Volume 11 - 2017 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fnhum.2017.00181">https://doi.org/10.3389/fnhum.2017.00181</a>
The lateral occipito-temporal cortex (LOTC)
is known to be involved in the perception of body parts
recent studies have demonstrated the role of the LOTC in higher-level body-related cognition in humans
This study consisted of two experiments (E1 and E2)
The first (E1) was an exploratory experiment to find the neural correlate of the mental manipulation of body part imagery
in which brain cerebral glucose metabolic rates and the performance of mental rotation of the hand were measured in 100 subjects who exhibited a range of symptoms of cognitive decline
we found that the level of glucose metabolism in the right LOTC was significantly correlated with performance in a task involving mental manipulation of the hand
controlled intervention study (clinical trial number: UMIN 000018310) in younger healthy adults to test whether right occipital (corresponding to the right LOTC) anodal stimulation using transcranial direct current stimulation (tDCS) could enhance the mental manipulation of the hand
we demonstrated a significant effect of tDCS on the accuracy rate in a task involving mental manipulation of the hand
Although further study is necessary to answer the question of whether these results are specific for the mental manipulation of body parts but not non-body parts
E1 demonstrated a possible role of the LOTC in carrying out the body mental manipulation task in patients with dementia
and E2 suggested the possible effect of tDCS on this task in healthy subjects
These findings suggest that the EBA not only receives sensory inputs regarding others&#x2019; body information but also represents the human body in a dynamic manner
including kinesthetic feedback for one&#x2019;s own actions
If the EBA receives kinesthetic information about self-actions
it is easy to speculate that the EBA would also be activated during the manipulation of body imagery
the inferior and middle occipital gyrus and the cerebellum
They also demonstrated that the mental rotation of bodily parts activates the cerebellum
the middle and inferior occipital and calcarine gyrus
the left inferior parietal lobe and the right supramarginal gyrus
the left precentral gyrus and the bilateral inferior frontal gyrus
the right middle frontal gyrus and the medial posterior frontal gyrus in addition to the right insula
not only motor areas but also visual areas
seem to be involved in the mental rotation task
With the aid of decompensated brains (i.e., elderly subjects mainly consisting of patients with dementia; n = 100), the first part of the present study (E1) intended to examine the brain region that is critical in cognitive decline (i.e., decompensation) in the mental rotation task (Figure <a href="#F1">1</a>)
we discovered that lower glucose metabolic ratios in the EBA and the FBA were correlated with lower performance on the mental manipulation of the hand imagery task
The participants were instructed to select the right or left picture that included a circle (i.e.
a target) by pressing a button with their right or left hand as soon as possible after a circle appeared on the screen
(B) During the recognition phase of the simple visual working memory task (WM task)
the participants were instructed to select the picture that depicted the same hand as that presented during the acquisition phase (non-flipped images)
(C) During the recognition phase of the visual working memory with mental rotation task (RWM task)
the participants were instructed to select the picture that depicted the same hand
which was flipped from the palm side (acquisition) to the back side (recognition)
Each condition consisted of 10 trials with different hand shape pictures and lasted for a duration of 3 min
we hypothesized that active stimulation of the LOTC using tDCS would enhance performance on the mental manipulation of the hand imagery task
we used the tDCS technique to perform a randomized
All participants underwent brain MRI and [18F] fluoro-deoxyglucose (FDG)-PET scans
The present study was approved by the Ethics Committee of Hamamatsu Medical Center (Heisei 8-1)
and written informed consent was obtained from all participants prior to enrollment
The methods were carried out in accordance with approved guidelines
Three visual tasks were presented on a liquid crystal screen in front of the subjects. As shown in Figure <a href="#F1">1</a>, one choice reaction time task (CRT task) and two types of working memory tasks were employed (<a href="#B24">Kikuchi et al., 2011</a>)
the participants were instructed to respond to a circle (i.e.
a target) that appeared on the screen by pressing a button with the hand that was located on the same side as the circle (either their right or their left hand) as soon as possible
One working memory task consisted of a simple visual working memory task (WM task) that involved pictures of various hand shapes (palm side only) and the other working memory task consisted of a visual working memory task with mental rotation (RWM task)
which used pictures of various hand shapes (palm and back side)
To control for speed&#x2013;accuracy trade-offs in the cognitive outcome data, we calculated a value [inverse efficiency (IE) score (<a href="#B57">Townsend and Ashby, 1983</a>; <a href="#B31">Ludwig et al., 2011</a>)] by dividing the median response time (RT) by the accuracy rate in each task condition
Because the RT was measured in ms and was divided by a unitless number
an average RT of 1000 ms and a 10% error rate would yield an IE value of 1111 ms (1000/(1&#x2013;0.1))
A lower IE score indicates better performance
All participants underwent 3-dimensional MRI immediately before the PET measurements. During this process, a static magnet (0.3 T MRP7000AD; Hitachi, Tokyo, Japan) was used in the 3-dimensional mode (<a href="#B42">Ouchi et al., 2001</a>)
The patients underwent a series of PET measurements after completing the battery of neuropsychological tests and the MRI examination. A high-resolution brain PET scanner was used (SHR12000; Hamamatsu Photonics K.K., Hamamatsu, Japan) (<a href="#B44">Ouchi et al., 1999</a>)
After a subject&#x2019;s head was fixed with a thermoplastic face mask and a 10-min transmission scan was acquired
a static 15-min PET scan was performed 45 min after an injection of a 1.2 MBq/kg dose of [18F] FDG
To evaluate glucose metabolism, a semiquantitative ratio index of [18F]FDG was calculated to obtain the standardized uptake value ratio (SUVR) (<a href="#B43">Ouchi et al., 2009</a>)
SPM8 was used for the voxel-wise analysis (voxel-size; 2 mm &#x00D7; 2 mm &#x00D7; 2 mm resolution)
All [18F]FDG-SUVR parametric images were first normalized to the MNI space and smoothed with an 8 mm isotropic Gaussian kernel
Voxel-based correlations were computed between [18F]FDG-SUVR parametric images and IE scores or accuracy rates in the three conditions using a multiple regression model with the statistical threshold set at p = 0.05 (corrected with FWE) for the peak height
These analyses were applied for the data from the healthy elderly subjects (n = 22) and all participants
including the cognitively impaired subjects (n = 100)
Based on the results from the first part of the present study
The study was registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (number UMIN000018310)
20&#x2013;43 years; all right-handed as assessed by the Edinburgh Handedness Inventory) performed the same tasks as described in the current PET study while receiving anodal tDCS to either the right occipital cortex covering the LOTC (i.e.
the region highlighted in the mental rotation task) or the right dorsolateral prefrontal cortex (DLPFC) (i.e.
the region used for the control condition) in combination with cathodal tDCS of the right DLPFC or the right occipital cortex (the former condition was referred to as occipital anodal/frontal cathodal stimulation and the latter condition was referred to as frontal anodal/occipital cathodal stimulation)
The participants were not taking any medications
had no history of neurological or psychiatric disease
and had normal physical and neurological examinations
Written informed consent was obtained prior to participation in the study
The Ethics Committee of Kanazawa University Hospital approved the methods and procedures and the methods were carried out in accordance with approved guidelines
The demographic data for all participants are presented in Supplementary Table S1
the electrodes were placed at the same positions used during active stimulation
but the stimulator was turned on for only 30 s
the participants may have experienced a tDCS-induced itching sensation at the beginning of the session but received no active current for the remaining stimulation period
LOTC and DLPFC] marked on the reconstructed scalp and brain surface of one subject in the active condition
(B) Schematic representation of the experimental design
and RWM tasks after receiving active or control stimulation for 10 min
The stimulation continued throughout the duration of the three tasks and for an additional 10 min
To determine whether anodal stimulation to the right occipital region affected the mental manipulation of body part imagery
we evaluated the performance of the subjects (IE score and accuracy) during the CRT
The performances of the subjects in the active group (i.e.
occipital anodal/frontal cathodal stimulation) were compared with those of the subjects in the control group (i.e.
a mixture of frontal anodal/occipital cathodal stimulation and sham stimulation
Based on the results of E1 in the present study
we hypothesized that the IE score will be lower and the accuracy rate will be higher during stimulation in the occipital anodal/frontal cathodal condition compared with the control condition
an unpaired t-test (one-sided) was used to compare the two groups (i.e.
a Wilcoxon rank-sum test (one-sided) was used to compare the two groups (i.e.
To avoid the risk of low statistical power caused by the smaller sample size
we added complementary analyses between sub-conditions (i.e.
we divided the control condition into two conditions) to test the difference between the active (occipital anodal/frontal cathodal stimulation; n = 20) and the reversed (frontal anodal/occipital cathodal stimulation; n = 10) or the sham conditions (n = 10)
a two-way ANOVA was performed (task &#x00D7; tDCS condition) for the IE score of the three tasks
The within-subjects factor was the task effect (CRT vs
RWM tasks) and the between-subjects factor was the tDCS effect (active vs
As shown in Supplementary Figure S1 and Table <a href="#T1">1</a>
the healthy elderly tended to show higher accuracy
and lower IE scores compared with the cognitively impaired patients in all tasks
The diversified data of the cognitive and brain metabolic profiles in the 22 healthy and 78 cognitively impaired subjects allowed us to conduct correlation analyses
Demographic characteristics of all subjects in the PET study
SPM analyses with multiple regression models in which the accuracy rate (A) or the inverse efficiency (IE) scores (B&#x2013;D) for the WM conditions were used as independent variables
(A) A decrease in the [18F]FDG-SUVR in the right frontal cortices was associated with poorer performance (i.e.
this significant association disappeared if we employed a conservative statistical threshold set at p = 0.05 (corrected with FWE)
(B,D) A decrease in the [18F]FDG-SUVR in the frontal cortices was associated with poorer performance (i.e.
(C) This significant correlation was still observed in the right frontal cortex if we employed a conservative statistical threshold set at p = 0.05 (corrected with FWE)
The yellow color bar indicates the T-value
SPM analyses with multiple regression models in which the accuracy rate (A) or the IE scores (B&#x2013;D) for the RWM conditions were used as independent variables
(A) A decrease in the [18F]FDG-SUVR in the occipital and parietal cortices was associated with poorer performance (i.e.
(B,D) A decrease in the [18F]FDG-SUVR in the occipital and parietal cortices was associated with poorer performance (i.e.
(C) This significant correlation was still observed in the bilateral occipito-temporal cortices if we employed a conservative statistical threshold set at p = 0.05 (corrected with FWE)
Brain regions in which a decrease in the [18F]FDG-SUVR was significantly associated with poorer task performance
Regarding the IE score, an unpaired t-test (one-sided) did not reveal significantly higher performance (i.e., a lower IE score) in the right occipital anodal stimulation group (i.e., active condition, n = 20) compared to the control group (n = 20) (t = 0.684, p-value > 0.05), which did not support our hypothesis. However, regarding the accuracy rate, as shown in Figure <a href="#F5">5A</a>
a Wilcoxon rank-sum test (one-sided) revealed a significantly higher performance (i.e.
higher accuracy rate) in the RWM task in the right occipital anodal stimulation group (i.e.
n = 20) than in the control group (W = 135.5
(A) Accuracy scores in the three tasks (i.e.
and RWM) with the application of active (red circles) and control (blue circles) transcranial direct current stimulation (tDCS)
The RWM task scores during right occipital anodal stimulation (i.e.
n = 20) were significantly higher than those during the control stimulation (n = 20)
(B) If the control condition was divided into two conditions
the reversed condition (frontal anodal/occipital cathodal stimulation; n = 10) (blue circles) and sham condition (n = 10) (green circles)
the RWM task scores during the active condition (n = 20) were significantly higher than those during the reversed condition (n = 10) but not than those during the sham condition (n = 10)
As complementary analyses for the IE score of the three repeated tasks
a two-way ANOVA (3 tasks &#x00D7; 2 tDCS conditions) revealed neither a significant main effect (tDCS condition; F = 0.173
p-value > 0.05) nor an interaction between the two factors (task &#x00D7; tDCS condition; F = 0.501
When we divided the control condition into two conditions
the reversed (n = 10) condition and the sham condition (n = 10)
a two-way ANOVA (3 tasks &#x00D7; 3 tDCS conditions) revealed neither a significant main effect (tDCS condition; F = 2.487
p-value > 0.05) nor an interaction between the two factors (task &#x00D7; tDCS condition; F = 1.691
we tested the hypothesis that active stimulation of the LOTC using tDCS would enhance performance on the mental manipulation in the hand imagery task
The results suggested a possible effect of tDCS on this task
Further study is necessary to clarify whether these results in E1 and E2 were specific for the mental manipulation of body parts but not non-body parts
which is consistent with our results from E1 (i.e.
functional deterioration in the LOTC induced the disability in mental rotation)
Although we did not know which strategy our participants preferred when rotating their hands in their mind in this study
One explanation is that internal hand imagery (possibly governed by the EBA) was necessary for the subjective view (i.e.
internal strategy) required to achieve mental rotation of the hands
The other explanation is that objective hand imagery (also possibly governed by the EBA) was required for an external object (i.e.
Although further studies are necessary to confirm whether anodal stimulation of these brain areas affects the mental manipulation of body part imagery
the right LOTC may be a possible candidate that plays a pivotal role not only in the mental recognition of body parts
but also in the mental manipulation of body part imagery
our data suggested the facilitative effect of tDCS (anodal stimuli for the right posterior brain area) on performance in the mental rotation task
This study was approved by the Ethics Committee of Hamamatsu University School of Medicine and Hamamatsu Medical Center and by the Ethics Committee of Kanazawa University Hospital in Japan
Written informed consent was obtained from all participants prior to enrollment
written informed consent was obtained from participants and their family prior to enrollment
direct and intellectual contribution to the work
The study was supported by the Center of Innovation Program of the Japan Science and Technology Agency
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
Masami Futatsubashi (Hamamatsu Photonics KK)
and Yutaka Naito (Japan Environment Research Corporation) for their support
The Supplementary Material for this article can be found online at: <a href="http://journal.frontiersin.org/article/10.3389/fnhum.2017.00181/full#supplementary-material">http://journal.frontiersin.org/article/10.3389/fnhum.2017.00181/full#supplementary-material</a>
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Minabe Y and Ouchi Y (2017) The Lateral Occipito-temporal Cortex Is Involved in the Mental Manipulation of Body Part Imagery
Copyright &#x00A9; 2017 Kikuchi, Takahashi, Hirosawa, Oboshi, Yoshikawa, Minabe and Ouchi. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
distribution or reproduction in other forums is permitted
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*Correspondence: Yasuomi Ouchi, <a id="encmail">b3VjaGlAaGFtYS1tZWQuYWMuanA=</a>
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Japan-based freelance writer and director Felicity Tillack explores the gastronomic delights of her former home on behalf of Wakayama Tourism
When people ask me where my favourite place in Japan is
only an hour-long train ride from Kansai International Airport
is home to everything a traveller would want to experience while visiting Japan: the clear waters and white sand of Shirahama; forested mountains that shelter Buddhist temples; natural hot springs that were enjoyed by emperors; friendly and hospitable people; and a knack for creating amazing food
(Credit: Minabe Town Tourism Center)Shirarahama Beach Next12For centuries
have been renowned across Japan and in addition
the prefecture is said to be the birthplace of Japanese soy sauce
While these traditional staples continue to be savoured
in recent years many restaurants in the region have been recognised with Michelin stars and Wakayama is keen to promote itself as a destination for international foodies
Having previously lived in Wakayama for four years
and with Wakayama&rsquo;s rising prominence in gastronomy
I felt inspired to revisit Wakayama&rsquo;s famous produce and set myself the delectable task of savouring its fruity&nbsp;ume&nbsp;preserves and wines
its mouthwatering&nbsp;miso&nbsp;and its prized wild tuna&nbsp;sashimi
Wakayama is home to many Unesco World Heritage sites
an ancient pilgrimage trail that stretches from the country&rsquo;s tallest waterfall at Nachi Taisha to the granite memorials and serene cedar forests of Koyasan
Travellers who walk both the Kumano Kodo and Spain&rsquo;s Camino de Santiago route can receive the title of &ldquo;dual pilgrims&rdquo;
The prefecture has taken great effort in producing beautiful and informative English language pamphlets and the excellent official guide to the Kumano Kodo to help visitors interested in hiking the sacred trails
 PreviousNachi WaterfallNachi Waterfall Next12For those with a car and a sense of adventure
I recommend driving the inland routes between the folds of Wakayama&rsquo;s steep
The roads are very twisty as they hug close to the edge of fast-flowing rivers
completely immersed in the naturally heated waters and more than 1000 years of history
Minabe is a small coastal town on Wakayama&rsquo;s East coast
its famous mountainside orchards blossom as almost a million&nbsp;ume&nbsp;trees bloom.&nbsp;Ume&nbsp;is a fruit that resembles
apricots but which must be preserved to consume
Wakayama produces most of Japan&rsquo;s&nbsp;ume&nbsp;and by June the fruit will be ready to harvest and made into&nbsp;umeshu
&ldquo;Minabe has worked hard to develop its special Nanko&nbsp;ume
They are widely considered to be high quality and juicy!&rdquo; says Iwamoto Keiko
a restaurateur who uses&nbsp;ume&nbsp;in her cooking
as well as an&nbsp;umeboshi&nbsp;shop owner
She is part of the community effort in Minabe to spread Wakayama&nbsp;ume&nbsp;to the world
She is also keen to demonstrate the multitude of ways that&nbsp;ume&nbsp;can be consumed.&nbsp;&nbsp;
(Credit: Minabe Town Tourism Center)The ume plums are checked for ripeness
and shochu flavoured with Wakayama’s famous ume plums
(Credit: Hidaka Kouiki Kankou Sinkou Kyougikai) Next13&ldquo;While many people have an image of&nbsp;umeboshi&nbsp;as being sour
&ldquo;It really matches the flavour of orange juice
we mix&nbsp;ume&nbsp;jam into demi-glace sauce or use&nbsp;umeboshi&nbsp;instead of salt in my garlic prawns
I even put a mix of sweet and salty&nbsp;ume&nbsp;on pizza.&rdquo;
Iwamoto suggested I mix some&nbsp;ume&nbsp;jam with yoghurt for breakfast
deep edge of flavour that quickly became addictive
Cravings for something more savoury were soothed by an&nbsp;onigiri&nbsp;rice ball with an orange flavoured&nbsp;umeboshi&nbsp;at its centre
and&nbsp;ume&nbsp;hot sauce added some tasty complexity to chicken wraps for dinner
A little further north of Minabe is the tiny town of Yuasa which retains much of its traditional charms.&nbsp; Rows of wooden homes with their ceramic tile roofs and bamboo window lattices give visitors the sense of slipping back through time
hand-painted plates hang in glass display cases along the walls for passersby to enjoy
a particularly tasty type of&nbsp;miso&nbsp;called,&nbsp;kinzanji miso
While a lesser-known Wakayama delicacy,&nbsp;kinzanji miso&nbsp;has a supersized claim to fame as the predecessor of Japanese soy sauce
The story goes that a Buddhist monk brought the original&nbsp;miso&nbsp;from China
the people of Yuasa discovered the umami filled tastiness of the savoury brown sauce by-product during the fermentation process
the town of Yuasa realised its delicious potential and began focusing on its production specifically
 PreviousYuasa townOta Kyusuke is a company that started producing soy sauce during the Edo period
and today specialises in making miso by hand.Making kinzanji misoMaking soy sauce Next14The&nbsp;kinzanji miso&nbsp;itself is made from fermented soybeans
barley and Japanese summer vegetables: eggplant
Unlike other forms of&nbsp;miso,&nbsp;kinzanji miso&nbsp;is not used as a soup base
but the condiment is also perfect to snack on by itself or as an accompaniment to a glass of sake.&nbsp;
freshly cooked rice.&nbsp;Kinzanji miso&nbsp;has a wonderfully deep aroma and each mouthful is a bit different: with spiciness from the chunks of ginger
rich and packed with all-important&nbsp;umami
&ldquo;This is rain country,&rdquo; he says
comes down through our rivers and flows into the ocean
I would like you to experience the mountains
Japan has a tradition of&nbsp;sei shoku,&nbsp;or raw food
so a high priority is placed on the freshness of the ingredients and the quality of their taste and texture
the sooner it is consumed the better and auctions start early in the morning so that the tuna can be enjoyed in high-end sushi restaurants
maintaining its taste and melt-in-your-mouth consistency
Nachi Katsuura&rsquo;s tuna market on Wakayama&rsquo;s Southeast coast takes in Japan&rsquo;s biggest tuna haul and late winter is their busiest season
Visitors can watch from a specially built viewing platform as the tasty tuna are auctioned
before trying some of the fresh catch themselves in the nearby restaurants
or buying some freshly prepared portions to take away
I sampled two perfect portions of fresh&nbsp;makajiki&nbsp;tuna
and Mr Wakiguchi recommended I try the tuna with a little soy sauce and chilli spice
the time had finally come to enjoy a delicious feast from Wakayama!&nbsp;
I carefully poured out some&nbsp;shoyu&nbsp;from Yuasa
fruity aroma akin to the&nbsp;kinzanji miso&nbsp;it was born from
and dipped in a slice of maroon coloured tuna
The&nbsp;makajiki&nbsp;tunawas soft but had a slight springiness which is prized in Japan
The flavour of the fish was subtle and creamy
It complemented the salty flavour of the soy sauce while the spice added a little kick that pleasantly lingered on my tongue
Then I sipped a little of the&nbsp;umeshu&nbsp;from Minabe
feeling almost instantly relaxed as the warming liqueur went smoothly down
I felt gratitude to the people of Wakayama who had taken the effort over generations to maintain traditions resulting in such high quality and delicious food
I thought back to my time living in Wakayama and hoped it would not be too long before I could try everything again
this would mean another trip to my beloved second home
or a calm hike along the paths of the Kumano Kodo
discover Japan&rsquo;s natural beauty and experience its gastronomic heritage
Experience Wakayama&rsquo;s unique culinary culture and amazing natural beauty
Build your appetite by visiting the sacred Kumano Kodo Trail and Koyasan
traditional Buddhist practices and mouthwatering cuisine
The 5th Conference of the East Asia Research Association for Agricultural Heritage Systems (ERAHS) was held from 26th to 29th August 2018&nbsp;in Wakayama Prefecture Minabe-Tanabe region
home to the "Minabe and Tanabe Ume System" which was designated as GIAHS
originated from the concept by United Nations University (UNU)
was established by the proposal of China to promote exchanging academic exchanges amongst designated GIAHS sites in China
The three countries take turns to host the annual conference and&nbsp;the United Nations University Institute for the Advanced Study of Sustainability (UNU-IAS) serves as the ERAHS Secretariat of Japan
Approximately 300 participants including about 100 from China and the Republic of Korea participated in this 5th Conference
and Director General of Kinki Regional Agricultural Administration Office graced the opening ceremony
and FAO GIAHS global coordinator Mr Yoshihide Endo
and FAO SAG member/Senior Visiting Professor of UNU-IAS Professor Kazuhiko Takeuchi gave the keynote presentations
Parallel discussions were then held in four sessions with themes on tourism
succession of GIAHS to the next generation
keynote presentations were also made by agriculture ministry’s representatives of Japan
then a reporting session was made on parallel sessions
GIAHS global coordinator Mr Endo commended the efforts of ERAHS and he would like to see such wonderful activities also extended to around the world
the participants made field visits to the GIAHS site where they visited the Wakayama prefecture Ume research center
two thematic sessions on monitoring and evaluation of GIAHS and biodiversity conservation were also held
this ERAHS conference emphasized and showcased the activities of young people such as university (graduate) students and regional cooperation of youths
Suggestions such as creating a GIAHS youth network and a special group meeting by young people at the next ERAHS conference were also proposed
exchange between domestic and foreign GIAHS designated sites was also active
It is expected that these exchanges and knowledge shared will be utilized to revitalize the rural area through the conservation of GIAHS
All the presentations can be accessed at the following link:
<a href="https://www.giahs-minabetanabe.jp/erahs/en/">https://www.giahs-minabetanabe.jp/erahs/en/</a>
The next ERAHS will be held from 19th May (Sunday) to 22nd (Wednesday) 2019 at the GIAHS site of Hadong (tea cultivation system) in the Republic of Korea
<a href="/articles/s41598-023-36727-8/metrics" data-track="click" data-track-action="view metrics" data-track-label="link" rel="nofollow">Metrics details</a>
Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations
and to prevent reproductive behaviors and diseases
This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy
The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1)
the neuropeptide that triggers and regulates pulsatile secretion of GnRH
Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2–3 weeks
Rats treated by either method did not exhibit estrous cyclicity
The EB-treated rats had fewer hypothalamic Kisspeptin neurons
but the GnRH-LH axis remained responsive to Kisspeptin stimulation
Because it would be desirable to use a biodegradable carrier that is also easier to handle
an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule
A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat
implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus
None of the treatments produced any concerning health effects
further development of this technology for sterilization in domestic female animals
such as dogs and cats is worthy of investigation
Reproductive sterilization is preferable to contraception in animals because it is permanent and irreversible; whereas in humans
contraception is preferred because of the desire to control timing of pregnancy
Surgical sterilization of animals is well accepted in the United States and other countries
but there has been a long-standing desire for a non-surgical alternative
The results reported here were obtained by adapting an old chemical
to a new approach of transient neonatal exposure to inhibit kisspeptin neuron development for inducing sterilization in female animals
these methods have received limited acceptance
partly because they provide only temporal inhibition
and require repeated dosing to maintain infertility throughout the animal’s reproductive life
we devised a method that targets neonatal development of the Kisspeptin neurons
with the potential to induce sterility in the female
which supports the keen interest in targeting these components of the HPG in the treatment of infertility
repeated injections of a treatment would not be optimal for promoting an alternative to ovariohysterectomy (OVX) in female animals
we tested the hypothesis that giving a single neonatal dose of EB in a sustained but temporary release carrier would inhibit Kisspeptin neurons sufficient to induce permanent infertility (sterility)
we employed conventional silicone capsules to deliver the EB for a duration exceeding 10 days in rats
and assessed the reproductive capabilities
we formulated poly lactic-co-glycolic acid (PLGA) microspheres to achieve a similar release pattern but in a biodegradable composition
a long-term goal is to use this type of method for inducing sterility in canines; therefore
a first-step study was performed to determine if neonatal EB would also inhibit KISS1 expression in the female dog
(A) Female rats on PND 0.5 were placed into one of the following treatment groups and followed for seven months: control (silicone capsule filled with oil); EBx11 (subcutaneous injection of estradiol benzoate
for 11 consecutive days for a total of 315 µg
PND 0.5–10); SC30 or SC300 (single subcutaneous impant of silicone capsules containing either 30 or 300 µg EB); EBMS300 (single injection of microspheres containing 300 µg of EB
some animals kept until PND 335 to determine late vaginal opening (Supplementary Fig
(B) Female Beagle dogs received SC9000 (silicone capsules containing 9000 µg EB) on PND 3 or 5
and their hypothalamic KISS1 expression was compared to that of control groups’ at PND 78
(C) Implants were made of silicone capsules with an inner diameter of 1.0 mm filled with EB and endcaps of silicone adhesive
(D) Biodegradable microspheres (EBMS300) consisted of matrix of poly lactic-co-glycolic acid (PLGA) forming spheres having less than 10 µm diameter and physically entrapping EB at 17.1% concentration
The rats were euthanized by CO2 asphyxiation followed by cervical dislocation
as approved by the University of Illinois Animal Care and Use Committee
as approved by the Ridglan Animal Care and Use Committee
Sigma) and loaded by syringe into the silicone capsules at a total dosage of either 30 or 300 μg and sealed with the adhesive
EB-loaded PLGA microspheres (EBMS) were synthesized by SpineThera (Minneapolis
with the final product of EBMS being in a dry powder form (Lot 6028–119
D99 = 8.72 µm) that was then reconstituted with PBS solution (pH 7.4) to 50 μl for injection by syringe
Several female rat experiments were performed using the following different treatment groups (A–D):
Control (Silicone capsule with sesame oil vehicle only
SC300 (High-dose single injection of EB in silicone capsule containing 300 μg EB in sesame oil
implanted on PND 0.5 at approximately 46 mg/kg BW);
SC30 (Low-dose single injection of EB in silicone capsule containing 30 μg EB in sesame oil
implanted on PND 0.5 at approximately 4.6 mg/kg BW)
The number of animals used per treatment group is listed for each experiment and associated endpoints
which was due to tissue sample availability
animal terminations for short-term studies and the number of female rats born for inclusion in a specific trial
Fertility of the female rats was determined at PND 180–210 (6–7 months)
a time point that would represent the middle age of their reproductive life span
and SC30 (n = 4) were housed continuously with proven breeder males for 14 days and fertility outcomes determined 21 days later
Percent fertility was calculated as the number of females that gave birth divided by the total number of females per group in each round of tests
control (n = 6) and SC300 (n = 5) were kept until PND 335 (11-month-old) to determine if a late vaginal opening had occurred
Experiment 2 included four treatment groups: Control (n = 7)
blood was collected from all animals by tail vein puncture for serum E2 and testosterone measurements (described below) and the females were euthanized on PND 75
The ovaries and uteri were collected for histological evaluation (described below)
Experiment 3 included four treatment groups: Control (n = 8)
Experiment 4 included two treatment groups: Control (n = 6) and SC300 (n = 5)
data were combined from both experiments: body and organ weights
body weights were determined and the stage of estrous cycle and vaginal opening were evaluated for each animal
Blood was then collected for serum chemistry
CBC and hormone measurements (described below) and the females were then euthanized
The anovaginal distance (AVD) was measured and the major organs removed and weighed
the uteri and livers were collected for histological evaluation and mammary gland tissues were collected for whole mount analysis (described below)
Blood cell population (CBC) was analyzed from 1 mL whole blood that was collected by cardiac puncture using BD Vacutainer® Plus blood collection tubes coated with a spray-dried K2 EDTA (Becton Dickinson
samples were gently inverted several times and complete blood count (CBC) analyses were conducted at the University of Illinois Veterinary Medicine Diagnostics Laboratory (Urbana
Brain slices were treated with blocking buffer for 1 h and then incubated in 1 ml pre-adsorbed primary antibody solution (1:1000)
After 72 h of incubation with primary antibody
sections were washed 3 times with 5–10 ml PBS for 10 min
1:750 goat anti-rabbit Alexa fluor 488 plus (Invitrogen) in blocking buffer 1–2 ml was added and incubated for 2.5 h followed by addition of DAPI (Invitrogen) (1:100,000) for 30 min
Stained sections were washed 3 times with 5–10 ml PBS for 10 min
A final wash with 5 ml TBS for 10 min removed PBS precipitate
the sections were rocked gently on a shaker at RT
Brain sections were mounted on glass slides and air-dried before adding ProLong™ Gold Mount media (Invitrogen)
coverslipped and cured for 24 h at RT in the dark
Sections were imaged using a confocal microscope (A1
KISS1 immunoreactivity (KISS1-ir) was quantified relative to DAPI signal (nuclear staining) in confocal images from PND 75 rats
All images were subjected to the same threshold to remove any background
a ratio of total DAPI to Alexa fluor 488-positive pixels was calculated using Image J (NIH) to give a relative expression of KISS1 in each image
An average of 7–8 images were normalized to control data
Blood cell population in control and treated rats were analyzed by collecting 1 mL whole blood by cardiac puncture using BD Vacutainer® Plus blood collection tubes coated with a spray-dried K2 EDTA (Becton Dickinson
it was hypothesized that biodegradable microspheres with embedded EB could be easier to handle for giving precise dosages to animals
and that EB delivery in this form would have the same inhibitory effect on female reproduction
biodegradable poly(lactic-co-glycolic) acid (PLGA) microspheres with embedded EB (EBMS) were developed for single injection
This experiment included two treatment groups: Control (n = 8) and EBMS300 (300 µg EB) (n = 8)
Blank PLGA microspheres without EB were unavailable; therefore
The EBMS powder was reconstituted with PBS solution (pH 7.4) to provide an average EB dosage of 46 mg/kg body weight
Neonatal animals were treated on the same day as in the previous rat experiments (PND 0.5) by subcutaneous injection in the back of the nape area
To assess the impact on reproductive status
including the onset of puberty and fertility
treated female rats were examined for vaginal opening and estrous cycle stage on PND 45 (1.5 months)
Fertility was tested in two rounds of breeding
The females were housed with proven breeder males from PND 54 to 105
Then on PND 120 (4 months) the rats were euthanized and the ovaries were collected for histological evaluation (described below)
Ovarian tissue volumes were calculated by measuring the length and width using a caliper
and collected their ovaries and hypothalami
Ovaries were immediately fixed in 4% paraformaldehyde (PFA) for 24 h and processed for histological observation (methods described below)
Hematoxylin and eosin-stained ovary slides were analyzed to compare follicle status between the control group and the SC9000 group
The hypothalamus was dissected after carefully removing the brain from the skull
Total RNA was extracted from the isolated hypothalmic tissues and analyzed for KISS1 expression using Reat-time PCR (methods described below)
The stage of the estrous cycle was ascertained by conducting vaginal cytology from female control (n = 8)
Aqueous vaginal smears were applied to glass microscope slides and inspected using light microscopy to identify the estrous cycle stage
The vaginal opening was observed in same rats from PND 28 to 74
When rats exhibit complete canalization of the vagina
it was determined as complete vaginal opening
Anovaginal distance was measured using a caliper
NJ) with a reportable range of 1.40–200 pg/mL was used to measure circulating 17β-E2
DRG Diagnostic) with a reportable range of 0.083–16 ng/mL was used to measure circulating Testosterone
The intra- and inter-assay coefficients of variability were less than 10%
The slides were imaged with an Olympus BX51 microscope (Melville
NY) to determine mammary tissue changes in branching and terminal end bud formation
To determine hypothalamic KISS1 expression
hypothalamic tissues isolated from female dogs were subjected to RT-PCR analysis
Total RNA was extracted using RNAqueous®-Micro Kit (Ambion® by Life Technologies™
CA) according to the manufacturer’s protocol
RNA concentrations were measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific
The RNA was diluted to equal concentrations before being reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosynthesis
PCR reactions were performed with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol
and Kiss1-R: 5’-GGTTTCTTGGCAGCTAATGCT-3’ were used for quantification of dog GAPDH (housekeeping; product size: 100 bp) and KISS1 (product size: 70 bp) mRNA levels
Fluorescence was measured using the ABI prism 7500 quantitative real-time thermocycler (Applied Biosystems)
Results are expressed as fold differences in relative gene expression with respect to control or equivalent group
Data analyses were performed using Microsoft Excel
Continuous data were analyzed for normal distribution by a Shapiro–Wilk test
All normally distributed continuous data were analyzed with parametric tests (Student’s t-test or ANOVA)
Data are graphically presented as the mean ± standard deviation (SD) or ± standard error (SE)
and statistically significant differences were accepted when P-value is lower than 0.05
The rat study protocols were approved by the University of Illinois Animal Care and Use Committee (Protocols: 20027)
The dog study protocol was reviewed and approved by Institutional Animal Care and Use Committee of Ridglan Farms
AAALAC accredited research facility (Protocol: IN-100-001)
and all efforts were made to minimize animal suffering
</a>Estradiol (E2) concentrations in serum and hypothalamus after estradiol benzoate (EB) capsule implantation
(A) E2 levels in the sera (pg/mL) from PND 0.5 to PND 30
(B) E2 levels in homogenized hypothalamic tissue (µg/mL) from PND 0.5 to PND 10
The number of animals for each date are provided in Supplementary Table 1
</a>Neonatal estradiol benzoate (EB) inhibited reproductive organ development and ovarian function in female rats
Controls received silicone capsules with oil only
The EBx11 group received EB injections on 11 consecutive days from birth (total of 315 µg EB)
The EB300 and EB30 groups received a single implant of a silicone capsule containing 300 or 30 µg EB (SC300 or SC30)
(A) Estrous cyclicity in representative rats in each group from PND 62 to PND 69
(B) Ovary weight/body weight ratio; uterus weight/body weight ratio by EB treatment at PND 178–211 (6–7 months)
and Anovaginal distance (AVD; mm) (Control
There was a significantly lower ovary/body weight ratio in all EB treatment groups compared to control; P-values indicate significant differences (One-way ANOVA with Tukey post hoc test)
(C) Representative histology of control ovaries at PND 60–75 (2.5 months) (Control
n = 3) or PND 178–211 (6–7 months) (Control
but numerous anovulatory follicles (*) are present
indicating the lack of maturation in external reproductive organs
and SC30 treatment groups produced offspring
</a>Serum testosterone and estradiol levels in adult control and SC300 (300 μg estradiol benzoate) treated rats and the effect of KP-10 injection on serum LH
Serum concentrations of (A) testosterone and (B) estradiol in PND 75 (2.5 months of age) rats (Control
n = 4) and PND 178–211 (6–7 months of age) rats (Control
Significant differences between the Control and SC300 are indicated by P-values (Student’s t-test)
Serum LH levels in control (n = 4) and SC300 (n = 3) rats before and after the treatment of kisspeptin analog
After intraperitoneal injection of KP-10 (50 nmole/animal) in rats at 2–2.5 months of age
serum LH levels at 30 min were significantly increased
Significant differences are indicated by P-values (Two-way ANOVA with Tukey post hoc test)
</a>Effects of neonatal estradiol benzoate (EB) on gene expression profiles of Kiss1-clusters in Control and SC300 hypothalami
(A) The hypothalamic area used for scRNAseq is marked by dotted line
(B) Pooled hypothalamic cells of Control (4,049 total) and the SC300 group (4223 total) were UMAP clustered together
Cells were identified by marker gene expressions
Cluster 6 was used for identifying genes that were differentially expressed in SC300 and Control
(C) Down-regulated genes in the hypothalamus of SC300 compared to Control are listed with log fold-change of the average expression between the two groups (avg_logFC)
percentage of cells where the feature is detected in the first group or second group (Pct
(D) Down-regulated genes in SC300 are grouped according to functional categories
X-axis indicates the number of DEGs in each category
(E) The first panel shows the total number of cells in cluster 6 with Kiss1-expressing cells distinguished by orange color
The panel on the right plots the number of Kiss1-expressing cells in each group with Kiss1-expressing cells defined as Kiss1 LogExp &gt; 0.5 (red dotted line)
</a>Effects of neonatal estradiol benzoate (EB) on body and organ weights and structure of the mammary gland
The EB300 group received a single implant of a silicone capsule containing 300 µg EB
All organs and tissues were collected at PND 178–211 (6–7 months)
(C) The weight of essential organs is presented as the ratio of organ weight/BW
(D) Mammary gland morphology by whole mount imaging
Arrowheads indicate branching points of mammary ducts
Ductal end bud formation appears to be underdeveloped in the EBx11 and SC300 glands
with ductal width also being thinner than in control mammary gland tissues
Significant differences between groups are indicated by P-values (One-way ANOVA with Tukey post hoc test)
</a>Neonatal injection of biodegradable estradiol benzoate-microspheres (EBMS300) inhibited vaginal opening and ovarian function
The EBMS300 group received a single injection of 300 μg of EB in the reconstituted microspheres
(A) Vaginal opening of control and EBMS300 rats until PND 45
(B) Estrous cycle of control (n = 8) and EBMS300 (n = 5) rats from PND 44 to 50 (1.5 months)
(C) Representative histology of ovaries on PND 125
numerous corpora lutea (CL) are easily recognized as large homogenous structures
which resulted in a greatly enlarged organ
with only anovulatory follicles (*) being present in a visibly smaller organ
(D) Volume of the ovary in control and EBMS300 rats (PND 125)
Significant differences are indicated by P-values (Student’s t-test)
</a>Inhibited hypothalamic KISS1 expression and ovary development in female Beagle dogs after neonatal implant of estradiol benzoate (EB)-capsule
(A) KISS1 mRNA expression in the hypothalamus of control and SC9000 (9,000 μg EB) dogs on PND 78 (2.5 months)
P-values indicate significant differences (Student’s t-test)
(B) Representative histology of control and SC9000 ovaries on PND 78 (2.5 months) (n = 3)
Arrowheads and arrows indicate early primary follicles and late primary/secondary follicles
we tested the hypothesis that a permanent infertility (sterility) could be induced in female animals by a transient elevation of estrogen at a neonatal stage using a slow estrogen-release method
Data presented here support the hypothesis
as postnatal treatment-induced elevation of estrogen level using silicone capsule or microspheres induced complete infertility in female rats
neonatal implantation of an EB pellet in the female dog decreased hypothalamic KISS1 expression
suggesting that the neonatal estrogen treatment may be developed as a non-surgical alternative to surgically sterilizing female dogs
E2 removal was likely active in hypothalamic cells at this neonatal age
future investigation of their potential enzymatic activity and role in the clearance of E2 from the hypothalamus is warranted
the mRNA expression levels of 17β-HSDs in the hypothalamus of the PND 29 were not different between Control and SC300
indicating there was likely no lasting impact on the expression of those genes by the neonatal hypothalamic E2 elevation
administering a single dosing of EB subcutaneously was able to induce sterility
most likely due to the ability to induce a significant elevation of E2 in the hypothalamus for a longer period of time using the extended-release methods
Female rats that were neonatally treated with EB implant or microspheres did not exhibit any sign of pubertal onset nor display estrous cyclicity
treatment of those rats with a KISS analog instantly elevated circulating LH to the level that was comparative to intact animals
indicating that GnRH neurons and pituitary gonadotrophs retain their functional responsiveness
These findings suggest that SC300 has a limited direct effect on the uterus compared to EBx11
which is thought to be due to the slower and declining drug release compared to EBx11
it has been suggested that AVPV Kisspeptin neurons have a higher sensitivity to EB treatment than ARC Kisspeptin neurons
it is posited that the decreased KISS1-ir in the ARC
which has relatively low sensitivity to neonatal E2 treatment
would be accompanied by a decrease in Kisspeptin expression in the AVPV
KISS1 expression was assessed in the ARC by the intensity of KISS1-ir
The results revealed that the EB treated rats had lower KISS1-ir in the ARC on PND 29
it was important to determine if this was simply an inhibition of neuronal activity or if the reduction represented a reduced number of Kisspeptin neurons
The reduction in activity could be interpreted as a temporary effect
but the loss of neurons would likely result in a permanent effect
but this would only be temporary and not induce sterility
further research involving EB treatment in post-pubertal rats is needed to confirm this
SC300 treated females that were given KP-10 (a kisspeptin analog) showed a robust increase in serum LH
demonstrating that the neonatal estrogen treatment had not produced a permanent effect on the GnRH neurons or pituitary gonadotrophs
but rather was focused on the kisspeptin neurons in the hypothalamus
during estrus the control pituitary would be able to respond to the normal secretion of KISS1
would not do so because of the KISS1 deficiency
the underlying mechanisms by which neonatal treatment with exogenous E2 decreased the number of hypothalamic Kisspeptin neurons remains unknown
Although ALP and serum cholesterol levels increased to similar extents in both SC300 and OVX groups compared to the control group
SC300-treated rats were healthier than OVX rats in terms of obesity risk
no major health concerns were found in female rats receiving neonatal EB treatment
but extensive work is needed to address potential safety concerns in other species
This would imply that using PLGA microspheres to deliver EB could increase hypothalamic E2 levels in a pattern similar to that found with silicone capsules and induce infertility in female rats
the data demonstrated that PLGA microspheres could serve as a biodegradable material for EB delivery
A single neonatal subcutaneous administration of an estrogen via slow-release methods induced permanent infertility (sterility) in female rats
This supports the idea that a supraphysiological
increase in E2 levels during postnatal development can disrupt the HPG axis by permanently inhibiting Kiss1 expression
while leaving GnRH neurons and gonadotrophs intact
Neonatal treatment of female rats with EB reduced the number of Kisspeptin neurons and their immunoreactivity in the hypothalamus
Repeated dosing of the animals was not necessary
as a single capsule implant of EB or injection of EB-microspheres was sufficient to induce anovulation
Health safety parameters were found to be similar to surgical ovariectomy
Preliminary testing in female dogs showed similar suppression of KISS1 expression in the hypothalamus
further development of this estrogen-treatment method for sterilization in domesticated animals
such as female dogs and cats is worthy of investigation
Such research should begin by determining the appropriate age range for EB treatment to induce sterilization in dogs and cats
The code and test sets are available in the github repository <a href="https://github.com/cjpark85/Rat_Hypothalamus_scRNAseq">https://github.com/cjpark85/Rat_Hypothalamus_scRNAseq</a> and the NCBI GEO database (GSE221002)
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Japan) for providing the anti-rabbit kisspeptin polyclonal antibody (RRID: AB_2910199)
University of Illinois at Urbana-Champaign)
Tseng (HPCBio) for their assistance in performing single-cell RNA sequencing and analyzing data
This work was supported by Epivara’s TTA (CK) and NSF SBIR grant 2052603 (CJ)
University of Illinois at Urbana-Champaign
Iwate Tohoku Medical Megabank Organization
Department of Toxicology and Forensic Medicine
Department of Veterinary Clinical Medicine
All authors edited and approved the final manuscript
CheMyong Jay Ko is the founder and CEO of Epivara
and Arnon Gal have no conflict of interest to disclose
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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DOI: https://doi.org/10.1038/s41598-023-36727-8
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The walk up the hill of the <a href="https://en.visitwakayama.jp/venues/6IwzCRT1V6mQcaGyY8wUGe/">Minabe Bairin</a> ume orchards in Minabe
but the effort is worth the spectacular view
especially for those visiting in February: thousands of ume trees — whose fruit is often called a plum but is more closely related to an apricot — are in bloom
Across rolling hills covered in a soft carpet of pink petals
ume orchards alternate with patches of forest
The town of Minabe is visible at the bottom of the valley and
the sea glistens brightly.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); });
The serenity of the view contrasts with the hustle and bustle taking place on the hill
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a local tradition where rice cakes are tossed into a crowd for good luck
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<a href="https://all.accor.com/a/en.html" target="_blank" rel="noopener">Accor</a>
the region’s largest international hospitality group
is delighted to announce the opening of 22 hotels and over 6,000 rooms in Japan on Monday
the Group’s footprint in Japan will instantly double increasing Accor’s portfolio of hotels in Japan to 46 properties
comprising twelve Grand Mercure and ten Mercure hotels
range in location from the northernmost prefecture of Hokkaido to the southernmost prefecture of Okinawa and are located close to local hidden gems such as Cape Zanpa in Okinawa
Shiroi Koibito Park in Hokkaido and Senri Beach in Wakayama
and wellness facilities such as onsen (hot springs)
with select hotels also offering stunning views of the local landscape
appeals to a traveller’s insatiable quest for discovery
offering an immersive experience that celebrates national culture and stimulates the senses
Each hotel is unique and authentically reflects its destination by embracing the local way of life
infusing age-old traditions with modern flair
Grand Mercure makes its debut in Japan offering “Proudly Local” premium hotels
inspiring guests to experience their destination through local celebrations and multisensory experiences crafted by local hosts
is positioned within the midscale segment and focuses on unveiling the local neighbourhood treasures surrounding each address
Mercure hotels offer far more than just a place to sleep – they are a portal to the destination
a warm invitation to discover and explore the locale
Accor is proud to unveil the following 22 properties in Japan:
Notable properties include Grand Mercure Okinawa Cape Zanpa Resort and Grand Mercure Wakayama Minabe Resort &amp; Spa
Grand Mercure Okinawa Cape Zanpa Resort is a modern hotel located on Zanpa Beach in Okinawa
this resort-style hotel combines modern amenities with traditional Okinawan hospitality
Its strategic location allows guests to enjoy the beauty of the coastline
enjoy a variety of recreational activities such as water sports
The hotel has one of the largest pool areas in Okinawa
The hotel offers a variety of accommodations with 465 spacious guest rooms and suites
making it an ideal destination for leisure travellers and those looking to relax in a quiet seaside setting
Surrounded by the scenic beauty of southern Wakayama Prefecture and located in Wakayama Minabe
Grand Mercure Wakayama Minabe Resort &amp; Spa offers a sophisticated retreat that blends contemporary elegance with a serene environment
the hotel offers a sophisticated getaway for travellers seeking comfort amidst natural beauty
Accommodations at Grand Mercure Wakayama Minabe Resort &amp; Spa are the epitome of modern comfort with 323 spacious rooms and suites that are equipped with modern amenities and offer charming sea views
creating an atmosphere of relaxation and luxury
Guests can enjoy the diverse flavours of the buffet restaurant
where outstanding chefs use local ingredients to create exquisite dishes and showcase Wakayama's rich culinary traditions
Accor was operating 24 hotels (5,741 keys) in Japan
under eight brands: MGallery; Pullman; Swissôtel; Mercure; Novotel; ibis; ibis Styles and ibis budget
In addition to the 22 Grand Mercure and Mercure hotels joining Accor’s network on 1 April 2024
there are two more properties currently in the group’s pipeline
For more information or to make reservations for Grand Mercure and Mercure, visit <a href="http://www.accor-resorts-japan.jp/" target="_blank" rel="noopener">www.accor-resorts-japan.jp</a>
Link IconCopy linkFacebook LogoShare on FacebookXShare on XEmailShare via EmailLink copied to clipboardCrack that caused a fatal Southwest engine failure was missed six years agoAn engine fan blade tore off
leading to an April 17 emergency landing in Philadelphia and the death of one passenger
Seven months after a titanium blade snapped inside the engine of a Southwest Airlines jet
causing an emergency landing in Philadelphia and the death of one passenger
a federal investigation has zeroed in on two central questions:
And why weren't its shards contained by a protective sheath that surrounded the engine
By examining the wear patterns in the failed blade from Flight 1380
forensic experts determined that the initial crack had been present nearly six years earlier — undetected during a 2012 overhaul and inspection
&gt;&gt;READ MORE: <a class="relative z-1 text-blue-mid hover:shadow-lightmode" data-link-type="article-body" class="text-blue-mid hover:shadow-lightmode" href="http://www2.philly.com/philly/business/transportation/southwest-ntsb-engine-failure-philly-investigation-20181114.html">A shattering noise, then a deafening roar. New details from Southwest plane's engine failure over Pennsylvania.</a>
Inspectors at the time were not required to use ultrasound or other advanced equipment to detect possible cracks — only their eyes
aided by fluorescent dye to highlight flaws
a lead engineer at engine maker CFM International
a joint venture of GE Aviation and Safran Aircraft Engines of France
Metallurgical experts were able to trace the crack's history by looking at "striations" — estimating how fast the crack had grown over the thousands of flights
"If we look at the striation count and go backward
the size of the defect was about 1/16 of an inch," Habedank said
The engine blades were designed to last at least 100,000 flights
but the crack began to form at just 20,000 flights
is one of the CFM56 series of engines manufactured by a partnership  between General Electric and the French company Safran Aircraft Engines and used by about 300 different airlines worldwide
The findings of the NTSB investigation could have far-reaching consequences for the airline industry
CFM officials had determined that the roots on this type of fan blade suffered excessive friction in flight
and recommended that they be removed and lubricated every 3,000 flights
such blades must now be lubricated every 1,600 flights
as well as undergo crack detection with either ultrasound or an eddy-current device
which uses electricity to detect abnormalities
Eight more blades have been removed from service as the result of these stepped-up inspections
director of the NTSB's office of aviation safety
"So it's great that the inspection technique is pulling those parts from service
but that's a lot of blades that are cracking," DeLisi said at the hearing
Habedank and other witnesses stopped short of saying what caused the crack
a metallurgist with Robson Forensic in Philadelphia
posited that maintenance technicians may have been too aggressive in "grit-blasting" the fan blade during overhaul
Analysis of the blade showed at least one piece of grit lodged in the metal
and perhaps another such piece started the crack
she said after reviewing the documents at the request of the Inquirer and Daily News
"They're getting pieces of the grit that are left behind
because it is a source of contamination that could affect adhesion of the coating or serve as a site for crack initiation," she said
Then there was the second failure: When the blade tore through the engine
its protective sheath couldn't contain the shrapnel
Data collected by the NTSB described a fan blade break: Within 1/200 of a second
the fan blade fragments hit the fan case and cause a ripple
essentially a shock wave that emanates from the impact point
The disruption causes the airflow to surge
In the next two seconds the engine shuts down
When the engine used on the Southwest plane was tested in the mid-1990s
engineers believed the casing would contain a broken fan blade
a project engineer with United Technologies Aerospace Systems
so engineers added a shield to contain any blade thrown from a rotor spinning at more than 5,000 rotations per minute
the fan blade may have been thrown forward beyond the containment shield into the engine's inlet
a ring in front of the containment shield blades that ensures a smooth airflow into the engine
the metal shell that a plane's passengers see when they look out the window at an engine
<a class="relative z-1 text-blue-mid hover:shadow-lightmode" data-link-type="article-body" class="text-blue-mid hover:shadow-lightmode" href="http://www2.philly.com/philly/news/southwest-emergency-landing-engine-tammie-jo-shults-20180418.html">&gt;&gt;READ MORE: Twenty minutes of terror in the sky: the emergency landing of Southwest Flight 1380</a>
The FAA and Boeing have no standards for a window's ability to withstand impact
according to documents compiled by the NTSB
and there are no "directed inspections" for passenger windows
Investigators saw similar damage to the inlet in a nearly identical mishap involving the same model engine on a Southwest flight near Pensacola
It was a kind of damage experts had never seen
"Fragments should not have traveled forward of the containment shield," Minabe said
Victor Wicklund of the FAA's transport standards branch confirmed in the hearing that the damage seen in both the Philadelphia and Pensacola incidents was unlike anything seen before
is supposed to be able to withstand the impact of a fan blade to be certified for use
Witnesses testified that modern diagnostic tools allow a much better understanding of the ripple effect that occurs when a fan blade breaks
The Philadelphia and Pensacola incidents suggest the tests that engines undergo may not include all that can happen when something goes wrong in flight
"We are seeing on the Southwest events," he said
"that inlet retention is not assured even after a successful certification test
So isn't that a good opportunity to rethink what we're doing on the ground and how well it simulates that failure in flight?"
Addressing the problem would likely require engineers to consider changes in testing
an aerospace engineering professor at Embry-Riddle University in Daytona Beach
and those two things counteract each other."
Technology that is not quite ready for use will likely be a longer term help in ensuring incidents like this don't happen again
Researchers are designing sensors that would be able to detect cracks in materials
or temperature variations that are outside the norm in the engine
giving advance warning of part failures that doesn't require manual inspections
"The number of sensors on engines is continuously increasing," Ricklick said
DeLisi told the hearing that manufacturers of engine components didn't agree how to interpret some of the fragments from the accident
He asked how much longer the investigation would need
"There's so much data and the hardware is in such a state that it takes a lot of time in terms of understanding what you're looking at," Minabe said
"There are things that we might first look at and think of as one thing
This takes an enormous amount of reviewing of the data
which had taken off from New York City's LaGuardia Airport and was headed to Love Field in Dallas
about 20 minutes later at Philadelphia International Airport
with all those on board except Riordan surviving with minimal injuries."},{"_id":"DZKV4KG64VH5JCI2F3YJNEXLME","type":"text","additional_properties":{"_id":1548967125495},"content":"At the first half of the NTSB hearing Wednesday morning
questions focused on the testing that titanium fan blades undergo and the engine casing's ability to contain a catastrophic event
The NTSB documents note that for pieces of the inlet
and cowling to break free due to the fan blade failure was unexpected
The safety agency took testimony from officials from Southwest
drawing thousands of pounds of air into the engine
The amount of use a fan blade can have without inspection has been halved since the Philadelphia incident
asked whether fan blades should have a mandated age at which they are pulled from use."},{"_id":"5MZ3LTHUSZB7XMMHHPLYDMPS5A","type":"text","additional_properties":{"_id":1548967125498},"content":"It was something that officials were looking into
Eight blades have been pulled from service due to the discovery of cracks since the Philadelphia incident
the NTSB reported at the hearing."},{"_id":"XSD4IWLYCRA5BO3EMBNXYL4PXU","type":"text","additional_properties":{"_id":1548967125499},"content":"Also under investigation is why the inlet and the cowl that surrounds the engine didn't contain the damaged engine
and didn't prevent debris from spraying forward into the fuselage
A virtually identical fan-blade failure occurred on another Southwest flight
Investigators are looking at whether the angle of impact of the fan blade on the casing was the reason the Philadelphia incident turned deadly
and whether further testing and precautions are needed to secure the parts surrounding an engine."},{"_id":"BKKYZCXYGFBY7ABOXUFKE6EF4U","type":"text","additional_properties":{"_id":1548967125500},"content":"In a statement Wednesday
CFM International noted it had moved aggressively to address the concerns raised by the accident."},{"_id":"Q36OCNRVVBCQNIJVHSFA3TVORI","type":"text","additional_properties":{"_id":1548967125501},"content":"\"All of the CFM56-7B fan blades targeted by the various Airworthiness Directives were cleared by mid-August 2018
ahead of the August 31 deadline,\" wrote the company
a cooperative between the French company Safran Aircraft Engines and General Electric."},{"_id":"D4MEYJYNZZD2XGVU5I4EI23XNU","type":"text","additional_properties":{"_id":1548967125502},"content":"Pieces of the plane rained down on Berks County
others large sheets torn from the ruined engine
All the recovered parts landed in Bernville
a community of about 1,000 people."},{"_id":"WKKNX24P3BHKNCULFHKA4WIZOI","type":"text","additional_properties":{"_id":1548967125503},"content":"A piece of blue metal
later determined to be a section of the inboard fan cowl
landed on the women's tee of the 17th hole at the Heidelberg Country Club
Part of the fan-cowl frame was found in the tree line that separates the 9th and 16th fairways
A section of the outer engine barrel plummeted onto state land
where it was found by two Pennsylvania Game Commission employees
After Fernheimer helped pulled her back into the plane
the flight attendant caught a glance of the ruined engine
Blood covered the passenger windows outside."},{"_id":"DNKCKZEVRRBGNKE76BR3NMCJNA","type":"text","additional_properties":{"_id":1548967125506},"content":"The people helping Riordan laid her across the row of seats once they pulled her back in
began to perform CPR."},{"_id":"AFS32BWAHNGDPAJJJ57VPYVO54","type":"text","additional_properties":{"_id":1548967125507},"content":"Passengers kept asking: Would they make it
The captain's voice came over the speakers
announcing they would be landing in Philadelphia."},{"_id":"EPNDJD2DKJGBXBK2PABUQMD2YA","type":"text","additional_properties":{"_id":1548967125508},"content":"We're going to make it
Fernheimer insisted."},{"_id":"CRG35RNKEVCLHGMJXCYOV4OO24","type":"text","additional_properties":{"_id":1548967125509},"content":"She moved forward to check on more passengers
the paramedic and nurse were still performing chest compressions on the woman
The Philadelphia medical examiner would eventually find that her spine had been broken in two places
The cause of death was \"blunt trauma of the head
neck and torso.\""},{"_id":"YCCCDZZ3OVBJ3CC4IVUX6QSNHM","type":"text","additional_properties":{"_id":1548967125510},"content":"While passengers and flight attendants fought to save Riordan
the plane's captain and first officer were facing chaos and shock
Shults described losing a sense of time amid the chaos
She struggled with the plane's suddenly sluggish controls
was flying the craft at about 32,000 feet when the engine exploded
and almost immediately felt extreme vibration as the plane veered hard to the left."},{"_id":"6YMZ4DNBRJGHFMYK3YHMS7Q4HE","type":"text","additional_properties":{"_id":1548967125513},"content":"Fog and dust filled the cockpit
The sudden decompression took Ellisor's breath away
He told investigators it was the \"most memorable few seconds\" of his aviation career."},{"_id":"NDAAHR3XYVBIXBHL6IFAJULWMY","type":"text","additional_properties":{"_id":1548967125514},"content":"Unable to hear the captain or air traffic control
he disconnected the autopilot and automatic throttle
only to realize it was already beginning to descend on its own."},{"_id":"HYXF3A4HZ5D6TJGHREXMYYO77U","type":"text","additional_properties":{"_id":1548967125515},"content":"Within two minutes of the explosion
Ellisor turned his attention to a checklist to determine what had happened to the plane
The fog in the cockpit led the crew to think there was a fire
but its sensors did not detect a blaze."},{"_id":"3CV62VIDZNFOVGUJXBLAKJYRQM","type":"raw_html","additional_properties":{"src":"https://media.philly.com/storage/inquirer/script/pym.js","type":"text/javascript","_id":1548967125516},"content":"<\/script>"},{"_id":"3WOHOJMNKZAZVIZBONQAVVPCAY","type":"raw_html","additional_properties":{"type":"text/javascript","_id":1548967125517},"content":"\n\t\tdocument.addEventListener('DOMContentLoaded'
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\"http://media.philly.com/storage/inquirer/Graphics/AI_to_html/southwest_1380_3/index.html\"
1 directive in Southwest's emergency procedures: \"Maintain aircraft control.\""},{"_id":"A4GCZTYQ7RCQPCP6UIDGIGLZFU","type":"text","additional_properties":{"_id":1548967125520},"content":"The suggestion from air traffic control was that she land at Harrisburg International Airport in Middletown
Shults initially wanted the 20-mile final approach to give her and Ellisor time to work through their checklists
But news about the gravely injured passenger changed her mind
The goal now was to get on the ground as fast as possible."},{"_id":"UZGLCKVRANERVEFN5HAZOJPYTQ","type":"text","additional_properties":{"_id":1548967125521},"content":"Ellisor described the landing on Philadelphia's Runway 27 as \"great.\" Fire trucks greeted the plane
After several frustrating attempts to talk to rescue teams over radio
he eventually yelled to the fire chief out the plane's forward door."},{"_id":"24FSC4HSBNFDNLYZQ5BDWAG46E","type":"reference","referent":{"id":"24FSC4HSBNFDNLYZQ5BDWAG46E","provider":"","type":"image","referent_properties":{"caption":"From Wednesday’s NTSB hearing into the April 17
New details from Southwest plane’s engine failure over Pennsylvania.","mobile":"","native":"","print":"","tablet":"","web":"","meta_title":""},"owner":{"name":"The Philadelphia Inquirer
file photo National Transportation Safety Board investigators examine damage to the engine of the Southwest Airlines plane that made an emergency landing at Philadelphia International Airport in Philadelphia
college football fans now know which returning players to expect for the 2024 season
Two of the top names coming back to campus in the Fall - Alabama quarterback Jalen Milroe and Colorado wide receiver/defensive back Travis Hunter - will be among the brightest stars both on the field and in NIL, both potential Heisman Trophy candidates and expected <a class="tagStyle_16dbupz-o_O-style_57t8sw" href="/fannation/name-image-likeness/news/way-too-early-athlete-nil-rankings-for-2024-college-football-season-michael9">top earners</a> off the field
the metal trading cards will print to order through January 22 and are numbered to the final print run
Minabe, who counts the NBA and Bleacher Report as clients, previously collaborated with Leaf on a Muhammad Ali <a class="tagStyle_16dbupz-o_O-style_57t8sw" href="https://shop.leaftradingcards.com/products/2023-leaf-metal-anime-nation-muhammed-ali-print-to-order-through-1-16?_pos=3&amp;_sid=a7d8e7b35&amp;_ss=r" target="_blank">card</a>
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the Leaf team has successfully cultivated a new series of weekly releases
Leaf&#39;s Director Of Marketing and Licensing shared with Sports Illustrated
created with original artwork in the Anime style by Shion Minabe
offering collectors an exciting and enjoyable way to collect cards featuring their favorite athletes
The recent launch of Jalen Milroe and Travis Hunter editions reflects Leaf&#39;s ability to tap into the vast NIL fanbase within the evolving landscape of Name
Anticipate the unveiling of more incredible pieces from Shion Minabe in the near future.&#34;
With a new coach at Alabama in Kalen DeBoer - and likely offensive coordinator Ryan Grubb - Milroe and the Crimson Tide&#39;s offensive scheme will be one of the most talked about storylines of the new offseason
The former Washington coaching staff should take Milroe&#39;s game to the next level
if they decide to stick with their returning signal caller
The SEC Championship Game MVP threw for 2,834 yards
23 touchdowns and 6 interceptions this season
while leading the Crimson Tide to the Rose Bowl where they lost in overtime to eventual-National Champion
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Volume 9 - 2018 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fpsyt.2018.00036">https://doi.org/10.3389/fpsyt.2018.00036</a>
Research suggests that many individuals with autism spectrum disorder (ASD) often demonstrate challenges providing appropriate levels of information during conversational interchanges
Considering the preference of individuals with ASD
robotic systems may yield promise in promoting certain aspects of conversation and interaction such as self-disclosure of appropriate personal information
we evaluated personal disclosures of events with specific emotional content across two differing robotic systems (android and simplistic humanoid) and human interactions
Nineteen participants were enrolled in this study: 11 (2 women and 9 men) adolescents with ASD and 8 (4 women and 4 men) adolescents with TD
Each participant completed a sequence of three interactions in a random order
Results indicated differences regarding comfort level and length of disclosures between adolescents with ASD and typically developing (TD) controls in relation to system interactions
adolescents with ASD showed a preference for interacting with the robotic systems compared to TD controls and demonstrated lengthier disclosures when interacting with the visually simple humanoid robot compared to interacting with human interviewer
The findings suggest that robotic systems may be useful in eliciting and promoting aspects of social communication such as self-disclosure for some individuals with ASD
deploying such systems in meaningful interventions settings may represent a potential use of such technology
In order to better understand whether robotic systems might be helpful in promoting self-disclosure for individuals with ASD
we designed and tested a controlled interaction paradigm comparing different robotic systems (i.e.
visually simple robot and android robot) in relation to controlled human interaction
We compared the difference of impression (i.e.
reported preference) in communicating with two types of humanoid robots and human interviewer
and ratio of change in measured length of disclosure statements in order to examine potential differences between adolescents with ASD and typically developing (TD) controls
We hypothesized that adolescents with ASD would report a greater preference for communicating with the robotic systems than TD adolescents
and demonstrate lengthier disclosures within the paradigm
It is about 0.3-m tall and has a limited number of body parts consisting of head
although they are still expected to evoke humanness
The distinguishing feature of the robot is the high degree of freedom of the eyes
Each participant completed a sequence of three interaction conditions in random order, all of which were guided and took place in a standard clinical assessment room. We ensured a balance in the order of conditions to a certain extent. Please refer to Supplementary Materials for this order. Figure <a href="#F3">3</a> provides an example of how participants typically interacted with the robots. The person in Figure <a href="#F3">3</a> has given written and informed consent to publish this image
both robots and human interviewer were situated in individual booths divided by opaque room dividers
We involved a variety of human interviewers (e.g.
The two robots were operated by researchers seated in front of a terminal computer located in an adjacent observation room separated by a one-way mirror so that they were not visible during the session
Each trial lasted as long as the participants chose to converse around the presented topic and ended when the participant answered the question
or communicated that he/she did not wish to answer the question or discuss the topic
The average duration of each trial was approximately 5 min
The human interviewer and two robots followed a specific interview script and protocol to elicit self-disclosure on events or feelings across all interviews
The scripts followed the same basic structure
participants were asked to share the happiest
and most embarrassing thing that happened to them at home
Please refer to Supplementary Material for examples of scripts
participants completed a self-report survey using a 9-point Likert scale to rank their level of enjoyment
and boredom while communicating with each agent
Using audio recordings collected during the experimental session
the research team transcribed and totaled the number of words used in each exchange between participant and agent across conditions
We performed statistical analysis using SPSS version 24.0 (IBM
and SCQ score between the groups were analyzed using an independent samples t-test
The difference in gender proportion was analyzed using the &#x003C7;2-test
Differences in the self-reported preference ratings communicating with the two robots between individuals with ASD and TD was analyzed using the Mann&#x02013;Whitney U-test
as well as the ratio of number of words used in self-disclosure between interactions with each robot vs
to ascertain whether one or both robots promote more self-disclosure compared to a human interviewer
we compared the number of words in self-disclosure between each robot and human using a Wilcoxon signed-rank test
Spearman&#x02019;s rank correlation coefficients were used to explore the relationships between IQ and the number of words in self-disclosure to each robot and human
An alpha level of 0.05 was used for these analyses
Overall impressions of communicating with each robot
Ratio of number of words used in self-disclosure between interactions with each robot vs
The number of words in self-disclosure between each robot and human interviewer for adolescents with ASD and TD
We did not find any relationship between IQ and the number of words in self-disclosure to android robot (r &#x0003D; 0.20
and human interviewer (r &#x0003D; &#x02212;0.09
Previous studies have suggested that individuals with ASD show preference for certain interactions with robotic systems relative to confederate human interactions (<a href="#B31">31</a>). In this capacity it has been hypothesized that some individuals with ASD may gravitate toward simple, mechanical objects (<a href="#B32">32</a>)
it was predicted that adolescents with ASD would be expected to show a stronger affinity to visually simple robots
and in our sample ASD adolescents did report higher levels of enjoyment while conversing with the visually simple robot and demonstrated a greater level of self-disclosure with the visually simple robot compared to TD peers
Adolescents with ASD also reported high levels of enjoyment in conversing with the android robot, but they did not show higher rates of self-disclosure with the android. Previous research showing that individuals with ASD show a strong affinity for robots (<a href="#B33">33</a>) supports the current study&#x02019;s observation that the sophisticated technology of the android embodied in ACTROID-F might be reported as favorable by adolescents with ASD
in terms of potential for meaningful self-disclosure around personal topics and experiences
it is possible that many adolescents with ASD were so focused or interested in the life-like appearance and movement of the android
that motivation to consider and share personal experiences were lower with the android
as the ACTROID-F is highly human-like in appearance
it is possible that both the android and human interviewer shared similar limitations in eliciting communicative exchanges when compared to the visually simple robot
This may either be due to the comfort levels of the teens with ASD or that the android and the human interviewer created higher levels of sensory stimulation or larger numbers of social cues to manage in comparison to the simplicity of CommU
it is possible that the limited expressive behavior of the android robot had an effect on self-disclosure by the adolescents
Aside from length or content of the self-disclosure
the present study does support that interactions with the two robots were positive experience for adolescents with ASD
These results provide preliminary support on the utility of robots to capitalize on engagement and interest of teens with ASD to create a context to work toward improving or practicing skills of conversation
social reciprocity and relationship building
Spontaneous conversation with another person provides greater insight into the mental states in daily life (<a href="#B34">34</a>) and is important in fostering and maintaining social relationships. Spoken language through conversation can be a key factor to acquire an understanding of psychological states of oneself and others (<a href="#B35">35</a>)
Perhaps interventions using visually simple robots may assist adolescents with ASD to develop skills in self-awareness and communicating those insights verbally through conversation
In their guidelines for humanoid robot designs, Ricks and Colton state that individuals with ASD could begin therapy with a simplistic robot, and as comfort levels increase, introduce more realistic human-like robot to evaluate and move toward increased generalization of learned skills (<a href="#B32">32</a>)
The same may be true for skills involving self-disclosure; after adolescents with ASD communicate with visually simple robot over a period of time
the android robot may offer a step toward generation of self-disclosure skills between the visually simple robot and human peer or therapist
test generalization or habituation effects
it represents one of the first systematic investigations in self-disclosure using robots for adolescents with ASD
it would be important to evaluate habituation effects with the two robots by observing interactions over an extended range of time
characteristics of the human interviewer may certainly influence the quality and quantity of self-disclosure provided by the participating adolescents
Our aim was to involve human interviewers matched according to the age and sex of the android (young adults and female)
we enlisted research assistants working in our laboratory (Caucasian
Further investigation regarding characteristics of the human interviewer (age
and disposition) might yield interesting results
analyses of the current study were also somewhat limited by the small sample size (n &#x0003D; 19) and a larger sample in the ASD and TD groups would be useful for yielding broader and more applicable results
as well as for determining why children with ASD demonstrated lengthier disclosures when interacting with the visually simple robot
it is possible that the within-subject design can be prone to the &#x0201C;carryover effect,&#x0201D; which may have affected the results
all participants were able to complete study procedures
and results suggest differences of import between ASD and TD teens regarding enjoyment levels in communicating with robots
as well as differences among ASD participants showing higher rates of self-disclosure in interactions with the visually simple robot compared to interactions with a human therapist
As our capacity to utilize technology in intervention and therapeutic settings continues to become a viable option over time
perhaps we can continue to consider ways in which robots represent meaningful contributions to the promotion of conversation
and social engagement with others among those affected by autism
All procedures involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards
and MK conceived of the study and participated in its design and assisted with data collection and scoring of behavioral measures and analyzed and interpreted the data and were involved in drafting the manuscript and revised it critically for important intellectual content
MK was involved in giving final approval of the version to be published
All authors read and approved the final manuscript
YY and HI serve as consultants of Vstone Co
All other 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 reviewer MM and handling editor declared their shared affiliation
We sincerely thank the participants and all the families who participated in this study
This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17H05857)
ERATO ISHIGURO Symbiotic Human-Robot Interaction Project
and was partially supported by The Center of Innovation Program from the Japan Science and Technology Agency
The Supplementary Material for this article can be found online at <a href="http://www.frontiersin.org/articles/10.3389/fpsyt.2018.00036/full&#x00023;supplementary-material">http://www.frontiersin.org/articles/10.3389/fpsyt.2018.00036/full&#x00023;supplementary-material</a>
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Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution
we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site
CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf
Recombinant cells were enriched on the basis of expression of AcGFP
and two cell lines that contained the repaired allele were subcloned
We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows
five viable fetuses were retrieved at Days 34 and 36
PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning
We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34
Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint
not only by gene disruption but also by gene targeting through genome editing-assisted homologous recombination
The objective of the present study was to generate progeny in which the IARS gene had been repaired
The mutated nucleotide in bovine fetal fibroblast (BFF) cells derived from a homozygous mutant fetus was substituted with the correct nucleotide by homologous recombination using CRISPR/Cas9-assisted genome editing
we were able to obtain cloned fetuses by somatic cell nuclear transfer (SCNT) using recombinant cells in which the mutation had been repaired and foreign marker DNA had been deleted
From insemination of a carrier cow by a carrier bull
we derived three fetuses that were retrieved on Day 41 of pregnancy (see Methods)
One fetus was diagnosed as C/C (homozygous mutant; #820-1
the second as G/C (heterozygous mutant; #820-2
There were no obvious differences in morphology
body weight or length among the different genotypes
</a>Design of the CRISPR/Cas9 single guide RNA and assessment of target specificity
(a) Sequence of the region that flanks the mutation site in IARS
The base at the third position of the codon that encodes p.Val79 was changed in the donor DNA from thymine to cytosine
The PAM sequence (underlined) is located at nucleotides 236 A to 234 G on the bottom strand of the IARS gene and includes the substituted nucleotide 235 G
(b) Flow cytometric analysis of the target specificity of CRISPR/Cas9
The empty vector pX330 (black line) or pX330_IARS (red line) was introduced into HEK293T cells with the vector p2color that contained either the mutant
The histogram shows the expression of EGFP in tdsRed positive cells
(c) Comparison of the expression of EGFP for different target sequences
The values are shown as the expression ratios of EGFP when each p2color vector was introduced with the empty vector or pX330_IARS
(d) Cleavage frequencies in the T7E1 assay in BFF cells derived from #810 (G/G wild-type)
M indicates DNA size marker (100 bp ladder)
The frequency of cleavage in homozygous mutant BFF cells was slightly higher than in heterozygous mutant BFF cells
suggesting that the designed CRISPR/Cas9 selectively cleaved the target sequence of the mutated IARS gene and that an indel mutation occurred in the flanking target region
(b) Enrichment of AcGFP-positive cells by cell sorting
(c) Southern blot analysis of subcloned recombinant cell lines
#820-1 is the parent cell line of #820-1-2
(d) Day 6 SCNT embryos derived from #820-1-2
The image shown is a bright-field image merged with a fluorescent image
(e) Bright-field and fluorescent images of a Day 34 cloned fetus
</a>Analysis of cloned fetuses from the second round of SCNT
(a) Isolation of a population of cells with low levels of AcGFP expression from #829 cells into which piggyBac transposase mRNA had been introduced
(b) Bright-field and fluorescent images of a Day 34 cloned fetus from the second round
(c) Southern blot analysis of cloned fetuses from the first and second round of SCNT
(d) Sequence analysis of the region that flanks the repaired IARS locus
Left: Direct sequencing of PCR products that correspond to the mutation site
Amplification from the repaired allele in the #829 cells failed due to the presence of the AcGFP cassette
Middle: Direct sequencing of RT-PCR products
very little mRNA was transcribed from the repaired allele because of aberrant splicing
which might be caused by the AcGFP cassette
Right: Direct sequencing of PCR products that correspond to the piggyBac junction site
and the milk produced by these transgenic cows showed antiviral activity in vitro
These strategies show the feasibility of genome editing technologies in reducing the costs and requirement for animal healthcare in animal husbandry and also in creating new biological industries
and reconstitution of the germ line from genome-modified pluripotent stem cells in vitro would dramatically shorten the cycle for the improvement of livestock
our results show that the application of genome editing to livestock improvement could prevent the wastage of excellent genome resources
It also could provide a more rapid innovative method for livestock breeding
All procedures involving the care and use of animals were approved by the Animal Research Committee of the National Agriculture and Food Research Organization
Cows were treated in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education
Thawed embryos were transferred to a uterine horn ipsilateral to the corpus luteum (CL) of estrus-synchronized recipient cows
A total of six embryos were transferred and pregnancy was diagnosed by ultrasonography
two pregnant cows were sacrificed by administering an overdose of sodium pentobarbital and three fetuses were recovered
Fibroblast cells derived from the recovered fetuses were genotyped by PCR and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) under 5% CO2 and 95% air at 37 °C
Cleaved embryos were then transferred into CR1aa medium supplemented with 5% FBS and cultured until Day 6 or 7 (Day 0 = the day of SCNT)
Day 6 SCNT embryos with normal morphology were transferred to a uterine horn ipsilateral to the corpus luteum of estrus-synchronized recipient cows
Pregnancy was diagnosed by ultrasonography
and pregnant cows were sacrificed on Day 34 or 36 by administering an overdose of sodium pentobarbital
Fibroblast cells isolated from the retrieved fetuses were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% FBS under 5% CO2 and 95% air at 37 °C
The BFF cells derived from cloned fetuses from the first round of SCNT carried the AcGFP cassette for use in positive selection; to remove this cassette
BFF cells that had undergone less than three passages were trypsinized and suspended (1 × 106 cells) in 100 µl of Opti-MEM (Thermo Fisher Scientific) with 5 µg of Excision-only piggyBac transposase mRNA (System Biosciences)
Cells were placed in a 10-mm gap cuvette and subjected to electroporation using a NEPA21 electroporator (NEPA GENE)
The electroporation conditions were as follows: 1) poring pulse: input voltage
cells were diluted immediately with culture medium and plated onto 60-mm dishes
cells with low AcGFP expression were enriched using a MoFlo Astrios cell sorter and
cells that were negative for AcGFP were used for SCNT to generate a second round of clones
Hybridization and DIG detection were performed using the DIG detection system (Roche) in accordance with the manufacturer’s protocol
The CDP-Star Detection Reagent (Roche) was used to develop the membrane and the resulting chemiluminescent signal was detected using Amersham Hyperfilm ECL (GE Healthcare)
A correction to this article has been published and is linked from the HTML version of this paper
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This work was supported by a Grant-in-Aid 27010 A from Science and Technology Research Promotion Program for Agriculture
Institute of Livestock and Grassland Science
Okayama University Graduate School of Environmental and Life Science
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DOI: https://doi.org/10.1038/s41598-017-17968-w
In Vitro Cellular &amp; Developmental Biology - Animal (2019)
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Transcranial direct-current stimulation (tDCS) to the dorsolateral prefrontal cortex (DLPFC) has been established as an effective and noninvasive method to modulate cognitive function
the mechanisms causing those cognitive changes under the tDCS remain largely unknown
We strove to elucidate the cognito-biological relation under the tDCS condition by examining whether the dopamine system activated by tDCS is involved in cognitive changes in human participants
we used [11C]-raclopride positron emission tomography (PET) scanning: 20 healthy men underwent two [11C]-raclopride PET scans and subsequent neuropsychological tests
One scan was conducted after tDCS to the DLPFC
One was conducted after sham stimulation (control)
Results of [11C]-raclopride PET measurements demonstrate that tDCS to the DLPFC caused dopamine release in the right ventral striatum
Neuropsychological tests for attentiveness revealed that tDCS to the DLPFC-enhanced participants’ accuracy
this effect was correlated significantly with dopamine release
This finding provides clinico-biological evidence
demonstrating that enhancement of dopamine signaling by tDCS in the ventral striatum is associated with attention enhancement
The mechanisms underlying these cognitive changes that occur under the tDCS remain largely unknown
the putative relation between tDCS-induced change in dopaminergic transmission and cognitive enhancement remains untested
Their study demonstrated that tDCS on the DLPFC can increase the level of endogenous dopamine released in the striatum in humans
a more intriguing point lay unexplored in their study: cognitive alteration with respect to dopamine release under the tDCS condition
the dopamine system is related to attentional control and executive function
it can be readily inferred that cognitive tests for attentional control and executive function might be desirable
we specifically examined appropriate parts of the cognitive test and analytic approaches in the current study
we aimed to elucidate the cognito-biological relevance under the tDCS condition by examining whether the dopamine system activated by tDCS is involved in cognitive changes in human participants
Our hypothesis was the following: tDCS enhances striatal dopamine release
which improves attention and executive function
Participants with metal implants inside the body
known history of epilepsy or substance dependence or chronic headache
and prospective participants with a known psychiatric disorder or on central nervous system-acting medications were excluded from the study
Written informed consent was obtained before enrollment
The Ethics Committees of Kanazawa University Hospital and Hamamatsu University School of Medicine approved the methods and procedures used for this study
which were applied in accordance with the Declaration of Helsinki
The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (no
we had to have the effect size of tDCS on striatal dopamine release
there was no study focused on the topic at that time
we decided to refer to the effect size of repetitive transcranial magnetic stimulation (rTMS) on striatal dopamine release and adjusted it for tDCS
This study used a randomized, sham-controlled, double-blind, crossover design. Participants completed two sessions at least 1 month apart to control for carry-over effects. Participants then underwent 26 min of tDCS (either cathodal or sham) on DLPFC, after which PET/magnetic resonance imaging (MRI) and the cognitive function test were performed (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41398-019-0443-4#Fig1">1</a>).
</a>The placement of the tDCS electrodes was based on the international 10–20 EEG system: the anode at the F3 (left DLPFC) and the cathode at the F4 (right DLPFC) a
The type of stimulation (active or sham) was randomized in a double-blind manner
Thirteen-minutes duration tDCS was applied twice with an interval of 20 min
PET was performed at 50 min after initiation of tDCS
followed by MRI measurement at 140 min and cognitive tests at 200 min
Participants underwent twice experiments in a crossover fashion at least 1 month apart b
and a computer-generated list was used in the Innovative Clinical Research Center
The study staff did not have access to the allocation sequence until the end of the study
Via two soaked (NaCl 0.9%) electrodes (35 cm2) using a stimulator (DC-Stimulator Plus II; neuroCare Group GmbH
participants received either 13 min × 2 of anodal stimulation (2 mA) or sham stimulation to the left hemisphere
they received 13 min × 2 of active stimulation (2 mA) or sham stimulation to the right hemisphere of the brain
The stimulation type (active or sham) was randomized across sessions and was counterbalanced across participants: 10 completed sham then active; the remaining 10 completed active then sham
The sham stimulation was applied for 30 s before the PET scan
where some participants were aware of the initial tingling sensation
but none noticed the difference in active or sham stimulation
We used the Cambridge Neuropsychological Test Automated Battery (CANTAB)
a semi-automated computer interface for assessing cognitive function
participants completed tests evaluating attention control (Reaction time
we used a task for working memory (Spatial Working Memory task
five circles are presented on the upper screen
a yellow dot will appear in one of the circles
The participant must react as soon as possible
selecting the circle in which the dot appeared
Outcome measures include the reaction time and the accuracy scale
The reaction time measures the participant’s response latency
The accuracy scale is the total number of trials for which the response is correct
We performed PET scanning using a high-resolution brain PET scanner (SHR12000; Hamamatsu Photonics K.K.
Japan) with image resolution of 2.9 × 2.9 × 3.4 mm full-width at half-maximum
A thermoplastic face mask was used to fix the head in the same place during the scans
Then [11C]-raclopride examination was conducted of all participants
After a 10-min transmission scan for attenuation correction with a 68Ge/68Ga source
All participants underwent two scans with an interval of &gt; 1 month between stimulations
To ascertain the areas of the regions of concern for setting regions of interest (ROIs)
MRI was applied using a 3 T MRI device (3 T Ingenia; Philips Healthcare
The Netherlands) with the following acquisition parameters: three-dimensional mode sampling
TR shortest (6 ms) and TE shortest (2.7 ms)
Our mobile PET gantry enabled us to reconstruct PET images parallel to the estimated intercommissural line without reslicing
we were able to allocate ROIs on the target regions of the original PET images
the putamen was selected as the target region
The cerebellum was selected as the reference region for [11C]-raclopride
Time–activity curves (TACs) were extracted for each region under active and sham stimulation conditions
The final SPM images were then generated by masking out the extra-striatal white matter
The variation of BPND of [11C]raclopride PET data were not different between two groups
which allowed further analysis of SPM that was based on the BPND PET data
The ROIs were located bilaterally on the dopaminergic projection regions (the caudate
and dorsal striatum) displayed on the 5–10 PET slices
which indicates that ROIs from each region contained functional information of the region 9–18-mm-deep in the z direction
These ROIs or volumes of interest were transferred automatically onto corresponding [11C]-raclopride BPND parametric images
Regional [11C]-raclopride BPND derived from these ROIs proceeded using PMOD software
Data were analyzed using software (Statistical Package for Social Sciences ver
Paired t tests were applied to compare the levels of [11C]-raclopride BPND in the respective brain regions between active and sham stimulation conditions
statistical significance was inferred for p &lt; 0.05
our main objective was twofold: to evaluate the putative relation between the change in dopamine concentration at the striatum and the change in accuracy/reaction time in RTI
and to evaluate the putative effects of tDCS on those measures
we applied paired t tests with z-transformation modified data to offset physiological and psychological variations among participants for evaluation of the effects of tDCS on the scores of mean accuracy and standard deviation of reaction time in RTI controlling for age
A larger standard deviation implies that the reaction time is so irregular that participants have difficulty performing tasks efficiently
We applied simple regression analyses to evaluate relations between post-tDCS changes of these two scores and post-tDCS changes in [11C]-raclopride BPND at the ventral striatum
and interaction between age and [11C]-raclopride BPND
we entered [11C]-raclopride BPND or a stimulation type (i.e.
sham) and age along with their two-way interaction into the model
To evaluate the main effect of the value of [11C]-raclopride BPND or the type of stimulation under the existence of the interaction between them
Statistical significance was inferred for p &lt; 0.05
we evaluate the relation between the change in dopamine concentration at the striatum and the change in strategy score in SWM
The statistical procedures (paired t test and a linear mixed effect analysis) were the same as those described in the previous section
except for those related to cognitive data (strategy scores in SWM)
All statistical analyses were conducted using software (Stata ver
</a>No difference was found between TACs from the cerebellum a and putamen b except for the right ventral striatum c. In this region, the magnitude of percentage of reduction in [11C]-raclopride binding was found to be higher after active stimulation (*p = 0.002) d. The vertical axes show %dose/L. The red curve shows active stimulation. The blue curve shows sham stimulation
</a>The brain region superimposed on magnetic resonance images shows marked reduction in [11C]-raclopride binding after active stimulation
</a>Relation between changes in adjusted standard deviation of reaction time and in [11C]-raclopride BPND
No significant regression equation was found for the strategy scores (F(1,18) = 0.67
[11C]-raclopride BPND was not found to be a significant predictor of the strategy score in SWM (t = 0.82
These results imply that lower [11C]-raclopride BPND (i.e.
a higher concentration of dopamine) at the ventral striatum corresponds to higher accuracy and lower standard deviation of reaction time in RTI after controlling for the interaction term
We found a significant two-way interaction effect (stimulation × age) (z = 4.41, p &lt; 0.001) for accuracy: stimulation (z = 2.57, p = 0.01) and age (z = −5.48, p &lt; 0.001) (Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41398-019-0443-4#Tab3">3</a>)
suggesting that tDCS enhanced the accuracy in RTI
after controlling for the interaction term
and that the effect of tDCS varies depending on age
Although significant main effects for stimulation (z = −2.93, p = 0.003) on strategy scores were found in SWM, no other factor was found to be significant (Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41398-019-0443-4#Tab4">4</a>)
This finding suggests that participants under the tDCS might take more strategic processes to reach a goal
As opposed to the case for accuracy/standard deviation of reaction time
the relation between [11C]-raclopride BPND and strategy scores was not significant
Our study demonstrated that tDCS caused a significant release of a dopamine in the right ventral striatum in healthy human male participants
The neuropsychological test for attentiveness revealed that tDCS enhanced participants’ accuracy in the RTI task
This effect was correlated significantly with dopamine release
tDCS enhanced participant’s executive working memory
was not correlated significantly with dopamine release
dopamine release was observed in the striatum on the hemisphere contralateral to the anode attachment
Although we found a significant increase only in the right ventral striatum
found a significant increase in the right dorsal striatum and the left putamen
Those different results might be attributable to the different stimulation methods used in the respective studies
Participants in our study received 13 min of stimulation twice
those in Fonteneau’s study received 20 min of stimulation once
suggesting that single and longer simulation might induce dopamine release over a wider area
our results confirm that tDCS induces dopamine release in the striatum and confirm that the enhanced dopaminergic transmission in the striatum eventually enhances attention
an elderly person might be a good candidate for application of tDCS
Our results are based on data from a small sample of young people of a narrow age range
to confirm the effects of tDCS on people from a general population
further study must be conducted with larger samples that include elderly people
Some limitations of our study must be considered
we evaluated cognitive functions for only a short period immediately after the stimulation
it is not possible to form any inference about the persistence of aftereffects
the lack of women among the study participants prevents generalization of this conclusion beyond men
the statistical threshold with FWE corrections could not be applied in the current SPM analysis
further studies must be conducted with a larger sample size
this report is the first describing a study of an association between tDCS-induced dopamine increase and improvement in attention beyond a speed–accuracy tradeoff using single-session tDCS
This finding provides clinico-biological evidence demonstrating that enhancement of dopamine signaling by tDCS in the ventral striatum is associated with attention enhancement
Improvements in attention and decision-making following combined behavioral training and brain stimulation
Role of human prefrontal cortex in attention control
Effects of prefrontal tDCS on executive function: methodological considerations revealed by meta-analysis
Boosting focally induced brain plasticity by dopamine
Effects of transcranial direct current stimulation (tDCS) on executive functions: influence of COMT Val/Met polymorphism
Frontal transcranial direct current stimulation induces dopamine release in the ventral striatum in human
Cognitive impairment and the brain dopaminergic system in Parkinson disease: [18F]fluorodopa positron emission tomographic study
Effect of levodopa-carbidopa intestinal gel on non-motor symptoms in patients with advanced Parkinson’s disease
Depressed dopamine activity in caudate and preliminary evidence of limbic involvement in adults with attention-deficit/hyperactivity disorder
Imaging the effects of methylphenidate on brain dopamine: new model on its therapeutic actions for attention-deficit/hyperactivity disorder
Combined insular and striatal dopamine dysfunction are associated with executive deficits in Parkinson’s disease with mild cognitive impairment
Executive subprocesses in working memory: relationship to catechol-O-methyltransferase Val158Met genotype and schizophrenia
New evidence of association between COMT gene and prefrontal neurocognitive function in healthy individuals from sibling pairs discordant for psychosis
Estimation of premorbid IQ in individuals with Alzheimer’s disease using Japanese ideographic script (Kanji) compound words: Japanese version of National Adult Reading Test
Repetitive transcranial magnetic stimulation of the human prefrontal cortex induces dopamine release in the caudate nucleus
Repetitive transcranial magnetic stimulation or transcranial direct current stimulation
Effects of low versus high frequencies of repetitive transcranial magnetic stimulation on cognitive function and cortical excitablility in Alzheimer’s dementia
Temporal cortex direct current stimulation enhances performance on a visual recognition memory task in Alzheimer disease
G* Power 3: a flexible statistical power analysis program for the social
Induction of late LTP-like plasticity in the human motor cortex by repeated non-invasive brain stimulation
Planning and spatial working memory following frontal lobe lesions in man
Effect of subthalamic nucleus stimulation during exercise on the mesolimbocortical dopaminergic region in Parkinson’s disease: a positron emission tomography study
Effect of simple motor performance on regional dopamine release in the striatum in Parkinson disease patients and healthy subjects: a positron emission tomography study
A heteroskedasticity-consistent covariance matrix estimator and a direct test for heteroskedasticity
New computer-aided diagnosis of dementia using positron emission tomography: brain regional sensitivity-mapping method
Long-term stimulant treatment of children with attention-deficit hyperactivity disorder symptoms
Positive effects of transcranial direct current stimulation in adult patients with attention-deficit/hyperactivity disorder - a pilot randomized controlled study
Transcranial direct current stimulation improves clinical symptoms in adolescents with attention deficit hyperactivity disorder
The relationship between working memory capacity and executive functioning: evidence for a common executive attention construct
The neural and genetic basis of executive function: attention
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This research is supported by the Center of Innovation Program from Japan Science and Technology Agency
and partly by Scientific Research on Innovative Areas (JP16H06402)
We thank the staff of Hamamatsu Medical Imaging Center for their valuable support
Preeminent Medical Photonics Education and Research Center
Research Center for Child Mental Development
The authors declare that they have no conflict of interest
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DOI: https://doi.org/10.1038/s41398-019-0443-4
Alzheimer&#x27;s Research &amp; Therapy (2025)
Journal of NeuroEngineering and Rehabilitation (2024)
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we studied the effects of age and gender on the expression of brain-derived neurotrophic factor (BDNF) mRNA and heat shock protein hsp-70 mRNA in the rat retrosplenial cortex following administration of the noncompetitive NMDA receptor antagonist (+)-MK-801 (dizocilpine)
Male and female Sprague–Dawley rats (5 weeks
or 10 months old) were given a single intraperitoneal injection of saline (1 ml/kg) or dizocilpine (0.3
No expression of BDNF mRNA and hsp-70 mRNA was detected in the rat retrosplenial cortex after administration of saline (1 ml/kg
IP) caused a marked induction of BDNF mRNA and hsp-70 mRNA in the retrosplenial cortex of male and female rats
Female rats were more sensitive to the induction of BDNF mRNA and hsp-70 mRNA in the retrosplenial cortex by dizocilpine as compared to male rats
It was also found that adult (12 weeks old) and aged (10 months old) rats were more sensitive to the induction of hsp-70 mRNA and BDNF mRNA in the retrosplenial cortex by dizocilpine as compared to young (5 weeks old) rats
These results suggest that the age and gender differences observed in the expression of BDNF mRNA and hsp-70 mRNA in the retrosplenial cortex by dizocilpine may be associated with the differences in dizocilpine-induced neurotoxicity observed with gender and age within the same region
thus the expression of heat shock protein HSP 70 may be a compensatory response of these neurons to the neurotoxicity produced by NMDA receptor antagonists
it has been suggested that expression of BDNF may occur as a trophic response to neuronal injury
The aforementioned findings suggest that susceptibility to the neurotoxicity induced by NMDA receptor antagonists is dependent on the age and gender of rats and that expression of heat shock protein HSP-70 and BDNF may play a role in neuronal injury induced by NMDA receptor antagonists
the present study was undertaken to study the effects of age and gender on the expression of BDNF mRNA and hsp-70 mRNA in the rat retrosplenial cortex following administration of dizocilpine
Male and female Sprague–Dawley rats (Nihon Clea
5 or 12 weeks old and 10 months old) were housed separately under a 12-h light/12-h dark cycle with free access to food and water
All experiments were carried out in accordance with the NCNP Guide for the Care and Use of Laboratory Animals
Densitometric analysis of sections was carried out using Macintosh-based image analysis software (NIH image)
The data were initially analyzed using a three-way analysis of variance (ANOVA) (age × gender × treatment)
Where a significant interaction in the between-subjects variables (age
where a significant interaction for the two-way ANOVA was found
a subsequent one-way ANOVA was carried out
followed by a post-hoc comparison using Scheffe's test
The criterion of significance was p &lt; .01
</a>Expression of BDNF mRNA in the rat brain induced by administration of dizocilpine. Male and female SD rats (12 weeks olds) were injected intraperitoneally with saline (1 ml/kg) or dizocilpine (0.3, 1.0, or 3.0 mg/kg). Animals were killed 4 hours after treatment for in situ hybridization
</a>Expression of hsp-70 mRNA in the rat retrosplenial cortex induced by administration of dizocilpine
or 10 months olds) were injected intraperitoneally with saline (1 ml/kg) or dizocilpine (0.3
Animals were killed 4 hours after treatment for in situ hybridization
Densitometric analysis of sections was performed using Macintosh-based image analysis software (NIH image) as described in Methods
The values are the mean ± SEM of four rats
The three-way ANOVA indicated significant effects of age
and treatment on BDNF mRNA expression (age: F [2,72] = 113.8
p &lt; .001) with significant interactions (age × treatment: F [6,72] = 17.9
p &lt; .001; gender × treatment: F [3,72] = 23.0
The two-way ANOVA on the young groups demonstrated a significant effect of treatment (F [3,24] = 5.7
p &lt; .004) without a significant interaction
the subsequent one-way ANOVAs did not show a significant effect of treatment for either the young female group (F [3,12] = 2.624
p = .09) or the young male group (F [3,12] = 4.07
The two-way ANOVA on the adult groups demonstrated a significant effect of treatment (F [3,24] = 78.0
p &lt; .0001) with a significant interaction (gender × treatment: F [3,24] = 15.1
The subsequent one-way ANOVA on the adult female group indicated a significant effect of treatment (F [3,12] = 29.9
p &lt; .0001) and the post-hoc comparison using Scheffe's test demonstrated that dizocilpine had significant treatment effects at 0.3 mg/kg (p = .0001)
1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .001)
The one-way ANOVA on the adult male group also showed a significant effect of treatment (F [3,12] = 77.8
and the post-hoc comparison using Scheffe's test demonstrated that dizocilpine has significant treatment effects only at 1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .0001)
The two-way ANOVA on the aged groups demonstrated a significant effect of treatment (F [3,24] = 78.0
p &lt; .0001) with a significant interaction (gender × treatment: F [3,24] = 15.4
The subsequent one-way ANOVA on the aged female group showed a significant treatment effect (F [3,12] = 33.8
p &lt; .0001) and the post-hoc comparison using Scheffe's test indicated that dizocilpine had significant treatment effects at 0.3 mg/kg (p = .0001)
1.0 mg/kg (p = .0001) and 3.0 mg/kg (p &lt; .0001)
The one-way ANOVA on the aged male group also demonstrated that dizocilpine had significant treatment effects
but post-hoc comparison with Scheffe's test revealed significance only at 1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .0001)
and treatment on hsp70 mRNA expression (age: F [2,72] = 69.3
p &lt; .0001) with significant interactions (age × treatment: F [6,72] = 10.6
p &lt; .0001; gender × treatment: F [3,72] = 15.3
The two-way ANOVA on the young groups demonstrated a significant effect of treatment (F [3,24] = 6.5
p = .002) without a significant interaction
the subsequent one-way ANOVA did not show significant treatment effects in either the young female group (F [3,12] = 2.8
p = .08) or the young male group (F [3,12] = 4.0
The two-way ANOVA on the adult groups demonstrated significant effects of treatment and gender (gender: F [1,24] = 27.0
The subsequent one-way ANOVA on the adult female group indicated significance (F [3,12] = 41.5
and the post-hoc comparison using Scheffe's test demonstrated that dizocilpine had significant treatment effects at 0.3 mg/kg (p &lt; .0001)
1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .0001)
The one-way ANOVA on the adult male group also showed significant treatment effects (F [3,12] = 65.9
but the post-hoc comparison using Scheffe's test demonstrated that dizocilpine had significant treatment effects only at 1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .0001) but not 0.3 mg/kg
The two-way ANOVA on the aged groups demonstrated a significant effect of treatment (F [3,24] = 64.0
p &lt; .0001) with a significant interaction (gender × treatment: F [3,24] = 18.8
The subsequent one-way ANOVA on the aged female group showed a significant treatment effect (F [3,12] = 22.9
and the post-hoc comparison using Scheffe's test indicated that dizocilpine had significant treatment effects at 0.3 mg/kg (p = .0001)
The one-way ANOVA on the aged male group also demonstrated a significant effect of treatment
but the post-hoc comparison using Scheffe's test showed that dizocilpine has significant treatment effects only at 1.0 mg/kg (p &lt; .0001) and 3.0 mg/kg (p &lt; .0001) but not 0.3 mg/kg
it seems that the age-dependency for NMDA receptor antagonist-induced neurotoxicity in rat brain roughly parallels the age dependency for sensitivity to ketamine-induced psychosis and for the emergence of the symptoms of schizophrenia in humans
although age comparisons between humans and rats are inherently problematic
2 months) in rat hippocampus but that no senescence-related changes (25 vs
the age difference between young (5 weeks) rats and adult (12 weeks) or aged (10 months) rats was shown
it is likely that a difference in the density of NMDA receptor expression may
contribute to the age-dependent expression of BDNF mRNA and hsp-70 mRNA in rat retrosplenial cortex by dizocilpine
dizocilpine is also metabolized in the liver
and the pharmacokinetic differences between male and female rats may be explained by a low capacity for metabolism of dizocilpine by the liver in female rats
which would imply that the half life of dizocilpine in female rats is longer than that in male rats
dizocilpine may be active in the female rat brain at a higher concentration for a longer period of time
may exert greater behavioral and biochemical effects at lower doses
it is likely that the pharmacokinetic differences between male and female rats may contribute to the gender differences in the expression of BDNF mRNA and hsp-70 mRNA in the rat retrosplenial cortex induced by dizocilpine
which undermines the importance of any differences in the metabolism of dizocilpine
This suggests that gonadal hormones may play a more important role in gender differences in the expression of BDNF mRNA and hsp-70 mRNA in the rat retrosplenial cortex induced by dizocilpine
Further studies will be necessary to examine the role of gonadal hormones in modulating the response to dizocilpine
we found the age and gender differences in the expression of BDNF mRNA and hsp-70 mRNA in rat retrosplenial cortex following administration of dizocilpine (0.3
the present study suggests that age and gender differences in the expression of BDNF mRNA and hsp-70 mRNA in rat retrosplenial cortex by administration of dizocilpine might be associated with these differences in the dizocilpine-induced neuronal injury in the same region
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(1998b): Behavioral changes and expression of heat shock protein hsp-70 mRNA
and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid
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(1993): MK-801 induces immediate-early gene proteins and BDNF mRNA in rat cerebrocortical neurons
(1991): BDNF mRNA expression is increased in adult rat forebrain after limbic seizures: Temporal patterns of induction distinct from NGF
(1991): Recent advances in the phencyclidine model of schizophrenia
(1994): Subanesthetic effects of noncompetitive NMDA antagonist
(1995): Ketamine activates psychosis and alters limbic blood flow in schizophrenia
(1998): Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-D-aspartic acid receptor activity
(1994): Neurotrophic factors: From molecule to man
(1959): Study of a new schizophrenomimetic drug—sernyl
(1984): Sex-dependent differences in the pharmacological actions and pharmacokinetics of phencyclidine
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(1989): Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs
(1997): The Rat Brain in Stereotaxic Coordinates
(1989): Characterization of the effects of the acute and repeated administration of MK-801 on the release of adrenocorticotropin
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(1994): Alterations in gene expression as an index of neuronal injury: Heat shock and the immediate early gene response
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(1999): Effects of acute and chronic administration of methamphetamine or phencyclidine on plasma adrenocorticotropin
(1993): Posterior cingulate cortex and spatial memory: A microlimnology analysis
Neurobiology of Cingulate Cortex and Limbic Thalamus
(2000): Abnormal expression of brain-derived neurotrophic factor and its receptor in the corticolimbic system of schizophrenic patients
(1998): Schizophrenia and glutamatergic transmission
(1980): PCP intoxication in young children and infants
(1982): Ketamine—Its pharmacology and therapeutic uses
(1990): Activity- dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors
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This work was supported by a grant from the Ministry of Health and Welfare of Japan
National Center of Neurology and Psychiatry NCNP
Kenji Hashimoto Ph.D &amp; Yoshio Minabe MD
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DOI: https://doi.org/10.1016/S0893-133X(00)00246-3
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Pathophysiological role of endoplasmic reticulum (ER) stress response signaling has been suggested for bipolar disorder
The goal of this study was to test the genetic association between bipolar disorder and an ER chaperone gene
We tested the genetic association between bipolar disorder and HSP90B1 by case-control studies in two independent Japanese sample sets and by a transmission disequilibrium test (TDT) in NIMH Genetics initiative bipolar trio samples (NIMH trios)
We also performed gene expression analysis of HSP90B1 in lymphoblastoid cells
rs17034977 showed significant association in both Japanese sample sets
The frequency of the SNP was lower in NIMH samples than in Japanese samples and there was no significant association in NIMH trios
Gene expression analysis of HSP90B1 in lymphoblastoid cells suggested a possible relationship between the associated SNP and mRNA levels
HSP90B1 may have a pathophysiological role in bipolar disorder in the Japanese population
though further study will be needed to understand the underlying functional mechanisms
association of the ER stress related genes with bipolar disorder is still controversial
We also performed expression analysis in LB cells derived from patients and controls
interviewed them and they did not have major mental disorders
A highly conserved 33-bp sequence is observed in the upstream region
Number indicates the position in the UCSC Human Genome Browser
To identify the possible functional polymorphisms that are linked to the haplotype associated with bipolar disorder, we sequenced the upstream region, 5′-untraslated region (UTR), exon14, exon17 and exon18, in which the existence of missense polymorphisms has been shown in UCSC Human Genome Browser, March 2006 (<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a>)
and 3′-UTR of HSP90B1 in 44 patients with bipolar disorder
We also sequenced around the highly conserved region with Evi12
Primer sequences are as follows: promotor region and 5′-UTR
forward 5′-CTGACCAATCGGAAGGAG and reverse 5′-ATCACTCACCGAAGGTCA; exon14
forward 5′-GACAGTTGAAAGACAATTGCTC and reverse 5′-CGTAAACTTGTTGTCTACCT; exon17
forward 5′-GAAAGAATGCTTCGCCTCAG and reverse 5′-ACTCTGTCCTAACTTTGCCAGAAC; exon18 and 3′-UTR
forward 5′-CATGCACAAACCTGTGAG and reverse 5′-TCAGCCAAGTATTAGGTAGT; around Evi12
forward 5′-GCACATATCTCTACTGTAATCAGAACGAG and reverse 5′-CCTGGGGTTTCGTTGGAAGTG
real-time quantitative PCR using TaqMan chemistry was performed according to the manufacturer’s protocol (Applied Biosystems)
ABI7900HT was used and the relative ratios to GAPDH were calculated
The experiments were performed in quadliplicate
Primer and probe mixture of HSP90B1 and GAPDH was obtained from assay-on-demand products (Applied Biosystems)
T test was used for comparison of the expression levels between controls and patients in LB cells
Southern blotting analysis was performed to examine the insertion/deletion mutations around the HSP90B1 gene
Ten micrograms of genomic DNA derived from LB cells was digested by EcoRV (Takara; Otsu
hybridization was performed using two kinds of digoxigenin-labeled probes
PCR-products were cloned into pCR-2.1-TOPO Vector (Invitrogen)
and then internally labeled with digoxigenin using a PCR DIG Probe Synthesis Kit (Roche
Germany) according to the manufacturer’s protocol
which detect 4642 bp fragments on the upstream region of HSP90B1 (chromosome 12
which detect 6050 bp fragments (chromosome 12
Hybridization of the probes was detected by LAS-3000 luminoimage analyzer (Fujifilm; Tokyo
Japan) using a part of DIG High Prime DNA Labeling and Detection Starter Kit II (Roche)
</a>HSP90B1 mRNA levels in lymphoblastoid cells. Y-axis indicates the relative ratio of HSP90B1 to GAPDH. No significant difference was observed between the cases of bipolar disorder (BP) and controls (CT)
</a>Relationship between HSP90B1 mRNA levels and SNP5 (rs17034977) in lymphoblastoid cells
Y-axis indicates the relative ratio of HSP90B1 to GAPDH
A statistically significant difference of HSP90B1 levels was observed between the homozygous A allele carriers and the C allele carriers
Open square indicates homozygote for the C allele of SNP 5 (n = 3)
The bars show the average and standard deviations
a total of 13 SNPs were suggested to exist in the 500-bp upstream region (rs3216180
or as missense polymorphisms in exon14 (rs3209749)
rs3037197) and exon18 (rs11547722) of HSP90B1
there were two SNPs (rs17034917 and rs17034919)
rs2307842 and rs3209749 were not polymorphic
rs3216180 (mainly linked with haplotype 1 [CAATT])
rs17034921 (mainly linked with haplotype 1)
rs2070908 (linked with rs1165681) and rs3037197 (mostly linked with rs1882019) were polymorphic but not linked to haplotype 3 [CGCTT]
rs17034917 was not polymorphic and rs17034919 was linked with rs17034921
To rule out a possibility of large deletion or insertion affecting transcriptional activity
the upstream region spanning about 10 kb of HSP90B1 gene was examined by Southern blotting
suggesting the insertion or deletion in the DNA of 53 patients with bipolar disorder derived from lymphoblastoid cells (data not shown)
we showed the association of a SNP within the HSP90B1 locus (SNP5
rs17034977) with bipolar disorder in a Japanese population
No significant association was observed in NIMH bipolar disorder trio samples and Japanese schizophrenia samples
We examined the mRNA expression levels of HSP90B1
There was no significant difference between controls and patients
The C allele of SNP5 (rs17034977) was significantly associated with lower expression levels of HSP90B1
There has been no evidence that supports the integration of retrovirus in this site in humans
and the present results also excluded such a possibility
Although the results of haplotype analysis
and mRNA analysis suggest that SNP5 itself might be important
it is still not known whether SNP5 itself affects the mRNA levels
Further studies will be needed to identify the genetic polymorphism affecting the mRNA levels
it is unlikely that the observed difference is the artifact due to population stratification
it is possible that some control subjects might actually have bipolar disorder but have reported themselves as healthy
because patients with bipolar disorder often do not have insight into the illness
A possibility that SNP5 is also associated with schizophrenia cannot be ruled out
because a similar tendency of association was observed (P = 0.09)
it is not known whether gene expression in lymphoblastoid cells reflects the alteration in the brain
Transcriptional regulation may differ between lymphoblastoid cells and neuronal cells
it is too premature to assume that subjects carrying SNP5 have lower expression levels of HSP90B1 in neuronal cells
we found an association of rs17034977 in HSP90B1 with bipolar disorder in a Japanese population and suggested that the alteration of mRNA expression by this SNP could be an underlying mechanism for bipolar disorder
an ER chaperone with protein and peptide binding properties
Morita Y et al (2005) Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p
Tohyama M (2004) GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis
Young LT (2000) Increased temporal cortex ER stress proteins in depressed subjects who died by suicide
Green E et al (2004) Lack of support for a genetic association of the XBP1 promoter polymorphism with bipolar disorder in probands of European origin
Smyth C et al (2003) Genome scan of pedigrees multiply affected with bipolar disorder provides further support for the presence of a susceptibility locus on chromosome 12q23-q24
and suggests the presence of additional loci on 1p and 1q
Sham P et al (1995) Linkage studies of bipolar disorder in the region of the Darier’s disease gene on chromosome 12q23–24.1
Turner G et al (1999) A high-density genome scan detects evidence for a bipolar-disorder susceptibility locus on 13q32 and other potential loci on 1q32 and 18p11.2
Koenig ML (2003) The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro
Wang Z (2005) Transcriptional profiling of mefloquine-induced disruption of calcium homeostasis in neurons in vitro
Perola M et al (2003) Evidence of susceptibility loci on 4q32 and 16p12 for bipolar disorder
Lanouar K (2001) Bipolar disorder after mefloquine treatment
Kruse TA (1998) Significant linkage between bipolar affective disorder and chromosome 12q24
O’Donovan MC et al (2005) Localization of bipolar susceptibility locus by molecular genetic analysis of the chromosome 12q23-q24 region in two pedigrees with bipolar disorder and Darier’s disease
Chuang DM (2005) Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: roles of intracellular calcium
Hong CJ (2004) X-box binding protein 1 (XBP1) C–116G polymorphisms in bipolar disorders and age of onset
Cadet JL (2004) Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades
Lemire AL et al (2004) Comparison of microarray-based mRNA profiling technologies for identification of psychiatric disease and drug signatures
Kusumi I et al (2003) Impaired feedback regulation of XBP1 as a genetic risk factor for bipolar disorder
Nakamura K et al (2005) Functional polymorphisms of HSPA5: possible association with bipolar disorder
Kato T (2006) XBP1 induces WFS1 through an endoplasmic reticulum stress response element-like motif in SH-SY5Y cells
Nakamura K et al (2007) Association analysis of ATF4 and ATF5
Kato T (2007) Molecular genetics of bipolar disorder and depression
Kato N (2003) Mechanisms of altered Ca2+ signalling in transformed lymphoblastoid cells from patients with bipolar disorder
Werstuck GH (2005) Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3
Duman RS (2001) Impairments of neuroplasticity and cellular resilience in severe mood disorders: implications for the development of novel therapeutics
Montgrain N et al (2005) Shared and specific susceptibility loci for schizophrenia and bipolar disorder: a dense genome scan in Eastern Quebec families
Gagne B et al (1999) Genome-wide search for linkage of bipolar affective disorders in a very large pedigree derived from a homogeneous population in quebec points to a locus of major effect on chromosome 12q23-q24
Sherman M (1994) A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca(2+)-binding proteins and members of the thioredoxin superfamily
Kaufman RJ (2005) ER stress and the unfolded protein response
Wang JF (2006) Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells
Weiller E et al (1998) The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10
Li PP (2007) Impaired endoplasmic reticulum stress response in B-lymphoblasts from patients with bipolar-I disorder
Thorne N et al (2007) Relative impact of nucleotide and copy number variation on gene expression phenotypes
Wakizono T et al (2006) Behavioral stress and activated serotonergic neurotransmission induce XBP-1 splicing in the rat brain
Delwel R (1999) Retroviral insertions in Evi12
a novel common virus integration site upstream of Tra1/Grp94
frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2
Delwel R (2005) The common viral insertion site Evi12 is located in the 5’-noncoding region of Gnn
a novel gene with enhanced expression in two subclasses of human acute myeloid leukemia
Young LT (1999) Differential display PCR reveals novel targets for the mood-stabilizing drug valproate including the molecular chaperone GRP78
Young LT (2003) Valproate inhibits oxidative damage to lipid and protein in primary cultured rat cerebrocortical cells
Toyota T et al (2004) Association analysis of FEZ1 variants with schizophrenia in Japanese cohorts
Yoshida H (2004) Molecular biology of the ER stress response
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This research was supported by a grant for Laboratory for Molecular Dynamics of Mental Disorders
a Grant-in-Aid from Japanese Ministry of Health and Labor
and a Grant-in-Aid from the Japanese Ministry of Education
The authors declare no conflict of interest
Laboratory for Molecular Dynamics of Mental Disorders
Department of Psychiatry and Genome Research Center
National Center of Neurology and Psychiatry
Kanazawa University Graduate School of Medical Science
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DOI: https://doi.org/10.1007/s10038-007-0188-4
As new Multi-Purpose rooms are being completed to welcome the student bodies of both Cloverland and Fair Oaks elementary schools
Magnolia Elementary is enjoying its own modern update
Earlier this school year the speakers were tweaked
a screen was dropped and training completed as the 76-year-old campus received a new state of the art Audio/Visual upgrade
“We wanted to make sure we delivered a high quality system that won’t be obsolete in a few years,” Oakdale Joint Unified School District Assistant Superintendent Larry Mendonca stated
“If you want to talk about the latest technology and highest quality
The new package was installed as a result of the “Facilities Update,” which was a wish of Magnolia staff and administrators
It includes a state of the art movie screen
surround sound speakers and a command center which can be managed remotely from an iPad
“The quality is extremely competitive,” Mendonca said
“We hired the folks that did the contract at the Gallo Theater for the Arts
“We are so proud to be able to have this system at our school site,” Magnolia School Principal Julie Minabe said of the school’s newest addition
“and are anxious to use it to its full potential
“The first time it was demonstrated to staff
“The real dynamic thing about this unit,” Mendonca added
The projection unit delivers a theater quality picture.”
Minabe shared that the Magnolia staff and student body are already reaping the benefits of the new improved system
it is being used most regularly in the way of indoor Physical Education and movie viewing in the late afternoon for the ASPIRE
“The idea to use if for PE came from the teachers who had originally been using P.E
videos in their classrooms,” the principal said
is perfect timing for the school to utilize the system
given the string of predicted triple digit temperature days
“Students are required to receive 100 minutes per week of P.E.
which is difficult to do outside during inclement weather,” Minabe shared in regards to factors including heat
we are able to get students moving in a more climate controlled environment.”
An estimated two to four classes can participate in a video streamed workout during the 30- to 45-minute time frame teaching staff may choose
Students begin with gentle stretching and transition to any and all types of workouts including Zumba
“The idea for the update actually came from the district,” Minabe said
We’ve also talked about the possibility of using the big screen to project pictures during the science fair
We used it for our Jog A Thon kick-off assembly
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Four traditional farming systems in Bangladesh and Japan have been designated today by FAO as &#8220;<a href="http://www.fao.org/giahs/giahs-home/en/" target="_top">Globally Important Agricultural Heritage Systems</a>.&#8221;
They include Bangladesh&#8217;s floating gardens
a unique hydroponics production system constructed with natural grasses and plants
which have been developed in flood areas; and a trio of sites in Japan: the sustainable river fisheries utilizing Sato-kawa system in Gifu
the Minabe-Tanabe Ume approach to growing apricots on nutrient-poor slopes in Wakayama; and the Takachihogo-Shiibayama mountainous agriculture and forestry system in Miyazaki which allows agricultural and forestry production in a steep mountainous area
The sites were officially recognized during a joint meeting of the GIAHS Steering and Scientific Committee at FAO headquarters in Rome
These new designations bring the number of Globally Important Agricultural Heritage Systems (GIAHS) systems to a total of 36 sites located in 15 countries in Africa
&#8220;In the context of today&#8217;s environmental and economic challenges and climate change
can offer real solutions for food security
the conservation of natural resources and sustainable rural development
if adequate policies and investment are directed to them&#8221;
said FAO Deputy Director-General Maria Helena Semedo
The Globally Important Agricultural Heritage Systems (GIAHS) which was launched by FAO in 2002 during the World Summit on Sustainable Development in Johannesburg
has been  recently endorsed by member countries
during the 39th Session of the FAO Conference
The new GIAHS sites include three in Japan and one in Bangladesh:
The Nagara River is one of the cleanest rivers in Japan that provides a number of ecosystem services
Various components of the system such as river
forests and farmlands are closely linked to each other
The sustainable inland fisheries of a specific type of fish (Ayu) benefit from clean waters of the Nagara River which are maintained through upstream forest management
Local communities have lived within this linked ecosystems and have developed their livelihoods and cultural practices
Minabe-Tanabe Ume System allows for the production of high-quality Ume (Japanese apricots) and various kinds of fruits on nutrient-poor slopes
Local communities have created a thriving Ume fruit production environment by maintaining upper coppice forests for landslide prevention and maintenance of water
By permitting the production of a diverse range of products
the system ensures stable livelihoods and makes communities more resilient to disasters
This site is located in a steep mountainous area where flat land is extremely scarce
local people have established a distinctive and sustainable system of agriculture and forestry which balances timber production with diverse farming activities &#8212; such as terraced rice growing
or tea cultivation.The forest is maintained as a &#8220;mosaic&#8221; of conifers and broadleaf trees using traditional practices
Farmers in some parts of  Bangladesh where flood waters can remain for a prolonged period of time have developed a unique hydroponics system in which plants can be grown on the water on floating organic bed of water hyacinth
This environmentally friendly traditional cultivation technique utilizes the natural resources of wetlands to grow vegetables and other crops almost all year round providing numerous social
agricultural and ecological benefits to the local population
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Colorado&#39;s two-way star Travis Hunter has inked an exclusive partnership with trading card company Leaf
He&#39;ll be featured in his own limited edition anime-style trading card available for purchase
They are print to order through January 22 and will be numbered to the final print run
offering collectors an exciting and enjoyable way to collect cards featuring their favorite athletes,&#34; CJ Breen
Leaf&#39;s Director Of Marketing and Licensing
&#34;The recent launch of Jalen Milroe and Travis Hunter editions reflects Leaf&#39;s ability to tap into the vast NIL fanbase within the evolving landscape of Name
Hunter is currently ranked seventh in On3.com&#39;s NIL Top 100
Hunter was a dominant force for the Buffaloes on both sides of the ball last season
three interceptions and five pass breakups
Hunter became the school&#39;s first consensus All-American since Nate Solder in 2010
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Accor is doubling its portfolio in Japan by opening 22 hotels and over 6,000 rooms
The additions will mean it has 46 properties in the country
range in location from the northernmost prefecture of Hokkaido to the southernmost prefecture of Okinawa and are located close to local&nbsp; gems such as Cape Zanpa in Okinawa
offers an immersive experience that celebrates national culture and stimulates the senses
Grand Mercure makes its debut in Japan offering &ldquo;Proudly Local&rdquo; premium hotels
Grand Mercure Nasu Highlands Resort &amp; Spa
Grand Mercure Yatsugatake Resort &amp; Spa
Grand Mercure Lake Hamana Resort &amp; Spa
Grand Mercure Wakayama Minabe Resort &amp; Spa
Grand Mercure Awaji Island Resort &amp; Spa
Mercure Nagano Matsushiro Resort &amp; Spa
Mercure Wakayama Kushimoto Resort &amp; Spa
where outstanding chefs use local ingredients to create exquisite dishes and showcase Wakayama&rsquo;s rich culinary traditions
&ldquo;As Japan continues to welcome back international travel and experiences a steady recovery of the tourism industry
we are excited to add these great hotels to our portfolio and in doing so we are doubling our group&rsquo;s presence in Japan
As the desire to connect with local culture and nature is increasingly in demand amongst travellers today
we offer our loyal guests just that; a range of local experiences offered in charming coastal towns and amongst nature
Osaka and Kyoto will always remain in high demand
however if you want to truly immerse yourself in the authentic culture of a country
sometimes you need to escape the big cities and head off the beaten path,&rdquo; said Garth Simmons
midscale and economy division for Accor in Asia
under eight brands: MGallery; Pullman; Swiss&ocirc;tel; Mercure; Novotel; ibis; ibis Styles and ibis budget
n addition to the 22 Grand Mercure and Mercure hotels joining Accor&rsquo;s network on 1 April 2024
there are two more properties currently in the group&rsquo;s pipeline
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