals
in that we found salivary gland uptake to be significantly decreased
in the medium-MA group compared with the high-MA group
They speculated that non–PSMA-mediated uptake mechanisms
such as megalin/cubilin-mediated tubular reabsorption
Xerostomia, caused by salivary gland uptake of the radioligand, is a major problem in targeted α-therapy using 225Ac-PSMA-617; it significantly impairs the quality of life and sometimes even requires discontinuation of PSMA-targeted treatment (<a id="xref-ref-17-1" class="xref-bibr" href="#ref-17">17</a>,<a id="xref-ref-18-1" class="xref-bibr" href="#ref-18">18</a>)
The present study revealed that adjusting the MA level of the injected radiotracer can reduce salivary gland uptake while maintaining tumor uptake
Such an adjustment may become one practical option for preventing xerostomia during PSMA-targeted radionuclide therapy
18F-PSMA-1007 PET studies with different MA levels in the solution should be performed before 225Ac-PSMA therapy to optimize the uptake ratio between tumor and salivary gland
Renal dysfunction is a major problem in 177Lu-DOTATATE treatment because of the high kidney accumulation (<a id="xref-ref-19-1" class="xref-bibr" href="#ref-19">19</a>). In 177Lu-PSMA-617 and 225Ac-PSMA-617 therapy, renal dysfunction has not been a major problem so far despite this high accumulation (<a id="xref-ref-20-1" class="xref-bibr" href="#ref-20">20</a>,<a id="xref-ref-21-1" class="xref-bibr" href="#ref-21">21</a>)
The present study revealed a high kidney accumulation of 18F-PSMA-1007
It would be difficult to adjust the MA level to decrease kidney accumulation while maintaining tumor accumulation
renal dysfunction should be carefully monitored
particular before PSMA-targeted radionuclide therapy
This study revealed that a decrease in the MA level of the injected 18F-PSMA-1007 solution resulted in decreased uptake in tumor and normal organs as seen on PET imaging and tissue sampling
Salivary gland was more sensitive than tumor to the decrease in MA level
This finding suggests the possibility of obtaining a therapeutic effect in tumor while minimizing adverse effects on salivary gland by setting an appropriate MA level in PSMA-targeted therapy
The optimal tracer dose might vary from patient to patient
depending on the tumor load and physiologic accumulation
Klaus Kopka and Frederik Giesel are listed as inventors on a PSMA-1007–relevant patent
This study was partly supported by collaborative research funds from Nihon Medi-Physics Co.
No other potential conflict of interest relevant to this article was reported
QUESTION: Does 18F-PSMA-1007 uptake in tumor and normal organs depend on its molar activity (MA) or peptide concentration of the injected solution
PERTINENT FINDINGS: The whole-body distribution of 18F-PSMA-1007 on mouse xenograft models was evaluated when solutions with different peptide concentrations were used
A decrease in the MA level resulted in decreased uptake in tumor and
IMPLICATIONS FOR PATIENT CARE: There is a possibility of obtaining a therapeutic effect in tumor while minimizing adverse effects in salivary gland by setting an appropriate MA level in PSMA-targeted therapy
We thank the PET Drug Synthesis Department at Osaka University Hospital for preparing the tracers
the Medical Imaging Center for Translational Research for providing excellent technical assistance
the Department of Nuclear Medicine and Tracer Kinetics for supporting the experiments
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<a href=http://www.soeda-a.net target=_blank rel=noopener>soeda and associates architects</a> developed &lsquo;ciel&rsquo; in an area where houses and low-rise stores coexist
and on a site surrounded by houses on three sides except for the front road
the wooden building encloses four tenant spaces that are planned for restaurants and offices
the overall form of the building was determined by adding a common terrace that also serves as a roof over two of the parking spaces
 &lsquo;in planning a building to be used by many people on a site surrounded by houses
our first priority was to consider possible impacts on the neighboring houses,&rsquo; notes soeda and associates architects
in order to be exempted from the local sun shadow regulation
the building height was kept below 10 meters
while its form is developed to blend with the residential surroundings
we came up with elevations in the shape of a gable roof house on all four sides by lowering the four vertexes of the upper part of the cuboid,&rsquo; adds the japanese architecture studio
&lsquo;the height of the eaves is aligned with the neighboring houses to reduce impacts on the neighbors.&rsquo;
 by making the four sides into the shape of a gable roof house
the roof takes on the shape of a hyperbolic paraboloid (HP) shell
the curved surface of the HP shell was assembled by installing 20mm-thick wood members on 60mm-thick rafters in a twisted manner and fastening curved plywood with nails
the entire roof is finished with gradually curving standing seam roofing
a large opening on the roof above the terrace brings in natural light and air
forming a shared outdoor free space for a variety of uses that remains walled off from the surrounding environment
architect: <a href=http://www.soeda-a.net target=_blank rel=noopener>soeda and associates architects</a>
structure engineer: ryotaro sakata /ryotaro sakata structural engineers
AXOR presents three bathroom concepts that are not merely places of function
but destinations in themselves — sanctuaries of style
<a href="/articles/s41598-024-65949-7/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics details</a>
the pathological hallmark in tau-related neurodegenerative disease
tau monomers aggregate into a diverse range of oligomers
consisting of approximately 40 tau protein molecules
are present in the prefrontal cortex of patients at Braak stages I-II
preclinical stages of Alzheimer’s disease (AD)
Antibodies to granular tau oligomers as antigens have not been reported
we generated new rat monoclonal antibodies by immunization with granular tau oligomers
Three antibodies from different hybridoma clones showed stronger immunoreactivity to granular tau oligomers and tau fibrils compared with monomeric tau
2D6-2C6 showed 3000-fold greater immunoreactivity in P301L-tau transgenic (rTg4510) mice than in non-transgenic mice
which detects pathological conformations of tau
These results suggest that 2D6-2C6 recognizes aggregates more specifically than MC1
2D6-2C6 recognized neurofibrillary tangles and pretangles
and co-localized within AT8-positive cells containing phosphorylated tau aggregates
The epitope of 2D6-2C6 is the 423–430 amino acid (AA) sequence of C-terminal regions
generated by immunization with granular tau oligomers binds to tau aggregates at the 423–430 AA sequence
These previous papers indicate that the extracellular tau oligomer released from cells bearing abnormal aggregated tau is a candidate for the tau seed
visualization tools to detect oligomeric tau are useful for elucidating the disease mechanism of tauopathies
the development of tools to detect tau oligomers is insufficient
and analyzed the binding of the antibodies to tau aggregates in vitro and in vivo
</a>Binding of tau aggregation antibodies in an in vivo model
The TBS-soluble fraction was obtained from rTg4510 mice overexpressing human P301L mutant tau (0N4R) and from non-transgenic mice (non-Tg) at 10 months of age
This fraction was then subjected to dot blot (A–D)
(A–D) The dot blot analysis detected tau in the occipital cortex homogenates from rTg4510 mice
The following antibodies were used: MC1 (0.74 μg/ml)
which reacts with conformational tau aggregates (A
pan-tau mouse monoclonal antibody (tau5; A
and tau aggregation antibodies 2D6-2C6 (0.74 μg/ml) (B)
Densitometry of tau immunoreactivity was quantified
and 8D6-1F7-reactive tau were normalized by the corresponding tau5-reactive tau levels
Quantitative data are presented as a percentage of non-Tg mice (mean ± SD of 4–5 mice)
P values were determined using Student’s t tests
Significance is indicated by *(p &lt; 0.05) shown on black columns comparing non-Tg with rTg4510 mice
F) Immunohistochemistry using the 2D6-2C6 antibody highlighted accumulated tau aggregates (arrows) in the CA3 region (left panels)
and entorhinal cortex (right panels) of rTg4510 mice (E)
both 2D6-2C6 (left panel) and tau5 (right panel) detected endogenous tau in non-Tg mice
</a>Immunohistochemistry and double immunofluorescence using 2D6-2C6 antibody on autopsied AD cases
(A–F) Immunohistochemistry using 2D6-2C6 antibody on autopsied AD cases
The panels (A–C) display CA1 of autopsied samples
B) demonstrated 2D6-2C6-immunopositivity in the CA1
whereas a neurologically healthy control (C) did not show dense aggregates
The 2D6-2C6 antibody immunolabeled NFTs (D)
Black scale bars = 100 μm (A–C) and 20 μm (D–F)
(G–L) Double immunofluorescence using 2D6-2C6 antibody combined with other anti-tau antibodies
All panels here are taken at the CA1 from the same AD case
Panels (G–I) indicate a combination of 2D6-2C6 and AT8
whereas panels (J–L) indicate a combination of 2D6-2C6 and T22
The 2D6-2C6 fluorescent signals were mostly merged with AT8 signals for NFT (G)
The 2D6-2C6 and T22 antibodies also exhibited colocalization (J–L)
Pretangle (K) was more sensitively detected by 2D6-2C6 antibody compared to T22
2D6-2C6 is identified as a novel tau monoclonal antibody that is immunoreactive for tau aggregates including granular tau oligomers
with greater sensitivity than the MC1 antibody
and effectively stained human pretangles and NFTs
(A) Schematic representation of human wild-type (WT) tau protein and its deletion mutants
The full-length 2N4R tau consists of an N-terminal region that includes a proline-rich region and N-terminal inserts
ΔR and ΔC were transfected into cultured COS-7 cells
Tau proteins in the cell lysate were detected with the pan-tau antibody JM (B
right panel) using SDS‒PAGE western blot analysis
(C) Schematic representation of peptides with partial sequences of the C-terminal region
(D–H) The peptides were dotted onto a nitrocellulose membrane and then reacted with 2D6-2C6
</a>Binding of other antibodies against C-terminal regions with tau aggregates
(A) The sequence at amino acid residues 417–441 in the tau C-terminal region is illustrated
The epitope of 2D6-2C6 covers residues 423–430
(B) Peptides (417–441 and 423–430) were dotted onto a nitrocellulose membrane and probed with the 2D6-2C6 antibody
as well as other C-terminal monoclonal antibodies: RTM38 (immunogen: 417–441) and tau46 (epitope: 404–441)
(C–F) Recombinant human full-length tau was polymerized using heparin and then fractionated
and longer fibrils are observed in Fraction 1
A dot blot analysis detected tau in all three fractions using the pan-tau rabbit polyclonal antibody (JM)
Tau immunoreactivity was quantified by densitometry (using ImageJ software)
and tau46 (F)-reactive tau were normalized by the corresponding JM-reactive tau levels
Quantitative data are presented as a percentage of Fraction 1 (mean ± SD of 4 experiments)
P values were determined using one-way ANOVA followed by Tukey’s multiple comparisons test
2D6-2C6 detected tau aggregates in the brains of AD subjects and in an in vivo mouse model
The epitope of 2D6-2C6 is located at AAs 423–430 in the C-terminus region of tau
While several antibodies targeting tau oligomers have been reported previously
this study is the first to identify an antibody that recognizes the C-terminal region of tau and binds tau aggregates specifically
an optimal strategy might employ a combined panel of N-terminal
there are no tau aggregation antibodies that recognize the C-terminus
This may have hindered optimal research strategies for studying the mechanism of tau aggregation
The 2D6-2C6 antibody found in this study can help advance this area of research
which detects phosphorylated tau aggregates
indicating that tau 423–430 AA residues recognized by 2D6-2C6 are externally exposed during tau aggregation in human samples
These observations suggest that the negative charge from phosphorylation at S422 may facilitate exposure of the 423–430 AA sequence in tau
the 2D6-2C6 antibody against granular tau oligomers may be useful for the detection of preclinical biomarkers for AD
2D6-2C6 is not a human-specific antibody because it also reacts with mouse tau
has previously been reported to show nonselective binding in mouse brain samples
This implies that even if the antibody reacts with mouse tau
it does not completely negate the significance of the antibody in detecting human pathology
2D6-2C6 is significantly more reactive to aggregated tau than to non-aggregated tau
though it is not completely insensitive to monomeric tau
the S/N ratio of 2D6-2C6 is better in vivo than in vitro
This result may be attributed to in vivo conditions
which include factors such as phosphorylation and interaction with other molecules
The T427 AA residue lies within the epitope of 2D6-2C6 (423–430 AA sequence)
targeting the phosphorylation of the 2D6-2C6 epitope may be a diagnostic and therapeutic strategy for tauopathies by removing toxic tau oligomers but not normal tau
our results show that the C-terminal 423–430 AAs of tau are positioned outward during the formation of granular tau oligomers
2D6-2C6 antibodies targeting the 423–430 AA sequence may be promising for the early diagnosis of tauopathies
Peptides (&gt; 50% purity) were obtained from Cosmo Bio Co.
Chemical reagents were purchased from Nacalai Tesque Inc
Tau protein concentration was determined with Coomassie brilliant blue (CBB) stain using bovine serum albumin (BSA) as a protein standard
Recombinant 2N4R tau protein (10 μM) and thioflavin T (10 μM)
which can detect β sheet-rich amyloid structures
were mixed in buffer containing HEPES (10 mM
Tau polymerization was induced by the addition of heparin (0.06 mg/ml; Acros Organics
Fluorescence resulting from the binding of thioflavin T with aggregated tau was monitored using a BioTek Synergy HTX multimode reader (excitation wavelength
Samples were recovered at various times after tau polymerization reactions
a sucrose gradient solution consisting of four layers (20
40 and 50% sucrose) was prepared in buffer containing HEPES (10 mM
The polymerized tau was layered on top of the sucrose gradient solution
for 2 h using either an MLS50 rotor (Beckman Coulter
Each layer was collected as Fractions 1 through 5
while the pellet was recovered as Fraction 6
Initial screening of the antibodies derived from hybridoma colonies for binding to granular tau oligomers was performed using ELISA
After completing the tau polymerization reaction
the recovered tau aggregates were diluted to a concentration of 1 μM in Milli-Q water
The samples were then deposited onto mica sheets and incubated for 10 min in a moisture box
the tau aggregates were visualized using a cantilever (either OMCL-TR400PSA or BL-AC40TS-C2; Olympus
Japan) on an SPM 9700 instrument (Shimadzu
rTg4510 mouse<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Santacruz, K. et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 309, 476–481. &#xA;                  https://doi.org/10.1126/science.1113694&#xA;                  &#xA;                 (2005)." href="/articles/s41598-024-65949-7#ref-CR6" id="ref-link-section-d494513054e1653">6</a>
expresses human tau 0N4R isoform containing the frontotemporal dementia associated P301L mutation
The rTg4510 mice were generated by crossing the responder line (#015815
carrying a tauP301L cDNA downstream of a tetracycline operon–responsive element (TRE)
expressing a tetracycline-controlled transactivator (tTA) under the control of the CaMKIIα promoter
was maintained on the C57BL/6 J background
The rTg4510 and non-transgenic mice (FVB/N-C57BL/6 J)
were individually housed and received food ad libitum
The mice were kept under standard conditions with a constant temperature of approximately 25 ℃ and a 12 h:12 h light/dark cycle
n = 5) were anesthetized with isoflurane and sacrificed
The left hemisphere was placed in 10% formalin
The occipital cortex was extracted from the right hemisphere for biochemical assays and stored at -80 ℃
The occipital cortex from rTg4510 mice was homogenized in cold TBS buffer containing protease inhibitors and phosphatase inhibitors
4 ℃) using a TLA55 rotor (Beckman Coulter)
The supernatant was stored at −80 ℃ until use in biochemical experiments
We included two autopsied cases with AD (two males who died at the age of 84 and 87, respectively) and a control without any neurological disorders (a male who died of pneumonia at the age of 67). The demography of included subjects was summarized in Supplemental Table <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/s41598-024-65949-7#MOESM1">1</a>
Autopsies were undertaken after obtaining of written informed consent from family members in accordance with ethical committee of Aichi Medical University
The brains were fixed in 20% formalin for at least a month
followed by standardized trimming and paraffin embedding
Sections of hippocampus at the lateral geniculate nucleus underwent immunohistochemical analyses
The 2D6-2C6 anti-tau oligomer antibody was used with 1:100 dilution overnight
and antigen retrieval was taken with 98℃ citrate buffer (pH6.0) for 25 min
We undertook secondary immunolabeling using a standard avidin-biotin method
We also performed double immunofluorescence using 2D6-2C6 antibody coupled with an anti-hyperphosphorylated tau mouse monoclonal antibody (AT8
MA) or an anti-tau-oligomer rabbit polyclonal antibody (T22
or rabbit IgG coupled with Alexa 488 or 568 was used for secondary immunolabeling
Immunofluorescent specimens were observed using a laser-confocal microscope (FV-3000
Tokyo Japan) under the same settings for each coupling of primary antibodies
The results are expressed as the means ± SDs
The significance of differences between two groups was evaluated using Student’s or Welch’s t tests
For comparisons involving three or more groups
one-way analysis of variance followed by Tukey’s multiple comparisons test was employed
All analyses were performed using PRISM9 (GraphPad Software Inc.
p &lt; 0.05 was considered statistically significant
All animal experiments and procedures were reviewed and approved by the Gakushuin University Animal Experimentation Committee (Permit No
All animal experiments were performed in accordance with Japanese and university guidelines and regulations for the care and use of experimental vertebrate animals
The authors have complied with the ARRIVE guidelines 2.0 for reporting
All recombinant DNA experiments were reviewed and approved by the Gakushuin University Recombinant DNA Experiment Safety Committee (Permit No
Human experiment was performed in line with the regulations outlined in the Declaration of Helsinki (WMA
and the research-purpose archiving of autopsied subjects was approved by the Ethical Committee of Aichi Medical University (AKBRC 2019-M019)
The datasets used and/or analysed during this study available from the corresponding author on reasonable request
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Takaichi, Y. et al. Amyloid β and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus). Acta Neuropathol. Commun. 9, 10. <a href="https://doi.org/10.1186/s40478-020-01104-3" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1186/s40478-020-01104-3">https://doi.org/10.1186/s40478-020-01104-3</a> (2021)
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This work was supported by JSPS KAKENHI (Grant Number 20K06896 and 23K05993) to Y.S.; Takeda Science Foundation to Y.S.; Abe Yoshishige Foundation to Y.S.; and AMED (Grant Number JP21wm0425016) to A.T
Department of Diagnostics and Therapeutics for Brain Disease
Shinsuke Ishigaki &amp; Moniruzzaman Mohammad
Graduate School of Agricultural and Life Sciences
Graduate School of Engineering Division of Science and Engineering for Materials
and epitope mapping experiments and drafted this article
generated rat monoclonal tau aggregation antibodies
immunostained mouse brain sections using the 2D6-2C6 antibody
conducted part of the immunostaining of mouse brain sections and critically supported the drafting of the article
critically discussed the design of the experiments and the article
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DOI: https://doi.org/10.1038/s41598-024-65949-7
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composed of hyperphosphorylated tau fibrils
are a pathological hallmark of Alzheimer’s disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease
A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss
Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene
we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules
we show that orally administered DL-isoproterenol
an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain
reduces the levels of detergent-insoluble tau
neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction
compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer’s disease and other tauopathies
link neuronal death to the tau aggregation process
we screened a small-molecule library for compounds with the potential to inhibit the formation of granular tau oligomers
We report here that compounds containing 1,2-dihydroxybenzene inhibit granular tau oligomer formation by modifying the Cys residues of tau
thereby reducing Sarkosyl-insoluble tau levels
neuronal death and brain dysfunction in P301L tau-transgenic mice
pyrocatechol violet (b,e,h) and lobaric acid (c,f,i) were screened as tau aggregation inhibitors
Inhibitory effects of tau aggregation were determined by fluorescence of thioflavin T (d,e,f) and pelleting assay (g,h,i) of heparin-induced tau polymerization incubated with various concentrations of compounds (1
Dimethyl sulfoxide was used as the vehicle
Thioflavin T fluorescence was measured at the indicated time
and results were represented as percentage of maximum thioflavin T fluorescence (d,e,f;mean±s.d
</a>Neuro-2a cells expressing human 2N4R tau (P301L) were treated with 0 (milliQ water)
10 mM lithium chloride as a positive control as an inhibitor of glycogen synthase kinase and 10 mM sodium chloride as a negative control for 48 h
upper part of panel) and RIPA-soluble fractions (a
middle part of panel) were obtained from the cell homogenates and were subjected to immunoblot analysis with JM antibody that recognized total tau
and levels of SDS-insoluble tau were normalized by corresponding RIPA-soluble tau
Results are represented as percentage of control (mean±s.d
of 3–6 experiments; 10 mM sodium chloride (n=3)
***P&lt;0.001; ****P&lt;0.0001 (one-way analysis of variance (ANOVA)
(b) Neuro-2a cells expressing human 2N4R tau (P301L) were pretreated with 0
1 and 10 μM propranolol 30 min before treatment with 0 (milliQ water)
10 mM lithium chloride and 10 mM sodium chloride for 48 h
middle part of panel) fractions were obtained and subjected to immunoblot using JM antibody
lower part of panel) and levels of SDS-insoluble tau were normalized to corresponding RIPA-soluble tau
of 2–3 experiments; 10 mM sodium chloride (n=2)
10 μM propranolol/1 μM isoproterenol (n=3))
</a>Heparin-induced tau aggregation mixture (120 h incubation) was subjected to sucrose density gradient centrifugation (a) and AFM observation (b)
Tau aggregation mixture was separated into six fractions
and levels of tau in each fraction were analysed by western blot using tau5 antibody that recognized total tau
three images were obtained from different areas (2 × 2 μm2) of the mica and the number and size of tau aggregates was quantitated using the image analysis programme
Sizes of tau aggregates are 0–9.9 nm (arrowhead)
40.0–79.9 nm (black dotted circle) and not less than 80 nm (black solid square)
**P&lt;0.01; ***P&lt;0.001; ****P&lt;0.0001 (unpaired Student’s t-test)
In first 60 min of heparin-induced tau aggregation
higher order tau oligomer was formed in soluble fraction with longer incubation time (c
but growth of tau oligomer was not seen in the presence of isoproterenol (c
Tau oligomer in soluble fraction was detected by non-reducing condition using tau5 antibody
</a>ISO and tau binding were performed according to the protocol described in <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/ncomms10216#MOESM1762">Supplementary Fig. 1</a> and ISO-bound tau was recovered in KCl
Each fraction was subjected to immunoblot analysis with tau5 antibody (a)
tau monomer and oligomer were detected from an eluate of FG beads with ISO (ISO (+)) and without ISO (ISO (−); b
Levels of tau monomer and oligomer were quantified (b
right part of panel) and shown as mean±s.d
*P&lt;0.05; ****P&lt;0.0001 (unpaired Student’s t-test)
FG beads with ISO (ISO (+)) and without ISO (ISO (−)) were reacted with ΔMTBR-tau
Pretreated ISO with R1 (tapvpmpdlknvkskigstenlkhqpgggk)
R3 (vqivykpvdlskvtskcgslgnihhkpgggq) and R4 (vevksekldfkdrvqskigsldnithvpgggn) peptides was incubated with wild-type (WT) 2N4R tau
Pretreated IS0 with R2 and R3 could not bind to tau
322A-2N4R tau and C322A-2N3R tau did not show tau band as well as tau incubating beads without ISO
ΔPHF6-tau and ΔPHF6*-tau showed tau band as ISO-bound tau (e)
indicating that ISO binds to sites localized in the R2 and/or R3 regions of tau
</a>Vehicle (milliQ water; a) and 1-hydroxybenzene-containing octopamine (OCT; MW=153.2; d) were used as negative controls
Compounds containing 1,2-dihydroxybenzene isoproterenol (ISO; MW=211.3; b) and pyrocatechol (CAT; MW=110.1; c) incubated with tau R3 partial peptide
R3’ (skvtskcgslgn; MW=1,180.3) showed ISO/R3’ and CAT/R3 signal in mass spectrum (b,c)
Mutant C→A R3’ peptide (skvtskagslgn; MW=1,148.2; e–h) incubated with vehicle (e)
CAT (g) and OCT (h) only showed mutant R3’ signal
Data are normalized to the each maximum mass spectrometry signal
where ISO (1.5 mg g−1 chow for 3 months) is seen to prevent age-related reductions in neuron numbers in the entorhinal cortex
temporal lobe and basolateral amygdala of aged P301L tau-transgenic mice; the drug did not influence neuron numbers in any of these areas in non-transgenic mice
Growing evidence for the key role of aggregates of tau protein in AD and FTDP-17 has spurred efforts to explore pharmacological means to maintain tau in its soluble form and thus
and thus increasing the propensity to form β-sheets and inhibiting the formation of seeds for the aggregation of toxic tau
The monoclonal anti-total tau antibody (tau5) was purchased from Invitrogen
All other reagents were of analytical grade and purchased from Nacalai Tesque Inc.
after blocking with 1% skimmed milk in 10 mM HEPES/100 mM NaCl for 1 h at room temperature
the slides were treated with recombinant tau protein in 10 mM HEPES/100 mM NaCl/0.05% Tween for 16 h at 4 °C
The slides were then probed with anti-tau antibody (JM) for 4 h
followed by incubation with a Alexa 633-labelled secondary antibody for 1 h at room temperature
Signal detection was performed with a GenePix 4100A microarray scanner (Molecular Devices)
equipped with a 635-nm laser and 655–695 nm band-pass emission filter
When Fraction 1 (soluble tau) was used as bait
86 out of 6,788 compounds showed potential association with tau
none of the compounds tested showed associations either Fraction 3 (granular tau oligomer) or Fraction 6 (fibrilar tau)
coli expressing tau was sonicated and boiled
recombinant tau proteins in the heat-stable fraction was purified by ion-exchange chromatography (P11; GE Healthcare
gel filtration chromatography (NAP10 column; GE Healthcare) and reverse phase-HPLC (COSMOSIL Protein-R Waters; Nacalai Tesque Inc.)
recombinant tau proteins were dissolved in milliQ water and stored at −80 °C as a stock solution
ThT binding was measured with modified method reported previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Maeda, S. et al. Granular tau oligomers as intermediates of tau filaments. Biochemistry 46, 3856–3861 (2007)." href="/articles/ncomms10216#ref-CR33" id="ref-link-section-d500130505e1912">33</a>
compounds (indicated concentration) and ThT (10 μM) were mixed in the HEPES buffer (10 mM HEPES
and incubated with heparin (0.06 mg ml−1; Acros Organics) at 37 °C
fluorescence generated by the binding of ThT to tau aggregates was measured (excitation wavelength: 444 nm; emission wavelength: 485 nm)
The tau aggregation mixture was collected 120 h after incubation
sucrose density gradient centrifugation or AFM
Sucrose density gradient centrifugation was performed as described previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Maeda, S. et al. Granular tau oligomers as intermediates of tau filaments. Biochemistry 46, 3856–3861 (2007)." href="/articles/ncomms10216#ref-CR33" id="ref-link-section-d500130505e1927">33</a>
Tau aggregation mixture (1 ml) was layered on top of sucrose step gradients (each 1 ml of 10
40 and 50% sucrose in HEPES buffer (pH=7.4)) was centrifuged (50,000 r.p.m.
2 h) in a MLS50 rotor (Beckman Coulter) and separated into fractions
Pellet (Pel; Fraction 6) was suspended in 1 ml of buffer containing HEPES buffer
and the recovered tau in each fraction was evaluated with western blotting
Pelleting assay was performed by modified method reported previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 54" title="Pickhardt, M. et al. Screening for inhibitors of tau polymerization. Curr. Alzheimer Res. 2, 219–226 (2005)." href="/articles/ncomms10216#ref-CR54" id="ref-link-section-d500130505e1939">54</a>
samples including tau aggregates were centrifuged (70,000 r.p.m.
2 h) in TLA100.4 or TLA110 rotors (Beckman Coulter) and separated into supernatant and pellet
After the pellet was suspended and sonicated in Laemmli SB including 2-mercaptoethanol
Cells were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum
5 μg ml−1 puromycin and 1 mg ml−1 G418 at 37 °C under 5% CO2
Cells at 80–90% confluency were plated in mediums without puromycin and G418
lithium chloride (10 mM) as a positive control that is a glycogen synthase kinase inhibitor
or sodium chloride (10 mM) as a negative control
To confirm the relationship between tau aggregation with β adrenergic effect by ISO
30 min after pretreating with or without 1 or 10 μM propranolol
which is a competitive adrenergic β blocker
Cells were sonicated in modified RIPA buffer containing 50 mM Tris (pH=7.4)
1 mM ethylene glycol tetraacetic acid (EGTA)
2 μg ml−1 aprotinin and 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride)
and phosphatase inhibitors (1 mM okadaic acid
1 mM Na3VO4 and 1 mM NaF) 48 h after treatment
The lysates (1 ml) were layered onto 0.32 M sucrose containing 10 mM Tris (pH=7.4)
The upper part (1 ml) was transferred to 1.5-ml tubes (357448; Beckman Coulter)
4 °C) in a TLA55 rotor (Beckman Coulter) and separated into supernatant (RIPA-soluble fraction) and pellet
After the pellets were sonicated in 1% SDS/RIPA and 1% SDS/TBS buffers
4 °C) in a TLA55 rotor and separated into supernatant and pellet
Pel (SDS-insoluble fraction) was dissolved in 70% formic acid and air-dried
Samples from RIPA-soluble and SDS-insoluble fractions were dissolved in Laemmli SB including 2-mercaptoethanol
Morphology of the recombinant tau aggregate was observed under AFM<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 33" title="Maeda, S. et al. Granular tau oligomers as intermediates of tau filaments. Biochemistry 46, 3856–3861 (2007)." href="/articles/ncomms10216#ref-CR33" id="ref-link-section-d500130505e1982">33</a>
samples including tau aggregates were loaded to mica and incubated at room temperature for 30 min in a moist box
The mica was washed with milliQ water and then cantilever (OMCL-TR400PSA; Olympus) detected tau aggregate with 3D-Stand Alone AFM (Asylum Research) under the tapping mode
Major and minor axis of tau aggregates and the number of tau aggregates were determined by image analysis using Matlab-based software (MathWorks Co
KCl and Pel were solubilized or resuspended in Laemmli SB
Tau protein in the samples was detected with western blotting
R3 (vqivykpvdlskvtskcgslgnihhkpgggq) or R4 (vevksekldfkdrvqskigsldnithvpgggn) peptides (120 μM) were pretreated with the avidin-biotin complexes before incubation with recombinant wild-type 2N4R tau
Recombinant R3’ (skvtskcgslgn) and mutant Cys→ Ala R3’ (skvtskagslgn) peptides (10 μM) were incubated with compounds (100 μM) for 5 days at 37 °C
The samples were diluted 20 times with milliQ water and loaded to spot under presence of α-Cyano-4-hydroxycinnamic acid (10 mg ml−1)
the samples were analysed using 4800 plus MALDI Tof/Tof analyzer (Applied Biosystems)
All experimental procedures used in this study were approved by the Committee of Animal Experiments at the National Center for Geriatrics and Gerontology
P301L tau-transgenic mice were anaesthetized and killed after treatment with the compounds; the hippocampus and cerebral cortex were collected
The tissues were homogenized in TBS buffer containing 50 mM Tris (pH=7.4)
protease inhibitors and phosphatase inhibitors
The homogenates were centrifuged (23,000 r.p.m.
4 °C) in a TLA55 rotor and separated into supernatant (TBS-soluble fraction) and pellet
Pellets were resuspended in 0.32 M sucrose containing 10 mM Tris (pH 7.4)
0.8 M NaCl and 1 mM EGTA and centrifuged (23,000 r.p.m.
Supernatants were collected and treated with 1% Sarkosyl for 1 h at 37 °C
4 °C) in a TLA100.4 or 110 rotors and separated into supernatant and pellet (Sarkosyl-insoluble fraction)
Samples from TBS-soluble and Sarkosyl-insoluble fractions were dissolved in Laemmli SB including 2-mercaptoethanol
Number of neuronal cells in the brain was measured with modified method reported previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 26" title="Kimura, T. et al. Aggregation of detergent-insoluble tau is involved in neuronal loss but not in synaptic loss. J. Biol. Chem. 285, 38692–38699 (2010)." href="/articles/ncomms10216#ref-CR26" id="ref-link-section-d500130505e2067">26</a>
After P301L tau-transgenic mice were anaesthetized and transcardially perfused with 10% formalin
brains were fixed in 10% formalin solutions for 48 h
Coronal sections (4 μm) were produced from brains embedded by paraffin
We counted the number of neuronal cells in the sections using microscope linked to a Neurolucida tracing system (MicroBrightField Inc.)
Samples dissolved in Laemmli SB were separated by Novex 3–8% Tris-Acetate Gel (binding assay and tau oligomerization assay; Invitrogen) or SuperSep Ace 5–20% gel (other assays; WAKO Pure Chemical Industries) and transferred onto membranes with semi-dry transfer systems (Bio-Rad Laboratory)
The membrane was blocked with 5% milk in PBS-T for 1 h at room temperature
They were probed with antibodies overnight at 4 °C
blots were incubated with horseradish peroxidase-linked second antibodies and then examined by enhanced chemiluminescence detection on Las3000 or Las4000 (GE Healthcare)
Mice were placed in the centre of an open field apparatus (50 × 50 × 40 cm3; O’Hara Co., Ltd.), and their locomotor activity was monitored with a CCD camera; digital data of real-time images were recorded using the public domain NIH Image J software (<a href="http://rsb.info.nih.gov/nih-image/">http://rsb.info.nih.gov/nih-image/</a>)
Data were analysed using customized Matlab-based software
using an image analysis tool box (Mathworks Co
the sequential position of the mouse was determined in each video frame from which locomotor speed was calculated
Mn-enhanced MRI was performed as previously described<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 55" title="Kimura, T. et al. Hyperphosphorylated tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau. EMBO J. 26, 5143–5152 (2007)." href="/articles/ncomms10216#ref-CR55" id="ref-link-section-d500130505e2105">55</a>
Mice were given an intraperitoneal injection of 30 mM MnCl2 (100 μmol kg−1) and returned to their home cages
mice were exposed successively to three different novel places separated by a small (diameter
30 cm) transparent plastic wall and was allowed to explore for 120 min
mice were returned to their home cages for 1 h before being anaesthetized with isoflurane (0.5–1.5% in air) and placement in a Bruker 3T MR scanner; during scanning
breathing and depth of anaesthesia was continuously monitored
with breathing rate maintained at 80–100 breaths per min
Mn-enhanced MRI images were visualized with open-source Osirix software (version 2.5) that allowed navigation through multidimensional DICOM images; relative regional brain activity was determined by measuring MR signal intensities
normalized to the mean signal intensity in the dorsal striatum
Male C57BL/6J mice (n=7) were administered ISO (1.5 mg g−1 chow) for 2 weeks before sacrifice when blood plasma and whole brain were collected
The samples were deproteinized using acetonitrile before determination of ISO concentrations using an LC-MS instrument (UPLC/Quattro Premier XE; Waters)
The significance of differences between two groups was assessed by Student’s or Welch’s t-tests
and differences between multiple groups were assessed by one-way analysis of variance and Tukey’s multiple comparisons test
P&lt;0.05 was considered statistically significant
Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups
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Tau-based therapies for Alzheimer’s disease: wave of the future
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Traveling the tau pathway: a personal account
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Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses
Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models
Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model
Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised
Alzheimer disease therapy--moving from amyloid-beta to tau
Clinical and pathological correlates of apolipoprotein E epsilon 4 in Alzheimer's disease
Neurofibrillary degeneration and neuronal loss in Alzheimer's disease
Neurofibrillary pathology--correlation with hippocampal formation atrophy in Alzheimer disease
Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17
Tau is a candidate gene for chromosome 17 frontotemporal dementia
Mutation in the tau gene in familial multiple system tauopathy with presenile dementia
Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model
Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein
amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein
Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau
Tau filament formation and associative memory deficit in aged mice expressing mutant (R406W) human tau
Modelling tauopathies in Drosophila: insights from the fruit fly
Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors
Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain
Implications for neurofibrillary degeneration in Alzheimer's disease
Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model
Increased levels of granular tau oligomers: an early sign of brain aging and Alzheimer's disease
Effects of isoproterenol on blood-brain barrier permeability in rats
Isoprenaline increases brain concentrations of administered L-dopa and L-tryptophan in the rat
U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy
Covalent binding of catechols to Src family SH2 domains
Pathologically activated therapeutics for neuroprotection
Electrochemical reduction of quinones in different media: a review
Effects of alpha-tocopherol on an animal model of tauopathies
Oxidative stress mediates tau-induced neurodegeneration in Drosophila
Alternative conformations of the Tau repeat domain in complex with an engineered binding protein
and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases
Presenilin 1 associates with glycogen synthase kinase-3beta and its substrate tau
Discovery of novel antiviral agents directed against the influenza A virus nucleoprotein using photo-cross-linked chemical arrays
Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions
Robust and systematic drug screening method using chemical arrays and the protein library: identification of novel inhibitors of carbonic anhydrase II
Screening for inhibitors of tau polymerization
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/ncomms10216?format=refman&amp;flavour=references">Download references</a>
We thank Drs Tamio Saito and Aya Asami (RIKEN
Japan) for kindly providing 6,788 test compounds; Dr Toshihide Hashimoto (Eisai Co.
Japan) for kindly providing biotinylated ISO; Ms
Japan) and Mr Shunji Yamashita (O'HARA &amp; Co.
Tokyo Japan) for valuable technical advice; and Ms Midori Yamamoto (National Center for Geriatrics and Gerontology
Japan) and Mr Tatsuya Mizoroki (Institute of Immunology Co.
Japan) for assistance in the maintenance of mice
This work was supported by JSPS KAKENHI Grant Number 23790313 (to Y.S.)
Scientific Research on Innovation Area (Brain Protein Aging and Dementia control (to A.T.)
Brain environment (to A.T.)) and Strategic Research Program for Brain Science (‘Integrated Research on Neuropsychiatric Disorders’) from Japan Agency for Medical Research and development
and H.S.) and Intramural grant of NCGG (to A.T.)
Present address: Present address: Study Promotion Strategy Section/Clinical Research Center
National Center for Geriatrics and Gerontology
Department of Stress Neurology and Neurogenesis
Gladstone Institute of Neurological Disease
RIKEN Center for Sustainable Resource Science (CSRS)
Osaka Electro-communication University (OECU)
Department of Clinical and Experimental Neuroimaging
Center for Development of Advanced Medicine for Dementia
Division of Regeneration and Advanced Medical Science
Gifu University Graduate School of Medicine
provided critical suggestions during writing of the manuscript
performed the chemical array screening and evaluated the data
calculated major axis and number of tau aggregates in images obtained by AFM using Matlab
provided critical techniques for ThT assay and cultured cell analysis
The authors declare no competing financial interests
Supplementary Tables 1-2 and Supplementary References (PDF 977 kb)
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Molecular and Cellular Biochemistry (2021)
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A <a href="https://doi.org/10.1038/srep09630" class="relation-link" data-track="click" data-track-action="view linked article" data-track-label="link">Corrigendum</a> to this article was published on 26 May 2015
This article has been <a href="#change-history">updated</a>
Cancers are composed of heterogeneous combinations of cells that exhibit distinct phenotypic characteristics and proliferative potentials
cancer stem cells (CSCs) must generate phenotypically diverse progenies including mature CSCs that can self-renew indefinitely and differentiated cancer cells that possess limited proliferative potential
no convincing evidence exists to suggest that only single CSCs are representative of patients' tumors
we established 4 subclones from a glioblastoma patient
These subclones were subsequently propagated and analyzed
the self-renewal and proliferative capacities of the subclones differed
Fluorescence-activated cell sorting and cDNA-microarray analyses revealed that each subclone was composed of distinct populations of cells
the sensitivities of the subclones to an inhibitor of epidermal growth factor receptor were dissimilar
In a mouse model featuring xenografts of the subclones
the progression and invasion of tumors and animal survival were also different
we present clear evidence that a brain tumor contains heterogeneous subclones that exhibit dissimilar morphologies and self-renewal and proliferative capacities
Our results suggest that single cell-derived subclones from a patient can produce phenotypically heterogeneous self-renewing progenies in both in vitro and in vivo settings
functionally analyzing the individual types of the heterogeneous cells and determining their role in tumor pathogenesis are critical
we established 4 subclones from a glioblastoma patient and demonstrated clear evidence that a brain tumor contains heterogeneous subclones that exhibit dissimilar morphologies
proliferative capacities and therapeutic sensitivities
(a) Distinct cellular morphologies observed after plating dissociated tumor cells in culture dishes
(b) Procedures used in establishing the 4 clones
Immediately after tumor spheres were formed from the patient's tissue
mechanically dissociated single cells were plated in small culture dishes
(c) The clones #2 and #4 formed sphere-like aggregates
whereas #3 and #5 attached to the uncoated culture dishes
(d) The 4 clones expressed the stem-cell marker nestin (red
</a>Dissimilar tumorigenesis in the in vivo animal model
(a) Kaplan-Meier survival plots; 3 mice were used for cells of each clone
(b) Representative brain tumors of NOD-SCID mice harboring xenografts of the clones #2 (upper left)
#4 (upper right) and #5 (lower right); H.E
(c) A representative xenograft of the clone #4; nestin-positive cells infiltrated the contralateral hemisphere (arrows)
</a>Analysis of cell-surface markers and cDNA arrays
Flow-cytometry data were collected in at least triplicate and at distinct culture periods in order to avoid ongoing culture selection
ORI: original cells not sorted into single cells; DIF: differentiated cells obtained using 10% serum
(a) CD133 expression (no statistically significant difference)
Fold-changes in gene expression were calculated as the ratio of the signal values of the original cells to the value of each of the clonal cells after duplication
These data suggest that COL1A1 and IGFBP7 may play critical roles in the development and tissue heterogeneity of gliomas
Since our four clones are insufficient number for analyzing p- or q-values
further evaluations of single cell derived clones may support our hypothesis
</a>Inhibition of EGF-EGFR/PI3K/Akt and/or MAPK-Erk1/2 pathways by an EGFR inhibitor
(a) Cell numbers determined after treatment with the EGFR inhibitor gefitinib (1 μM); cells were counted after one week. *P &lt; 0.01. Cont: control; EGFRi: EGFR inhibitor. (b) Phosphorylation of Akt, Stat3, Erk1/2 and Smad1/5 in cells incubated with 1 μM gefitinib for 1 h. C: control, Ei: EGFR inhibitor. Full-length blots are shown in <a data-track="click" data-track-label="link" data-track-action="supplementary material anchor" href="/articles/srep07979#MOESM1">Supplementary Fig. 4</a>
Although we have not conclusively identified the mechanism that can explain such differences
the findings suggest that such mechanisms include distinct RTK signaling pathways that are responsible for GBM proliferation
Further evaluating the underlying metabolic events in heterogeneous cancer cells and assessing the biological features of each distinct type of cancer cells will yield valuable information on the in situ behavior of cancers and help identify optimal cell-specific therapies in the future
Tumorigenicity was determined by injecting tumor cells orthotopically into non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice (SLC
Cells were injected into the brain of ketamine-anesthetized NOD-SCID mice; 2 μL of a cell suspension (1 × 108 cells/mL) in proliferation medium were delivered into the right striatum (1 μL/min) by using a stereotactic instrument (SR-60
The injection coordinates were 3 mm to the right of the midline and 3 mm anterior to the lambda
We injected 3 mice each with the cells of each clone and all of the mice were monitored daily for signs of morbidity such as weight loss
posturing and nasal and/or periorbital hemorrhage; the mice were sacrificed at the first sign of morbidity and the collected brains were examined histologically for the presence of tumor
Tumor samples were fixed in 4% paraformaldehyde
embedded in paraffin and cut into 3-μm sections
sections were first stained with Mayer's hematoxylin (1 min) and then counterstained with alcoholic eosin
deparaffinized sections were washed in Tris-buffered saline (TBS) and endogenous peroxidase was neutralized using 3% H2O2 in methanol (15 min) after 15-min antigen retrieval in citrate buffer in a microwave at 500 W
Sections were blocked with 1% bovine serum albumin in TBS and then treated overnight at 4°C with the following primary antibodies: anti-human nestin (mouse monoclonal antibody (mAb)
After treatment with biotinylated secondary antibodies and horseradish peroxidase (HRP)-linked streptavidin (LSAB2 kit
color reactions were performed using the peroxidase substrate 3,3′-diaminobenzidine (DAB
All sections were counter-stained with Mayer's hematoxylin
Cells were evaluated on a Coulter EPICS cytometer (Beckman Coulter
each sample was labeled with phycoerythrin (PE)-conjugated anti-human CD24
PE-conjugated CD133/1 (AC133) (Miltenyi Biotec
CA) according to the manufacturer's recommendation
All data used unstained control and used Cellquest Pro software/FlowJo for data acquisition and analysis including adequate fluorescence labeling
compensation and calibration at flow cytometry facilities in University of Pittsburgh and/or University of Virginia
All experiments were performed in triplicate
Western blot analyses were performed as described previously<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 6" title="Soeda, A. et al. Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells. J Biol Chem 283, 10958–10966 (2008)." href="/articles/srep07979#ref-CR6" id="ref-link-section-d18179526e920">6</a>
Antibodies against the following molecules were used: actin and phospho-STAT3 (P-Ser727) (Santa Cruz Biotechnology
phospho-ERK1/2 (P-Thr202/Tyr204) and phospho-Smad1/5 (P-Ser463/465) (Cell Signaling Technology
tumor cells were lysed in a buffer consisting of 20 mM Tris-HCl
1 μg/mL leupeptin and 1 mM phenylmethylsulfonyl fluoride
lysates were clarified by centrifugation at 12,000 × g for 10 min at 4°C and the protein content in the supernatant was measured according to the Bradford method
Aliquots (40 μg of protein per lane) of total protein were separated using SDS-polyacrylamide gel electrophoresis (7.5% gels) and blotted onto nitrocellulose transfer membranes (0.2 μm; Amersham Biosciences
Each membrane was blocked with 5% non-fat dry milk in TBS-T (20 mM Tris-HCl
137 mM NaCl and 0.01% Tween-20) for 1 h at room temperature and then incubated with the appropriate primary antibodies overnight at 4°C
room temperature) with HRP-conjugated anti-rabbit
or anti-goat secondary antibodies diluted (1:1,000) in TBS-T containing 5% non-fat dry milk
Immunoreactive bands were detected using an enhanced chemiluminescence reagent (Amersham Biosciences)
hybridization and scanning of the microarrays were performed according to the manufacturer's protocol (Affymetrix
CA); analysis was on both a spectrophotometer and an Agilent 2100 Bioanalyzer (Agilent Technologies
Total RNA was extracted from each sample by using TRIZOL reagent (Invitrogen); this was followed by passage through an RNeasy spin column (Qiagen
CA) and amplification with RiboAmp RNA Kits (Arcturus Engineering
CA) according to the manufacturer's protocol
Amplified RNA (7.5 μg) was labeled with Cy5-dUTP (experimental RNA) or Cy3-dUTP (Stratagene
CA) by using Superscript II reverse transcriptase (Invitrogen)
The labeled cRNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 Genechip
with the use of 60-rpm rotation for 16 h at 45°C
the microarrays were washed in a buffer containing biotinylated anti-streptavidin antibodies (Vector Laboratories
CA) and stained (10 min at 25°C) with streptavidin-PE (final concentration 10 μg/mL; Molecular Probes
restained with streptavidin-PE and washed again before measuring fluorescence at 570 nm in the Affymetrix GeneChip Scanner 3000
All of the microarrays were examined for surface defects
housekeeping gene expression and the 3′/5′ ratio of probe sets from genes of varying length (signal 3′/5′ ratio &lt; 3)
Initial data analysis was performed using Affymetrix Microarray Suite 5.0 to determine gene expression levels
Our study was approved by the Medical Review Boards of University of Pittsburgh
University of Virginia and Gifu University School of Medicine
Our experimental procedures involving animals also followed the guidelines of the Animal Experimental Committee of Gifu University
Statistical analysis was performed using Student's t tests; P &lt; 0.05 was considered statistically significant
A correction has been published and is appended to both the HTML and PDF versions of this paper
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma
The WHO classification of tumors of the nervous system
Cancer stem cells in gliomas: identifying and understanding the apex cell in cancer's hierarchy
Identification of human brain tumour initiating cells
Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cells
Targeting cancer stem cells through L1CAM suppresses glioma growth
Long-term maintenance of brain tumor stem cell properties under at non-adherent and adherent culture conditions
Glioma stem cells promote radioresistance by preferential activation of the DNA damage response
Removal of right cerebral hemisphere for certain tumors with hemiplegia: Preliminary report
Surface protein dynamics in glioma stem cells
The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation
Alteration of serum and tumoral neural cell adhesion molecule (NCAM) isoforms in patients with brain tumors
Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells
Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system
CD166/activated leukocyte cell adhesion molecule is expressed on glioblastoma progenitor cells and involved in the regulation of tumor cell invasion
Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors
A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors
Comprehensive genomic characterization defines human glioblastoma genes and core pathways
IGFBP7 downregulation is associated with tumor progression and clinical outcome in hepatocellular carcinoma
Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma
<a data-track="click" data-track-action="download citation references" data-track-label="link" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/srep07979?format=refman&amp;flavour=references">Download references</a>
This work was supported by grants from JSPS KAKENHI (Grant Number 24659647) and the Department of Neurosurgery
Department of Tissue and Organ Regeneration
designed and performed experiments and A.H
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Cellular and Molecular Life Sciences (2023)
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Laura Robson will face Belgium's Kirsten Flipkens in the women's draw
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Andy Murray was drawn against Japan's Go Soeda in the first round of the Australian Open but it was the pairing of world number one Rafael Nadal with home hope Bernard Tomic that really captured the imagination.
There were gasps at Melbourne Park when the unseeded players were placed in the draw and the blockbuster match-up emerged.
Tomic is a mercurial talent who will relish taking on Nadal, with the Spaniard back in Melbourne for the first time in two years after missing the tournament 12 months ago as he recovered from knee problems.
Murray is seeded fourth for his first grand slam since undergoing back surgery in September and there was mixed news for the Scot from the draw.
Soeda, who he has never played before, is a 29-year-old ranked 112th in the world and should not present any real problems.
Should Murray prevail he would then play a qualifier before potentially meeting familiar foe Feliciano Lopez in the third round.
Given Murray's lack of matches, a kind first few rounds would have been his main hope, but from there things get a lot trickier.
The draw is markedly top heavy and, after John Isner in the fourth round, Murray could find himself having to beat Roger Federer, Nadal and Novak Djokovic if he is to lift a third grand slam title.
Second seed Djokovic, who has won the title for the last three years, will play Slovakia's Lukas Lacko in the first round while sixth seed Federer meets Australian wild card James Duckworth.
Djokovic, who travelled to the draw on a tram with reigning women's champion Victoria Azarenka, is playing his first grand slam since hiring two-time Australian Open champion Boris Becker as his head coach.
The Serbian said: "For me it's an honour to have him alongside all my other team members. We'll do our best to make a success of our partnership and it's just the start."
Laura Robson, Britain's only direct entrant into the women's draw, was given a tough first-round tie against Belgium's Kirsten Flipkens.
Flipkens is seeded 18th and had a surprise run to the semi-finals at Wimbledon last year.
Robson has yet to complete a match this season because of a left wrist problem but has been practising at Melbourne Park.
There was also an enticing match-up at the top of the women's draw, with world number one Serena Williams playing Australian wild card Ashleigh Barty.
Barty, 17, made two grand slam doubles finals last year and is ranked 153rd in singles having won the Junior Wimbledon title in 2011.
Williams is in the same half of the draw as fourth seed Li Na, who meets a qualifier first up, while second seed Azarenka plays Johanna Larsson of Sweden.
Also in the bottom half is third seed Maria Sharapova, who has returned to the tour after shoulder problems brought a premature end to her 2013 season.
The Russian has a potentially tricky draw against American Bethanie Mattek-Sands.
Play is due to begin at Melbourne Park on Monday but the weather could cause delays, with projected temperatures of more than 40C.
The Australian Open has an extreme heat policy, and tournament director Craig Tiley revealed there are expected to be six days when the temperature tops 30C.
Join thought-provoking conversations, follow other Independent readers and see their replies
Roger Federer and Novak Djokovic","description":"Laura Robson will face Belgium's Kirsten Flipkens in the women's draw
Deus Ex Machina runs stores and cafes all over the world
the brand has broadened its appeal on a global scale
we two-wheeled maniacs know Deus as stylish custom shops
the collective pumps out some of the most sophisticated builds in the entire industry
The Deus Ex Machina Japan chapter is no different, and builder Tomoyuki Soeda shows off that tasteful distinction with his Green Envy and Beluga XM projects. Starting with a <a href="https://www.rideapart.com/honda/" data-inline-widget="internal-links" data-type-id="2" data-params="%7B%22alias%22%3A%22honda%22%7D">Honda</a> FT400
Soeda adopted a simple guiding principle: keep it cheap
That approach didn’t leave room for much custom work
but the builder completely transformed the standard into a scrambler nevertheless
It’s addition by subtraction with Green Envy
as Soeda exposes the subframe by ripping away the side panels and replacing the tank with an older unit
He also ditches the dual shock setup for a monoshock configuration
and a SuperTrapp exhaust complete the scrambler metamorphosis
For Soeda’s second project, he turned to a much more modern donor bike. The <a href="https://www.rideapart.com/ktm/" data-inline-widget="internal-links" data-type-id="2" data-params="%7B%22alias%22%3A%22ktm%22%7D">KTM</a> 200 Duke’s electric fuel injection gave Soeda the confidence to turn the fifth-liter naked bike into a little tourer
He replaces KTM’s sharp and aggressive bodywork with a smooth
white shell that earns the project its Beluga XM name
The build’s Bridgestone AX41 adventure tires open new paths to the little roadster
and a detachable surf rack helps riders catch a few waves along the way
Both Green Envy and Beluga XM benefit from an intuitive modular luggage system devised by the Deus Ex Machina team
The Honda FT400 and KTM 200 Duke boast a removeable rear platform
but the Beluga takes that convenience even further with a front rack
Riders and non-riders may know Deus for its elevated style
but Soeda proves that the brand’s style can still be approachable
There's a Rumor Can-Am Owner Wants to Buy KTM
Kawasaki is Building a Supercharged 250 Horsepower UTV
KTM's Parent Company Lost Nearly 1.2 Billion Euros Last Year
This Limited-Edition Motorcycle Helmet Celebrates the Gnarliest Race on the Planet
KTM’s Factory Racing Tour Is Somehow Still Happening
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<a href="/ajw/national_report/">National Report</a>
Osaka Prefecture—Tran Thi Hien’s skills are in heavy demand these days
She is a Vietnamese interpreter who is now increasingly helping young compatriots suspected of committing crimes in Japan
Around 450,000 Vietnamese are working nationwide mainly under the technical trainee program
The intern program is supposed to provide trainees with skills that they can use after they return to their home countries
complaints are rife that employers are forcing the interns to do menial or dangerous tasks under terrible working conditions
Some employers have also been accused of withholding wages from the interns or abusing them
said the number of interpretation requests is constantly increasing
a Vietnamese trainee fled a workplace because of bullying and
“Flaws in the government’s framework (for the intern program) may be part of the reason they end up resorting to criminal acts,” Tran said
She said the program should be abolished because rampant misuse has deviated it from its initial purpose
Tran lived in her hometown in Vietnam’s Thai Binh province until she graduated from high school
“I wanted to leave the countryside to learn what the world is like,” she recalled
Tran joined a new local subsidiary of Honda Motor Co
was asked by the Justice Ministry to translate past court rulings
She has since served as an interpreter for tourists
Tran has also immersed herself in helping Vietnamese in court trials and police investigations
she is sometimes involved in three separate court trials on a single day
she was rushed to a police station in a patrol car for interpretation work after the last train run
Japan and Vietnam established diplomatic relations in 1973
The countries will mark the 50th anniversary of bilateral ties next year
Tran says people from both the countries must share their honest feelings with each other
“I want to contribute to the mental bonds between people as well as economic and diplomatic affairs,” she said
Rumor leads to dead baby and trial for trainee from Vietnam
Vietnamese priest prays for souls of his compatriots 
Multilingual tour guides struggle as pandemic upends industry
Vietnamese trainee sentenced for abandoning stillborn twins
Vietnamese trainee endured 2 years of physical abuse
Group: Use ‘plain Japanese’ in vaccine info for foreign residents
Information on the latest cherry blossom conditions
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A series based on diplomatic documents declassified by Japan’s Foreign Ministry
Here is a collection of first-hand accounts by “hibakusha” atomic bomb survivors
chefs and others involved in the field of food introduce their special recipes intertwined with their paths in life
A series about Japanese-Americans and their memories of World War II
<a href="/ajw/inhouse_news/">In-house News and Messages</a>
No reproduction or republication without written permission
Japan (Reuters) -     Japan warned of more rain to come
especially in the southwest island of Kyushu
as rescue teams searched for people missing in landslides and residents grew weary of the continuing rain
Heavy rains lashed much of Japan over the weekend
submerging streets and triggering landslides that killed four people in Nagano and Nagasaki prefectures according to local media
Kyushu and other areas of Japan have seen record levels of rainfall
Residents of the small town of Soeda on Kyushu island spoke of their weariness of rains that have been pummelling them for the past few days
so I've been really worried," said Ryoji Watakabe
and he has closely watched the river that flows past his shop for signs it would overflow
"I was trembling with fear as I monitored the river" over the past few days
which lies in a mountainous area along a river
a 75-year-old woman was rescued from a landslide that happened earlier in the day
Local resident Hideki Fujikawa said the rain is unsettling
"I can hear the rocks in the river rolling on the riverbed at night
and it's very hard to sleep," said Fujikawa
brown water flowed fast in the river next to his house
He checks on the river every so often to make sure the water hasn't risen too high
He said he thinks his safety isn't at risk for now
                (Reporting by Sakura Murakami and Joseph Campbell; Editing by Tom Hogue)
<a href='/en/ncommon/contents/news/1008550/1008550.jpg' class='fancybox' rel='gallery' data-libid='1008550'></a>FILE PHOTO: Rescue workers search for missing people at a landslide site caused by heavy rainfall in Unzen
in this handout image taken and released by Unzen City August 15
<a href='/en/ncommon/contents/news/1008551/1008551.jpg' class='fancybox' rel='gallery' data-libid='1008551'></a>A landslide that left a 75-year-old woman injured is pictured after heavy rain in Soeda
<a href='/en/ncommon/contents/news/1008552/1008552.jpg' class='fancybox' rel='gallery' data-libid='1008552'></a>Workers clear a site where a landslide left a 75-year-old woman injured after heavy rain in Soeda
<a href='/en/ncommon/contents/news/1008553/1008553.jpg' class='fancybox' rel='gallery' data-libid='1008553'></a>Workers clear a site where a landslide left a 75-year-old woman injured after heavy rain in Soeda
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a former Amagasaki mayor in Hyogo Prefecture
8 that she will run for the prefectural governor's seat that was recently vacated by scandal-ridden incumbent Motohiko Saito
Inamura held a news conference that day at the prefectural government's building
Her candidacy follows Saito losing the position after the prefectural assembly unanimously passed a no-confidence motion against him on Sept
The election campaign period will officially start Oct
who also previously served as a prefectural assembly member
said she decided to run due to a whistleblower document scandal involving Saito
which accused him and others of abusive and corrupt actions
“I am determined not to let the confusion and stagnation in the prefectural government continue as is,” she said
Inamura said she was personally approached by people related to the prefectural assembly and others about running for office
She said she will be committed to examining the response to the whistleblower document issue and enacting an ordinance to prevent power harassment
which would also apply to the governor and vice governor
Regarding free tuition at prefectural universities
“There may be room to consider a scholarship system that covers a wider range of young people,” assuming that those already eligible for the scholarship are not disadvantaged
Inamura is originally from Nara Prefecture
After serving two terms as a Hyogo prefectural assembly member
she ran in Amagasaki city's 2010 mayoral election and won
making her the youngest female mayor in Japan at the time
at least six candidates including Saito and Inamura have declared their intention to run
Nippon Ishin no Kai (Japan Innovation Party) is considering supporting Takayuki Shimizu
is endorsed by the Japanese Communist Party
a former mayor of Kasai city in the prefecture
Embattled Hyogo governor to lose post but will run in next election
Inaction and fear allow abuses in local government to go unchecked 
Campaigning starts in nine elections for governor
Major parties take aim at Osaka Ishin in mayor election
Pressure rises on Hyogo governor after death of ‘whistleblower’
KOBE--A Buddhist priest here has consoled the souls of about 50 Vietnamese people
including students and trainees killed in accidents and incidents in Japan
and sent the remains to their bereaved families
has received no compensation for his support
as 80 percent of those he looked after were in their 20s
“I cannot substitute for their family members but I want to stay close to them until the last moments,” said Tri
Tri came to Japan in 2015 to refine his skills as a web designer
Shortly after he began studying Japanese in Kyoto Prefecture
Tri learned that a high school classmate died from overwork in Mie Prefecture under Japan’s foreign intern program
The system is intended to provide job skills that trainees can use after they return to their home countries
complaints are rife about employers overworking the interns or burdening them with menial tasks under horrific working conditions
Tri heard the classmate’s bereaved family could not visit Japan for financial reasons so the chief priest of the Vietnam-style temple had the body cremated
“The classmate left our homeland and worked hard his entire life and had dreams just like I have,” said Tri
Tri since learned Buddhism to enter the priesthood
He became the new head of Chua Hoa Lac three years ago
When a Vietnamese man was beaten and pushed into the Dotonbori river in a busy commercial area in Osaka last summer and died
a friend of the victim who was “at a loss about what to do” contacted Tri for advice
Tri was commissioned by his bereaved family to pick the body up from Osaka prefectural police so a funeral could be held in Osaka with more than 100 Vietnamese and others in attendance
His relatives were unable to enter Japan due to the travel restrictions stemming from the ongoing novel coronavirus pandemic
and they watched the service through a video link
The man's remains were cremated and delivered to his family
Chua Hoa Lac also offers accommodations and meals for young people plagued by human relationships and jobs
More individuals are currently coming to the temple amid the virus outbreak
“My happiness is everyone becoming happy,” Tri said
Saitama temple takes in exploited Vietnamese trainees
Buddhists urge calm in videos: ‘Don’t fight over toilet paper’
Vietnamese find lifeline at temple in Nagoya after losing jobs
Memorial service held for Sri Lankan who died in detention
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<a href="//mainichi.jp/articles/20230826/k00/00m/040/131000c">Japanese version</a>
A growing number of Japanese companies are on a mission to hire a more diverse range of workers
including people with conditions like autism and ADHD
to ensure a broader range of perspectives and ways of thinking among their employees
They say increasing neurodiversity in the workplace can help drive innovation
Yoshida Soryu is on the autism spectrum and lives with an intellectual disability
The company is trying to diversify its workplace
Employees with certain disabilities or disorders go through a rigorous training period
the company can better understand their strengths and needs
While he loves talking with his colleagues
Soeda says answering the phone is really hard for him because he is very sensitive to sound
I would be reprimanded if I didn't perform well
because I have colleagues who understand me."
The company says finding each employee a position where they can excel and be happy creates a win-win situation
"We support both them and their coworkers even after the placement," says Nagai Yuko
"It means the employees stay with the company longer."
Marui Group is one of many companies across the world that are now taking this approach
US tech giants have spent years courting neurodivergent employees
Microsoft launched its hiring program in 2015 with the idea that different ways of thinking could help fuel innovation
a growing number of companies are embracing the same philosophy
the Japan Research Institute think-tank teamed up with seven firms to launch an initiative to hone their management practices and recruit neurodivergent candidates
"We don't know what we can specifically do to increase neurodiversity on our digital teams," says Sato Kazuaki
an executive with one of the companies that took part
The firm joined the initiative in the hope of getting some ideas
That's leading employers to broaden their hiring outlook
and also prompting some to embrace more flexible work practices
Senior Development Manager at the Japan Research Institute
says neurodivergent employees "can reach their full potential if there are adjustments to the work environment."
"There might be roles that match their talents well
It's important to leverage untapped talent and make it a driving force of corporate growth."
says people with the same diagnoses can have very different strengths and needs
He says reasonable accommodations are required to support them in the workplace
"Some people have enormous ability but struggle when working in a team or interacting with people
More companies are now modifying their work environments to help them
for example to allow for communication by email."
Umenaga says employers can improve the performance of their overall business if they recognize the learning styles and unique abilities of their employees and provide appropriate support
As the Japanese work environment becomes more flexible
neurodivergent employees are inspiring companies with fresh ideas and new perspectives
Add articles to your saved list and come back to them any time
FICTIONThe City And Its Uncertain WallsHaruki MurakamiPenguin $49.99
Shortly before the release of A Wild Sheep Chase
the gleefully madcap debut that shot him to instant stardom
Haruki Murakami published a novella in a Japanese literary magazine
It went mostly unnoticed and has been the subject of little discussion in the 40-plus years since
at the tail end of a stellar career – one that has seen his name regularly bandied about for the Nobel Prize – Murakami has returned to that novella
expanded it substantially and lumped us with another hefty tome that might appeal to diehard fans but is otherwise just one more in a long line of recent disappointments
That the first part of The City and Its Uncertain Walls stems from Murakami’s creative juvenilia is obvious
that I half expected Prince to come back from the dead to sing about it
It’s a strange juxtaposition; this conceptual precursor to one of my favourite Murakami novels
Hard-Boiled Wonderland and the End of the World
scribbled down by someone neck-deep in a barrel of adjectives
If you can step over the “delicate silvery fish” and tune out the “cries of the invisible night birds”
there is much to like in this youthful stab at magical realism
An unnamed narrator recalls his first love
its only entrance guarded by a fearsome gatekeeper
an opportunity for him to pass through arises.The town library requires a new dream reader
He meets the gatekeeper who rips away his shadow and cuts his eyes
Had The City and Its Uncertain Walls ended there
it would have made for an enchanting piece of Murakami ephemera
as he escapes Tokyo to become the head librarian in a small country town
He is completely unqualified for the job and fumbles through the day-to-day necessities
The only regular visitor to the library is a teenager in a Yellow Submarine parka who spends his days wolfing down books
There is little substance to these characters
opportunities for Murakami to wax lyrical – at length – on his favourite topics
Murakami’s latest novel might be for diehard fans only.Credit: Nathan Bajar
lectures the narrator on the fundamental difference between the corporeal self and the soul
described in classic Murakami fashion in terms of her body and sexual allure (she has “healthy-looking calves”)
explains the history of the library and the tragic life and death of Mr Koyasu
who one day draws a map of the walled-in town of the narrator’s youth
He has “savant syndrome … like the character in the movie Rain Man”
There’s a fine line between representation and exploitation
By leaning into tired tropes that reduce the boy to a narrative device that bridges the real and magical worlds
Murakami shows little insight into where that line might lie
I got the sense that The City and Its Uncertain Walls was a book deeply at odds with itself
it struck me as a hodgepodge of the literary titans who informed Murakami’s early sensibilities and the cultural trends that have bubbled up around him over the last four decades
Murakami has the girl in the dream library declare that she only existed for the narrator and now that he is leaving
Add to it all the frankly undergraduate philosophical ruminations on the soul
and I was left to wonder what – other than the boredom of pandemic lockdown and the self-confessed fear that he had run out of stories to tell – compelled Murakami to write it
a relatively unknown Murakami has just published a delightful novella about a town with impregnable
and a man who arrives there to become the resident dream-reader
The Booklist is a weekly newsletter for book lovers from Jason Steger.<a href="https://www.brisbanetimes.com.au/newsletter-signup?newsletter=the-booklist"> Get it delivered every Friday</a>
at the tail end of a stellar career \\u2013 one that has seen his name regularly bandied about for the Nobel Prize \\u2013 Murakami has returned to that novella
That the first part of The City and Its Uncertain Walls stems from Murakami\\u2019s creative juvenilia is obvious
It\\u2019s a strange juxtaposition; this conceptual precursor to one of my favourite Murakami novels
If you can step over the \\u201Cdelicate silvery fish\\u201D and tune out the \\u201Ccries of the invisible night birds\\u201D
opportunities for Murakami to wax lyrical \\u2013 at length \\u2013 on his favourite topics
described in classic Murakami fashion in terms of her body and sexual allure (she has \\u201Chealthy-looking calves\\u201D)
who one day draws a map of the walled-in town of the narrator\\u2019s youth
He has \\u201Csavant syndrome \\u2026 like the character in the movie Rain Man\\u201D
There\\u2019s a fine line between representation and exploitation
There\\u2019s an awkward tilt at pandemic lit
it struck me as a hodgepodge of the literary titans who informed Murakami\\u2019s early sensibilities and the cultural trends that have bubbled up around him over the last four decades
Not that he trusts in the intelligence of his readers to connect the dots
we get an entire primer on magical realism
including excerpts from Marquez\\u2019s Love in the Time of Cholera
there is the repeated suggestion that he was \\u201Cspirited away\\u201D
as if the multiple nods to  weren\\u2019t obvious enough
in case the episode with the gatekeeper didn\\u2019t conjure \\u2019s Before the Law in the reader\\u2019s mind
and I was left to wonder what \\u2013 other than the boredom of pandemic lockdown and the self-confessed fear that he had run out of stories to tell \\u2013 compelled Murakami to write it
The Booklist is a weekly newsletter for book lovers from Jason Steger.
NEWSPAPER SECTION: <a title="NEWSPAPER SECTION: Sports" href="https://search.bangkokpost.com/search/result_advanced?category=news&xNewsSection=Sports">Sports</a>
Thailand's No.1 Danai Udomchoke crashed out in the first round of the PTT Thailand Open after losing to Japan's Go Soeda 6-4
Danai Udomchoke hits a forehand return during his first round match against Go Soeda
His defeat ended local interest in the singles competition of the US$551,000 tournament
who received a wild card into the tournament
became the only player to have played in all 10 editions of the Thailand Open
''I knew I was the underdog coming into this match,'' said Danai
''I knew that I had to be more defensive and go for passing shots but he returned so well and I made too many mistakes
It was very disappointing to go out so early.''
World No.50 Soeda will take on French fourth seed Gilles Simon in the second round
Serbian top seed Janko Tipsarevic became the first player to reach the quarter-finals after he beat Japanese qualifier Hiroki Moriya 6-4
will meet the winner between American Donald Young and fifth seed Fernando Verdasco of Spain
world No.56 Gael Monfils continued his strong return from injury with a 6-4
7-5 first-round victory over South African qualifier Kevin Anderson
The Frenchman needed two hours and 25 minutes to secure his progress
who had been out since May with right knee trouble but reached the semi-finals in his comeback event last week in Metz
said his injury woes had given him a ''new mindset'' now he is back in action
that is the biggest thing,'' said the unseeded Monfils
''It was four tough months for me _ I didn't know when I would be back on the court
''I've been in the top 10 and I'm motivated to be there again
Towering Croatian Ivo Karlovic reached the second round as he beat Australian Marinko Matosevic 6-3
Karlovic will next play Canadian third seed Milos Raonic
Raonic yesterday learnt some Muay Thai lessons from Thai Olympic silver medallist Kaew Pongprayoon and his boxing teammates Chatchai Butdee and Sailom Ardee
''This is my first time learning Thai boxing
I think it is not for me because I am very tall,'' said a smiling Raonic
Danai still stay alive in the doubles after teaming up with Lu Yen-hsun of Taiwan to win their first-round match yesterday
The Thai-Taiwanese wild-card combination defeated Johan Brunstrom of Sweden and Jonathan Marray of Great Britain 6-1
Danai and Lu will meet Britain's Jamie Murray and Brazil's Andre Sa in the quarter-finals
Murray and Sa upset second seeds Santiago Gonzalez of Mexico and Scott Lipsky of the United States 6-4
Singles first round: Ivo Karlovic (CRO) beat Marinko Matosevic (AUS) 6-3
Go Soeda (JPN) beat Danai Udomchoke (THA) 6-4
7-5; Gael Monfils (FRA) beat Kevin Anderson (RSA) 6-4
Second round: Janko Tipsarevic (SRB) bt Hiroki Moriya (JPN) 6-4
Doubles first round: Jamie Murray (GBR)/Andre Sa (BRA) beat Santiago Gonzalez (MEX)/Scott Lipsky (USA) 6-4
10-7; Leander Paes (IND)/Horia Tecau (ROU) beat Robin Haase (NED)/Jean-Julien Rojer (NED) 6-4
7-5; Christopher Kas (GER)/Viktor Troicki (SRB) beat Grigor Dimitrov (BUL)/Bernard Tomic (AUS) 6-4
7-5; Danai Udomchoke (THA)/Lu Yen-Hsun (TWN) beat Johan Brunstrom (SWE)/Jonathan Marray (GBR) 6-1
Quarter-final: Sonchat and Sanchai Ratiwatana (THA) beat David Marrero/Fernando Verdasco (ESP) 6-3
TODAY'S ORDER OF PLAYCentre Court (12 noon)
Bernard Tomic (AUS) vs Dudi Sela (ISR); Gael Monfils (FRA) vs Viktor Troicki (SRB); Donald Young (USA) vs Fernando Verdasco (ESP)
Ivo Karlovic (CRO) vs Milos Raonic (CAN); Grigor Dimitrov (BUL) vs Richard Gasquet (FRA)
Gilles Simon (FRA) vs Go Soeda (JPN); Lu Yen-Hsun (TWN)/Danai Udomchoke (THA) vs Jamie Murray (GBR)/Andre Sa (BRA); Leander Paes (IND)/Horia Tecau (ROU) vs Janko Tipsarevic (SRB)/Alexander Waske (GER)
Eric Butorac (USA)/Paul Hanley (AUS) vs Christopher Kas (GER)/Viktor Troicki (SRB)
Big-serving Ivo Karlovic of Croatia in action during his match against Marinko Matosevic of Australia
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OSAKA--Flying cars are one step closer to ferrying attendees of the 2025 Osaka Kansai Expo around the exhibition halls as a means of transportation
fulfilling the vision of local officials here
together with the Osaka prefectural government
14 signed a partnership agreement with SkyDrive Inc.
a Tokyo-based entrepreneurial venture that develops drones for logistics
Osaka Governor Hirofumi Yoshimura took a demonstration ride in an exhibited prototype that day
“Taking on a new challenge is one of Osaka’s strengths,” Yoshimura said
“The image of it flying in the sky above resembles a sci-fi movie
SkyDrive launched a project to develop a flying vehicle in 2018 and has conducted demonstration experiments and analyses as well as manned flying experiments
Its latest prototype is a single-seated vehicle 2 meters high and 4 meters in width and length
The flying vehicle is equipped with eight electric propellers and can fly at a maximum speed of 40 to 50 kph for 5 to 10 minutes at a time
Before such flying cars are put to work at the Expo
legislation still has to be passed allowing their use
New legislation gives companies legal rights to lunar resources
Osaka Kansai Expo in rush to entice pavilion operators
New humanoid robots closing in on humans with better dexterity
Comeback for futurist version of human washing machine
Director Izutsu uses yakuza’s life to portray ‘heat’ of Showa Era
Weird but eye-catching Osaka Expo logo raises public’s interest
<a href="//mainichi.jp/articles/20180613/k00/00e/040/210000c">Japanese version</a>
<a href="//mainichi.jp/articles/20190622/k00/00m/040/054000c">Japanese version</a>
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This Japanese-Muslim Revert In SG Shares About Her Interracial Marriage And Journey To Islam
This Japanese-Muslim Revert In SG Shares About Her Interracial Marriage And Journey To Islam  • Dec 26
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