This month’s Art Crawl highlights include a group show at Zeitgeist and a farewell to Josh Black  I’m hoping we’re already through the frozen days of deepest winter after last week’s cold snap because it feels like Nashville’s winter art scene is fully warmed up Now we’re all just preparing for the great and powerful seasonal rite when a clairvoyant rodent from rural Pennsylvania will cast his shadow-working divinations peering — Doctor Strange-like — into some future spacetime where/when spring will again kiss our amber waves and warm our purple mountains I’m predicting cozy crawling this Saturday night featuring some outstanding holdovers from January alongside several debuts These scenes are packed with recognizable trees but also more abstract spaces where viewers might discern other forms and figures Wilson’s canvases are more abstract overall but not entirely unrecognizable as landscapes animal forms and figures in obscure narratives Wilson is emblematic of contemporary Black formalists whose abstract styles are unburdened by activist slogans and homages to Jean-Michel Basquiat His work bucks gallery market trends that commodify readily recognizably Black art while also upsetting cultural expectations about the kind of work Black artists are “supposed to make.” Wilson’s paintings are timely and sharp aggressive marks and bundles of vibrant color but they’re there alongside mountain shapes under cloud blobs at implied horizon lines If you missed Jodi Hays’ Befores and Afters exhibition when it opened last month be sure to hit this reception on Saturday night Over the decades that I’ve followed Hays’ practice it’s evolved from more traditional painting into 2D works boasting multitudes of reused materials stepping well into collage but stopping short of assemblage on the painting-to-sculpture pipeline this show actually includes sculptural installations in the center of the gallery where Hays displays an arrangement of plants on stands along with a painting displayed lying flat on the ground (“Self-Portrait at 48”) I love the irreverence of installing a painting on the floor in a gallery full of people It’s a great lesson in how perspective can change perception — looming over a painting is a totally different experience from mooning at an illuminated work of art hanging on a wall from several feet away paintings on the floor break that sublime-sacred-space-spell that connects churchgoing and gallerygoing I’ve only seen crucifixes on floors in horror films Hays’ pieced-together installation isn’t meant to shock but it’s literally and figuratively grounding — these are humble works and they’re perfectly at home on the floor Single Mothers” is the show’s signature image It shouts out its titular vocational categories with colored text scrawled across a fractured kaleidoscopic surface of colored paper squares and diamonds As contemporary art trends toward the formal — that is away from content and messaging — Hays’ title alone might have sufficed here but spotlighting everyday people jibes with this show’s nothing-fancy vibes The text is also written in loose script using colors that tend to blend into their backgrounds It compels viewers to stop and really stare at the work as they try to make out what Hays has written Zeitgeist has always represented some of the best artists in the city but I’d argue it’s the gallery’s commitment to connecting with and nurturing the careers of emerging artists that’s kept it so relevant for so long Zeitgeist celebrated its 30th birthday in 2024 and its February First Saturday opening features a guest curator and a group show with a great lineup Rooted Chronicles is the dreamchild of local artist-curator Marteja Bailey Bailey had work hanging in Zeitgeist’s Rise Above exhibition during December and January and for February she’s assembled a large cast of Black artists working in various styles and mediums spotlighting new creators and established artists during Black History Month The show includes contributions from Alexis Jones stop by Julia Martin Gallery to see comedian Josh Black’s farewell to Nashville art exhibition The local funnyman has announced plans to move to Chicago in 2025 but not before one last hurrah in Wedgewood-Houston Email notifications are only sent once a day Your browser is out of date and potentially vulnerable to security risks.We recommend switching to one of the following browsers: , Strict regulations restrict how producers can use nitrogen fertilizer depending on the form it takes when applied Trucks show up at Han Langeler’s dairy farm in the Netherlands each spring and haul some of the cow manure up to 150 kilometres away it is spread on fields in an area with fewer livestock farms than Langeler’s area near Aalten Langeler then buys nitrogen fertilizer to top up the manure he was allowed to spread Trucking manure is part of the strange nitrogen mathematics used in Europe as it attempts to balance nutrient use and penalizes manure based only on its nitrogen effect soil organic matter and biological benefits Grain and oilseed millers are predicted to see sales volumes and margins decline in 2025 according to Farm Credit Canada’s food and beverage report The regulations led to farmer protests across Europe this winter and spring leading up to the recent European-wide elections Langeler spends about 10,000 euros (C$15,000) per year to send his manure north to an area with more crops and clay soil when the Netherlands decreases the amount of nitrogen allowed to be spread to 170 kilograms per hectare Langeler’s bill will increase to nearly 30,000 euros ($44,400) His farm can now spread 230 kilograms per hectare of nitrogen Farmers on clay soils can spread about 20 kg more per hectare “We found we have a lot of problems among farmers with it We said it is a little bit stupid because you must fertilize,” says Langeler during a visit to his farm on the dairy farm started by her grandmother They milk about 130 cows with two robotic milkers There are reasons the Netherlands is concerned about nitrogen The country is on the North Sea coast and has thousands of man-made and natural waterways The Netherlands has about 1.6 million dairy cattle but they are housed in an area about one-third the size of southern Ontario in the short distance between the house and the barn and points to several other dairy farms within a kilometre The farms are most often generational and started on small plots of land and their main crops are grown two kilometres away from the barn but that’s still not enough land to satisfy regulations that limit nitrogen The government is also pushing greater use of grass versus corn The Netherlands exports 60 to 70 per cent of its milk which has prompted the suggestion that it should aim for self-sufficiency That would require a significant reduction in dairy farmers and processors Most of the guidance on limits to nitrogen application comes from metrics set by European Union bureaucrats That’s resulted in what’s called “derogation” cracking down on areas where nitrogen is believed to be too high Both countries have growing dairy production When milk quota was phased out in 2015 in the Netherlands farmers took steps to secure their business futures by expanding and becoming more productive the government is using environmental regulations instead of quotas to control milk production Langeler says EU officials claim chemical nitrogen fertilizer is more available to plants and has less pollution potential than manure There isn’t strong research to support that stance and he says research is underway to provide better data on nutrient availability by nitrogen source Concern about the far-reaching implications of government regulations has resulted in protests by farmers in the EU It has become more than just a popular movement The Farmer-Citizen Movement party (known as BoerBurgerBeweging or the BBB) won the most seats in the Dutch Senate in March the BBB was one of four parties that formed a coalition government Langeler says it’s estimated that 10 to 20 per cent of a Dutch farmer’s time is spent on paperwork and regulation compliance he pointed to numerous management practices determined not by efficiency but by environmental or societal edicts on animal care Dutch farmers have had to inject their manure but a few hundred metres from Langeler’s farm and across the German border manure injection isn’t a requirement The demand doesn’t come just from government Processing companies have their own requirements adding to the complex web of sometimes contradictory subsidies and regulations Langeler’s farm qualifies as a low-carbon output farm mostly because it doesn’t use imported soybeans for feed Grass has to be about 80 per cent of the ration There’s concern that as fewer dairies graze their cows The low-carbon status gives Langeler a few cents more on his milk price to 0.48 euros (70 cents) There’s another two cents to be had by grazing cattle like keeping calves with their mothers for at least six months Significant pressure from the government will leave Langeler and his family with choices to make The government has a buyout plan that pays 25 per cent more than the market value of the farm the buildings would be levelled and the farmer would have to sign a legal agreement not to produce livestock in the future Newsletter Sign Up - Receive free Western Producer newsletters Breaking ag news stories and commodities markets snapshots delivered daily right to your inbox Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" John Greig is a senior editor with Glacier FarmMedia with responsibility for Technology Cattle markets hold value amidst uncertainty Drought preparation is better than reaction Producers must take foot-and-mouth disease seriously Farmer incorporates cattle into grain operation to boost soil health Terms and Conditions | Privacy Policy | © 2025 Western Producer Publications Limited Partnership "(Required)" indicates required fields Δdocument.getElementById( "ak_js_2" ).setAttribute( "value" This link leads to an external page in a new window a connection with Google is established and your personal data is forwarded to Google Conservation and preservation of historical monuments Project: City of the Future - Breathing & Moving Bocholt Economic Development and City Marketing Company The municipality of Aalten will become a border municipality for the first time on Monday 1 July 2024 This means that Dutch nationals living abroad will also be able to go to the municipality of Aalten to apply for or renew their passport or identity card from this date this was only possible in 11 other Dutch border municipalities and at Schiphol Airport Aalten's mayor Anton Stapelkamp is enthusiastic about this expansion of municipal services: "The municipality of Aalten is a border municipality and the relationship with our German neighbours is important to us Our police and fire brigade work closely together we have had a bus connection between Bocholt and Aalten again for several years (line C11) and it was possible to vote across the border in the European elections And now we are pleased to be able to offer Dutch citizens living in North Rhine-Westphalia the opportunity to apply for a passport or identity card at our municipal office at Hofstraat 8 in Aalten Around 10,000 people with Dutch citizenship live in the neighbouring district of Borken alone We therefore expect to be able to welcome many of them to the cosy centre of Aalten from 8 July." Aalten City Council works by appointment. You can easily make an appointment at www.aalten.nl or by calling +31 (0)543 49 33 33 visitors receive a bag with tips and tourist information about Aalten When they return to Aalten a few days later to collect their document they can combine this with a nice day trip to the German-Dutch border area The centre of Aalten has a lot to offer and the municipality would like to show this to its visitors The neighbouring German municipality of Bocholt is also just a "stone's throw" or a short bike ride away Further information can be found on the Alleten municipal website at www.aalten.nl. Stadtverwaltung BocholtKaiser-Wilhelm-Str. 52-5846395 BocholtTel.  +49 2871 953-0Fax. +49 2871 953-9530stadtverwaltung(at)bocholt(dot)de The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. SPIE is the independent European leader in multi-technical services in the areas of energy and communications Our 55,000 employees are committed to achieving the energy transition and responsible digital transformation alongside our customers SPIE aims to contribute to a more sustainable Do you want to use your skills to contribute to projects that help society a key player in the energy transition and digital transformation We are committed to the fight against climate change and mobilised for a responsible digital transformation intall and maintain energy-efficient and environmentaly-friendly facilities Find in this section all the useful information you can access SPIE's profile and key figures Would you like to interview an SPIE spokesperson 1 October 2024 - SPIE Nederland, the Dutch subsidiary of SPIE the independent European leader in multi-technical services in the areas of energy and communications, will integrate the Dutch company Achterhoek Antennebouw Aalten B.V. which is the Dutch part of the mainly German-based ICG Group was acquired by SPIE in April 2024 and will be transferred to SPIE Nederland on 1 October.  TripleA has extensive experience in the telecom services sector The company operates as a full-service provider for the major telecom operators in the Dutch market and also serves customers in Belgium and Germany By bringing the Aalten firm under the umbrella of SPIE Nederland they can form one SPIE company in the Netherlands; with a single face to the market and to their (future) clients sees many opportunities: “With the transfer we become a strong organisation with national coverage through the location in the east of the country it offers many opportunities for the entire SPIE organisation.” is also positive: “It is nice to have a strong organisation behind us and to serve the market together We look forward to getting to know the SPIE organisation in the Netherlands better.” Metrics details Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population In total 5−10% of ID cases are due to variants in genes located on the X chromosome variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families an essential enzyme that catalyses O-linked glycosylation with β-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins we compile the work from the last few years that clearly delineates a new syndromic form of ID which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG) We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions Model for the full-length human OGT produced by superposition of crystallographic models for the human OGT catalytic core (Protein Data Bank code 5C1D) and TPR domain (Protein Data Bank code 1W3B) and purple highlighted residues represent variants found in OGT-XLID patients The female twins have a full lower lip and twin 2 has a wide mouth OGT catalyses the transfer of O-GlcNAc moiety onto Ser/Thr residues of acceptor substrates This modification is removed by the OGA enzyme Variants in the OGT gene which lead to amino acid substitutions in the OGT protein may mediate the XLID pathophysiology via (1) downstream effect on the O-GlcNAc proteome (3) incorrect processing of the HCF1 transcriptional coregulatory HCF1 which is also encoded by an XLID-associated gene (HCFC1) (4) misfolding of OGT possibly leading to misfolded OGT aggregation Brain anomalies were a commonly observed clinical observation among XLID patients with an aberrant OGT (Table 1) Three probands with different OGT variants present with microcephaly while megacisterna magna was found in two patients Additional anatomical brain abnormalities reported among OGT-XLID patients include thin corpus callosum In patients with detailed clinical description coarse facial features with drooling are often described and genital defects were observed among many of the patients carrying OGT-XLID variants Six patients were reported to have behavioural problems and two patients presented with sleep abnormalities some patients display visual and hearing impairment were observed in a high proportion of patients with OGT-XLID variants while these characteristics are not common in XLID syndromes Patients are negative for glycosylated transferrin test results excluding most N-linked congenital disorders of glycosylation (CDGs) and coarse facial features with high forehead and triangular face are shared among the majority of patients with OGT-XLID variants we propose that this constitutes a novel syndrome while not currently supported by existing findings this hypothesis for OGT variants’ catalytic activities being impaired for specific substrates cannot be formally excluded suggesting a possible link between OGT-XLID variants and perturbations of OGA levels Taken together, these results suggest that there may be an association between OGT-XLID variants and HCF1 processing. However, while c.1701=/T>A p.(Asn567Lys) appears to affect HCF1 processing, current data across all variants suggest that HCF1 misprocessing is unlikely to be the general mechanism by which OGT variants lead to the observed XLID phenotypes (Table 2) despite reductions in the thermal stability of OGT-XLID mutants and some structural evidence for misfolding there is no evidence yet of formation of toxic aggregates in cultured mammalian cells it is possible that TPR variants in OGT interrupt specific substrate and/or adaptor protein interactions The loss of interaction with adaptor proteins and/or glycosylation of the target substrate could lead to functional consequences for the substrate protein that have yet to be defined the presence of CD variants in OGT that lead to XLID with similar phenotypes complicate this hypothesis the OGT interactome in appropriate tissues and/or differentiated cell lines must first be defined Comparisons can then be made between the wild-type and XLID variant OGT interactomes to identify differential interactors and evaluate impact on a subset of adaptor proteins and their binding partners that could easily be overlooked via western blotting of whole cell extracts Defining the O-GlcNAcome and O-GlcNAc cycling rates will be essential to evaluate the role of catalytic OGT variants in this hypothesis This review has discussed a number of hypotheses, some of which have been partially tested, as to the biological mechanisms underpinning the patient phenotypes (Fig. 3) The hypotheses of decreased OGT activity as a glycosyltransferase or protease are not supported as a common mechanism by currently existing data on multiple variants The contribution of decreased OGT/OGA levels due to decreased OGT stability the possibility for aberrant interactions between OGT-CDG variants and target substrates and alterations in O-GlcNAc cycling rates are compelling hypotheses that still need to be explored new approaches to examining the OGT interactome and the dynamics of the O-GlcNAc modification must be developed Generation and characterisation of CRISPR/Cas9 engineered stem cells and model organisms will significantly contribute to the elucidating of the biological processes that underlie the OGT-CDG pathology First attempts have been made with the generation of human and mouse ES cells and a fly model of the c.1701=/T>A p.(Asn567Lys) variant although further work is required including vertebrate models of the disorder such disease models and in-depth the understanding of the disease mechanism they offer will be instrumental in devising a potential therapeutic intervention strategy suggesting this may be a possible future therapeutic approach A suitable diagnostic test has to be devised to detect OGT-XLID in patients A commonly used CDG diagnostic test is isoelectrofocusing of serum transferrin it detects only N-glycosylation disorders associated with sialic acid deficiency and thus not OGT-CDG Since patient-derived cells show reduced OGA we propose western blotting for OGA protein levels as a rapid initial diagnostic test for OGT-CDG MD: American Associacion on Intellectual and Developmental Disabilities; 2010 The relation between intellectual functioning and adaptive behavior in the diagnosis of intellectual disability The epidemiology of developmental disabilities in low-income countries The epidemiology of mental retardation: challenges and opportunities in the new millennium X linked mental retardation: a clinical guide Chelly J, Khelfaoui M, Francis F, et al. Genetics and pathophysiology of mental retardation. Eur J Hum Genet 2006;14:701–13. https://doi.org/10.1038/sj.ejhg.5201595 Vissers LELM, Gilissen C, Veltman JA. 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Crit Rev Biochem Mol Biol. 2014;49:140–63. http://www.ncbi.nlm.nih.gov/pubmed/24524620 Download references This work was funded by a Wellcome Trust Senior Research Fellowship (WT087590MA) to DMFvA These authors contributed equally: Veronica M Division of Gene Regulation and Expression and School of Life Sciences Division of Cell and Developmental Biology Department of Biochemistry and Molecular Biology and Chemistry Adelaide Medical School and the Robinson Research Institute Institute of Molecular Cell and System Biology The authors declare that they have no conflict of interest Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41431-020-0589-9 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Volume 12 - 2019 | https://doi.org/10.3389/fnmol.2019.00163 This article is part of the Research TopicLithium and NeurodegenerationView all 5 articles Lithium has been used for decades to treat Bipolar Disorder Some of its therapeutic benefits may be through inhibition of Glycogen Synthase Kinase (GSK)-3 Enhanced GSK3 activity associates with development of Alzheimer’s disease (AD) therefore lithium is a currently used therapeutic with potential to be repurposed for prevention of Dementia An important step toward a clinical trial for AD prevention using lithium is to establish the dose of lithium that blocks GSK3 in Mild Cognitive Impairment (MCI) a high-risk condition for progression to AD and assessed biomarkers of GSK3 in MCI compared to control and after lithium treatment Drop out was not related to lithium blood concentration 33% of the withdrawals were in the first week of very low dose lithium Most made it through to the highest dose of lithium with no adverse events We analyzed 18 potential biomarkers of GSK3 biology in rat PBMCs but only four of these gave a robust reproducible baseline signal The only biomarker that was modified by acute lithium injection in the rat was the inhibitory phosphorylation of Ser9 of GSK3beta (enhanced in PBMCs) and this associated with reduced activity of GSK3beta In contrast to the rat PBMC preparations the protein quality of the human PBMC preparations was extremely variable There was no difference between GSK3 biomarkers in MCI and control PBMC preparations and no significant effect of chronic lithium on the robust GSK3 biomarkers indicating that the dose reached may not be sufficient to modify these markers suggest that it would be relatively straightforward and safe to recruit to a larger clinical trial within this dosing regimen it is clear that we will need an improved PBMC isolation process along with more robust in order to use GSK3 pathway regulation in human PBMC preparations as a biomarker of GSK3 inhibitor efficacy The pharmacological treatment of Dementia is limited to the treatment of symptoms, primarily due to the ever increasing number of failed clinical trials (Hardy, 2009; Wang et al., 2016; Amanatkar et al., 2017), while the paucity of non-amyloid targeting drugs in development is highly concerning (Cummings et al., 2017) there is increasing awareness of the importance of identifying high risk groups and developing Dementia prevention strategies GSK3 in human dementia is related to abnormally high GSK3 activity in adults and no GSK3 inhibitor toxicology studies have been reported in an adult preclinical model with excessive GSK3 activity have the potential to prevent conversion to dementia It seems reasonable therefore to suggest that lithium is a clinically viable GSK3 inhibitor that is available for immediate use in trials to evaluate GSK3 inhibition as an anti-dementia therapeutic agent The therapeutic range of lithium contains the IC50 (50% inhibition constant) for direct GSK3 inhibition in vitro [approx. 0.5 mM (Davies et al., 2000)], suggesting that pharmacologically relevant GSK3 inhibition would be achieved by the doses used to treat Bipolar disease. However, lithium regulates GSK3 activity by several distinct mechanisms (Sutherland and Duthie, 2015) which may mean that inhibition of GSK3 in patients will occur with even lower levels of lithium than predicted from in vitro obtained IC50 values experimentally evaluating the lithium concentration required to inhibit GSK3 in relevant human populations becomes a valuable exercise prior to balancing safety concerns with therapeutic benefits measuring changes in GSK3 activity in humans is challenging this depends on antibody availability and protein detection in the tissues of interest Potential molecular biomarkers of GSK3 activity classified by the relationship to GSK3 function The current study aimed to assess and validate many of the potential biomarkers for GSK3 activity in Table 1 in rodent Peripheral Blood Mononuclear cells (PBMCs) We then took robust biomarkers from that study to generate pilot information on inhibition of GSK3 by lithium in people with MCI Importantly we also aimed to extend the evidence that recruiting and retaining this population to a trial which involved taking lithium one where we gave rats an acute dose of lithium and measured GSK3 biomarkers in PBMCs one (Arm 1) where we compared GSK3 biomarkers in PBMCs between volunteers with MCI and age-matched controls and one (Arm 2) where we performed a step-wise increasing lithium dosing experiment in MCI volunteers over 12 weeks All experiments were performed using male Wistar rats (Harlan Ltd. Animals were housed in cages of four (12:12 h light: dark cycle) at an ambient temperature of 22 ± 1°C and 50% humidity All animal procedures were approved by the University of Dundee Ethical Review Process and were performed in accordance with UK Home Office regulations (under the auspices of Project License PIL60/4280) Animals were assigned at random to one of three treatment groups (i) Control (Saline) (ii) Low dose lithium chloride (2 nmoles/kg i.p.) or (iii) High dose lithium chloride (4 nmoles/kg i.p.) 3 h post injection animals were sacrificed by cervical dislocation and blood samples collected by cardiac puncture for the preparation of PBMCs and plasma lithium analysis and cerebellum) and liver samples were isolated and snap frozen in liquid nitrogen for subsequent biochemical analysis One milliliter of whole rat blood was collected from each animal on Sodium Heparin Samples were spun and serum transferred to 1.5 ml Eppendorf tubes prior to running on an AVL 9180 Electrolyte Analyser alongside an appropriate lithium standard curve Peripheral Blood Mononuclear cells were collected from the rat blood samples immediately after collection using OptiPrep density gradient medium using the flotation technique as per manufacturer’s instructions Protein was harvested from the cell pellet in lysis buffer [25 mM Tris–HCl (pH 7.4) protease inhibitor cocktail tablet (Roche 11836170001) and 0.27M sucrose] and snap frozen in liquid N2 prior to storage at -80°C The study was approved by the North of Scotland Research Ethics committee Eleven volunteers were given low dose (100 mg daily) for 3 weeks then medium dose (200 mg daily) for 3 weeks Blood samples were taken for safety measures PBMC preparation and Lithium measure at baseline (visit 1) 1 and 3 weeks after starting each dose (visit 2–7) Participants over age 55 and with a diagnosis of MCI were recruited from Tayside Old Age Psychiatry and Geriatric Medicine outpatient clinics and by using Join Dementia Research (JDR) Database. Of 830 clinic contacts screened via consultant caseloads, 67 MCI patients were identified and invited by letter plus 4 contacts identified through existing local research lists and 3 via JDR. MCI was defined according to Petersen’s criteria (Petersen et al., 2001) Two brief standardized questionnaires [Bristol Activities of Daily Living Schedule and Geriatric Depression Scale Short Form (15 point version)] were administered to check diagnosis and existing cognitive assessments were documented from case file The control group was recruited via two routes firstly through Join Dementia Research and secondly by approaching spouses/partners/relatives of similar age to the MCI group but who lack an MCI diagnosis Exclusions for the Study Arm 1 group were as follows; Dementia Rheumatoid Arthritis and Autoimmune Inflammatory disorders and for Study Arm 2 the following additional exclusion criteria were applied: contraindication to Lithium Carbonate (Priadel) according to Summary of Product Characteristics Moderate renal failure (defined by eGFR < 60 mL/min/1.73 m2) or severe Renal failure (defined by eGFR < 29 mL/min/1.73 m2) Screening included baseline blood tests for renal function and thyroid function Volunteer blood (13–18 ml) was collected in Na-heparin containing vacuettes kept at room temperature and processed within 6 h of collection In sterile test tubes 3.5 ml of blood was carefully layered on top of 3 ml Mono-poly resolving media 800 ×g in a swinging bucket rotor at room temperature without brake (Beckman Coulter Allegra X-12 centrifuge) The plasma layer at the top of the tube was then removed and discarded using a Pasteur’s pipette and the top layer of cells containing the mononuclear leucocytes was collected and combined in a clean test tube The cell suspension was washed with three volumes of PBS and centrifuged 10 min The cell pellet was washed in PBS and centrifuged again An equal volume of PBS was added to the pellet the cells were resuspended gently and the cell mixture transferred to a cryovial and snap frozen in liquid N2 prior to storage at -80°C The PBMC pellets were lysed on ice in lysis buffer Following centrifugation to remove insoluble material supernatants were collected and protein concentrations were determined using the Bradford method Lysates were made up to equal concentrations with lithium dodecyl sulfate sample buffer added and subjected to SDS-polyacrylamide gel electrophoresis on 4–12% NuPAGE polyacrylamide gels (Novex then either stained using Coomassie Blue (SimplyBlueTMSafe Stain incubated with primary antibodies overnight at 4°C and then Alexa-fluor secondary antibodies for 1 h at room temperature Proteins were visualized using the Licor Odyssey detection achieved with ECL and proteins visualized on film using different exposure times Protein bands were quantified using the Image Studio software (Licor) Odyssey images were quantified directly while ECL images on film were scanned using a desktop scanner and imported into the software The background was determined around each box individually Normalization was done by generating a ratio against a loading control (usually actin) or for Phosphorylation sites a ratio against the total protein signal of the protein of study where antibodies were available The differences between groups were determined by using a Student t test Analyses were performed using Excel (Microsoft Corp.) A total of 200 μl of (50:50) Protein A agarose beads in PBS were conjugated to 20 μg of either anti-GSK3β or anti-GSK3α by incubating for 1 h at room temperature with shaking Unbound antibody was removed by washing with lysis buffer and 5 μl of the (50:50) anti-GSK3β-beads incubated with 100 μl of 1 μg/μl cell lysate overnight at 4°C with shaking GSK3β immunocomplexes were isolated by centrifugation at 3000 ×g for 5 min Supernatant was incubated with 5 μl of (50:50) anti-GSK3α-beads for 4 h at 4°C prior to isolation of the GSK3α immunocomplexes by centrifugation at 3000 ×g for 5 min All immunocomplexes were washed with 1 ml of lysis buffer followed by 1 ml of kinase assay buffer (25 mM TRIS 5% glycerol) containing 0.5 M NaCl and then 1 ml of kinase assay buffer before resuspending to 10 μl in assay buffer Each washed immunocomplex was incubated with PGS2 peptide (10 μM final) 0.5 × 106 CPM/nmole) in assay buffer in a final volume of 50 μl at 30°C for 20 min The assay was stopped by spotting the assay mix onto P81 phosphocellulose paper and placing in 10 mM Phosphoric Acid (PA) Papers were washed 4× 5 min in PA then once in acetone and air dried Phosphate incorporation into the GSK3 substrate was quantified by counting the paper in a scintillation counter One unit of activity of each protein kinase was calculated as the amount required to transfer 1 nmole of phosphate/min The recruitment strategy is provided in Figure 2 Study Arm 1 contained six MCI and eleven controls (all providing two blood samples and Study Arm 2 recruited eleven MCI volunteers for lithium treatment (eight blood samples across 12 weeks) The number of participants excluded due to BMI above 30 was greater than anticipated from this age group and was the most common reason for exclusion Consort diagram detailing the recruitment process for the clinical studies Plasma lithium level (mmoles/l) during stepwise increase in lithium dose in volunteers There were no serious adverse events reported Six participants experienced side effects (54%) with two of these continuing with the lithium titration phase to completion Side effects reported at either moderate or severe intensity included: fatigue Feeling drowsy or lethargic during the day was the most commonly reported side effect with five of the six participants reporting this in either mild Only one individual of the eleven given lithium exhibited a change in renal function (mild reduction in eGFR to 57 mls/min with accompanying mild increase in creatinine) Examination of the case files revealed that while normal at trial initiation this individual had previously had similar mild impairment of renal function on blood testing There were no instances of electrolyte disturbance or other renal problems during the trial Our initial study was to investigate whether the GSK3 biomarkers in Table 1 could be detected in a PBMC preparation from rats and whether detectable markers respond to lithium administration we wanted to try to investigate whether any lithium induced biomarker responses in the PBMCs associated with changes in the brain panel of GSK3 biomarkers as a readout for GSK3 function in humans to investigate early dementia and for establishing efficacy of lithium dose the major GSK3 regulatory pathways and the best characterized GSK3 substrates to date all proved difficult to detect robustly in PBMCs by immunoblotting Biomarkers of GSK3 in rat tissues following lithium injection injection of vehicle or lithium chloride as detailed in section “Materials and Methods,” and tissues isolated 3 h later Representative examples of Western blots of rat protein lysates isolated from (A) rat PBMCs we also provide a comparison of vehicle treated rat PBMC lysate with rat brain cortex lysate with every antibody as evidence for the utility of each antibody (antibody control) The ratio of expression of GSK3 phosphorylation at the N-terminal inhibitory site to the overall isoform expression was quantified from the images as detailed in section “Materials and Methods,” and is provided for each tissue (±SD) in the table inserted into A The red box highlights the major biomarker changes in response to lithium although only the P-GSK3β-total GSK3β ratio was significantly increased by lithium (p < 0.05 GSK3 specific activity (measured by IP assay) in rat tissue lysates Expression of potential biomarkers of GSK3 function in rat tissues This may mean the brain does not get exposed to the same level of lithium as the blood or the time point we examined was not optimal for effects on the brain Rat plasma lithium levels achieved after a single i.p The next step in our study examined the GSK3 biomarkers In Study Arm 1 we wanted: (1) to establish that the GSK3 activity biomarkers detectable in rat PBMCs could also be detected in cells isolated from the human population of greatest interest (i.e. (2) to investigate the reproducibility of marker detection in two PBMC preparations generated from the same individual but taken 12 weeks apart (3) to ask whether there were major differences in any of these markers that associated with MCI diagnosis We recruited six patients with MCI (mean age 78 we had baseline samples from the 11 MCI volunteers recruited to Arm 2 below (making n = 17 for the MCI group) there was not a significant difference in the c-myc:actin ratio between controls and MCI (0.143 ± 0.1 controls This protein is degraded upon phosphorylation by GSK3 hence the data would suggest no significant increase in GSK3 activity in MCI PBMCs Comparison of Biomarkers of GSK3 in human PBMCs isolated from volunteers with MCI and controls (Study Arm 1) Protein lysates were prepared from PBMCs isolated from two groups of human volunteers In each case the volunteer provided two blood samples for PBMC preparation (A) Representative Coomassie protein staining of PBMC lysates from 8 of the 17 volunteers in Study Arm 1 where equal total protein was loaded on the gel (B) Representative immunoblots of biomarkers within the PBMCs of four of the volunteers in Study arm 1 (C) Immunoprecipitation and assay of GSK3 isoforms from 50 μg of protein lysate in each of the PBMC samples The specific activity was quantified for GSK3alpha and GSK3beta from all PBMC preparations and the average activity (±SE) for the MCI and control groups at each visit and this implies that 0.33–0.61 mM plasma lithium is not sufficient to regulate the inhibitory phosphorylation of GSK3β in human PBMCs These data emphasize the need to improve the standard PBMC isolation process and increase volunteer numbers to utilize PBMC preparations for monitoring GSK3 activity GSK3 inhibition is considered to have potential therapeutic value in all of these conditions easily attainable markers of GSK3 may have clinical utility in diagnosis prognosis and response to therapy (such as response to lithium) very few of them were detectable (by immunoblot) in PBMCs in rats or humans the quality and reproducibility of the signaling proteins (especially phosphoproteins) isolated from human PBMCs using standard protocols was disappointingly poor our study in the rats indicated that there may be a complex non-linear relationship between plasma lithium level GSK3 regulation in PBMCs and GSK3 regulation in brain and it may be sensible to move to quantitative proteomic technology rather than relying on immunoblotting We would also propose that improved protocols to isolate human PBMCs (or other blood cells) are required to permit quantification of metabolic signaling pathways Molecules such as GSK3 are regulated by a wide variety of extracellular cues This may include some of the chemicals involved in PBMC isolation or volunteer specific characteristics such as hormonal or diurnal cycles and dietary or stress influences on the day of blood collection the high degree of variation in our human PBMC preparations compared to the rat preparations would point to these latter influences as worthy of consideration Previously, increased GSK3 protein levels were found in white blood cells in AD and MCI patients (Hye et al., 2005), while reduced GSK3 phosphorylation was found in platelets of MCI patients (Forlenza et al., 2011b) We were not able to confirm these important findings possibly due to the poor quality of our PBMC protein preparations It is also worth noting that the relationship between plasma lithium and GSK3β phosphorylation and activity in rat PBMCs was unusual There was a similar effect on GSK3 inhibition at 0.83 and 1.6 mM plasma lithium This may mean that the maximal dose dependent effect on GSK3β phosphorylation was below 0.83 mM the degree of effect on GSK3 was not major (<twofold enhancement of phosphorylation and around 15% inhibition) This may be related to the fact we examined a single time point or the nature of this biomarker (phosphorylation rather than direct measure of GSK3 activity) if GSK3β was 50% phosphorylated in control animals then the maximum possible effect of lithium on phosphorylation would be a twofold increase the IP assay is perhaps not the ideal biomarker for effects of small molecules as lithium would be washed away during the IP process but it should still assess activity changes due to the inhibitory phosphorylation This is why we believe that the accurate assessment of the pharmacological relationship between plasma lithium and GSK3 regulation would be best achieved with biomarkers that directly monitor GSK3 specific targets rather than simply the phosphorylation of GSK3 It was concerning that we did not detect any effects of lithium on any GSK3 biomarkers in the brain of the rats despite achieving relatively high plasma levels of 1.6 mM lithium (albeit acutely). In this case we were monitoring several markers that we know are modified by GSK3 inhibition in cell culture (Cole and Sutherland, 2008), and in animal models of GSK3 deficiency (Soutar et al., 2010) This implies that this acute level of plasma lithium is not sufficient to regulate GSK3 in these brain areas it is possible that either the injection resulted in a very transient increase in plasma lithium that is not sufficient to alter these biomarkers or that the time point after injection was not optimal for effects on brain biomarkers In essence a much more extensive study is needed to truly establish the lithium dose required to modify GSK3 activity in these brain areas We should also keep in mind that lithium is a low-cost drug and even continuous monitoring for the known side effects is relatively cheap we provide further evidence that preforming an efficacy trial on the effect of lithium on progression from MCI to dementia is not only feasible but worthwhile Within that study one could expand the investigation of GSK3 biomarkers from a simple readout for diagnosis to investigate any association with conversion from MCI to dementia As such we are actively working to address the technical issues listed above The study was approved by the North of Scotland Research Ethics Committee AD was the Principle Clinical Investigator in Dundee and contributed to writing of the manuscript SL was the Principle Clinical Investigator in Oxford and JG were responsible for volunteer recruitment and volunteer safety monitoring as well as contributing to study design and JGall were responsible for all rodent and lab work and contributed to the writing of the manuscript This work was supported by the Chief Scientist Office 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 We are extremely grateful to all volunteers who gave up their time to participate in the study in particular to those volunteers who undertook the lithium dose study The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2019.00163/full#supplementary-material Analysis of recent failures of disease modifying therapies in Alzheimer’s disease suggesting a new methodology for future studies Inhibition of glycogen synthase kinase-3 ameliorates beta-amyloid pathology and restores lysosomal acidification and mammalian target of rapamycin activity in the Alzheimer disease mouse model: in vivo and in vitro studies Regulation of Th1 cells and experimental autoimmune encephalomyelitis by glycogen synthase kinase-3 Molecular actions and clinical pharmacogenetics of lithium therapy Lithium promotes longevity through GSK3/NRF2-dependent hormesis Lithium ameliorates open-field and elevated plus maze behaviors and brain phospho-glycogen synthase kinase 3-beta expression in fragile X syndrome model mice GSK3 inhibitors: 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perspectives and spatial learning deficits in mice with traumatic brain injury Long-term treatment with lithium alleviates memory deficits and reduces amyloid-beta production in an aged Alzheimer’s disease transgenic mouse model Lovestone S and Sutherland C (2019) Recruitment and Biomarkers of Response; A Pilot Trial of Lithium in Humans With Mild Cognitive Impairment Copyright © 2019 Duthie, van Aalten, MacDonald, McNeilly, Gallagher, Geddes, Lovestone and Sutherland. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Calum Sutherland, Yy5kLnN1dGhlcmxhbmRAZHVuZGVlLmFjLnVr †Present address: Simon Lovestone, Janssen-Cilag, High Wycombe, United Kingdom Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish. 2010 12:00 AM EDTTo a score of marching drums and pipes we see the expedition trudge across a snowy expanse and up the mountain the sun setting over the Anatolian hinterland below we go inside a dark cave and watch members of the expedition inspect what appears to be a solid wooden wall entombed within layers of glacial ice and volcanic rock A gnarled beam runs suspended from one part of the cavern to another There’s straw and bits of old rope on the ground; a structure is taking shape This is the footage of the alleged discovery of the biblical vessel perched more than 12,000 ft (4,000 m) high on Mount Ararat in eastern Turkey that was first shown to journalists on April 25 at a press conference in a fancy boutique hotel in Hong Kong composed largely of Hong Kong–based Evangelicals an art historian and a handful of Turkish academics and government officials They displayed specimens of objects recovered from the supposed ark which they say they encountered in seven dismembered compartments within the mountain: on show are pieces of petrified wood allegedly carbon-dated at 4,800 years old a chunk of crystal and a cluster of seed-like pellets “There is a tremendous amount of evidence that this structure is the ark of Noah,” said Gerrit Aalten a Dutch researcher of ark lore who was enlisted to evaluate the team’s findings (See the top 10 religious relics.) Reported sightings of the ark are almost as old as the biblical story itself God set about annihilating a “corrupt” and “violent” world once Noah built a wooden vessel to hold his family and two of “all living creatures from all flesh.” As the floodwaters abated the barque came to rest by “the mountains of Ararat.” Since then medieval travelers and Ottoman soldiers have all supposedly spotted this mythical ship amid the region’s peaks So too have a host of Christian explorers — most recently in 2006 when a Colorado-based Evangelical and amateur archaeologist claimed to have uncovered the ark petrified within Iran’s Alborz mountain range along the Turkish border geologists declared it to be just an oddly shaped variety of rock (See R. Crumb’s version of the Book of Genesis.) The Hong Kong team says it has been far more thorough heralded at the press conference as a “99.9%” success they have verified their findings with the insight of a few Turkish archaeologists and geologists edited video of their trek and discovery to the public Yeung says they have shown full footage of the ascent to colleagues in the cult field of Arkeology (They justify their secrecy by saying it’s for the integrity of the site which the local government in the area now intends to prepare as a tourist destination.) (See people finding God on YouTube.) The team has conducted missions to Mount Ararat since 2003 under the auspices of a Hong Kong–based organization dubbed Noah’s Ark Ministries International which is in turn linked with another well-funded Christian group in the city called Media Evangelism The latter drew attention in 2008 when it helped set up a park that now houses a life-size replica of the ark accompanied by models of animals on board as well as a vivid film depicting God’s wrath at a sinful mankind and the flood he sent to wash it away Yeung talks of the ark’s discovery in almost apocalyptic terms: “At this day it has a very special meaning when we see so many natural disasters and earthquakes,” he tells TIME “People should come and see the ark and think about their place in the world.” a prominent biblical archaeologist at George Washington University and author of the best-selling From Eden to Exile: Unraveling Mysteries of the Bible questions why this group made up mostly of amateurs in the field chose to announce their findings at a press conference rather than have them peer-reviewed and then published in a scholarly journal as is standard archaeological and scientific practice “You see these sorts of claims almost every other year,” he says “When people of faith go out looking for things it seems they almost always find them.” (See the top 10 religion stories of 2009.) Archaeologists on blogs and forums have suggested the structure up on Mount Ararat may well just be a hut or some other form of rudimentary shelter Cline also wonders why the ark over time would have been left intact at all “that a portion of the vessel still survives … on the mountains … and that persons carry off pieces of [it] Yeung and his colleagues are pressing ahead hoping to gain the support of UNESCO and spend the next few years deepening their analysis of the site Cline says this sort of work strays from the real purpose of biblical archaeology which is to bring to light the greater social realities of that ancient time rather than prove the truth of Christian doctrine with quests for biblical totems (Are the Bible’s stories true? The evidence of archaeology.) It also misses a larger point about the history of the myth The flood has echoes in legends from Central America to South Asia and it almost certainly predates Judeo-Christian times Scholars believe it was most likely transmitted to the Israelites from Mesopotamia: in the far older Epic of Gilgamesh a man chosen by the gods to live alone in a boat full of animals while the world around him ended in a deluge as the rains stopped he sent out both a dove and a raven to gauge whether the waters had receded “That’s why I tell my students,” says Cline “that if I am going to look for an ark Maybe it would be Utnapishtim’s.” See the top 10 Jesus films of all time. See 10 surprising facts about the world’s oldest Bible. Contact us at letters@time.com Moonachie-based women's clothing retailer Joyce Leslie received bankruptcy court approval Tuesday to conduct an expedited auction of its stores early next week which filed for bankruptcy earlier this month will either result in a buyer who wants to operate the company as a going concern A liquidation group led by SB Capital has submitted a stalking horse bid for the right to liquidate the company's stores the North Bergen-based women's clothing chain Madrag/10spot has made a proposal to buy up to 20 leases Joyce Leslie asked to defer a motion on that sale for one day and asked the court to consider the auction request as a separate motion Related:   Moonachie-based Joyce Leslie files for bankruptcy Nash said Tuesday in U.S. Bankruptcy Court in White Plains that Joyce Leslie executives still are hopeful that a buyer will be found who can keep the stores open and preserve jobs for employees. The company has 730 employees and 47 stores, including five in Bergen and Passaic counties. The 69-year-old company caters to women aged 15 to 35 living in urban areas. If a buyer doesn't surface, Joyce Leslie will present its going-out-of-business sale and liquidation plan to the court on Feb. 3, the day after the auction. Nash said. Seth Van Aalten, the attorney for the creditors committee, said it is in the interest of Joyce Leslie's creditors for any auction, sale or liquidation to be expedited. Van Aalten noted that Joyce Leslie is burning cash, saying the company has been called "a melting ice cube and it's half-melted." Every day the stores remain open is costing the company "in the six figures," he said, without being specific. U.S. Bankruptcy Court Judge Robert Drain agreed that shortening the amount of notice required for an auction is warranted. Documents filed in U.S. Bankruptcy Court in the Southern District of New York reported assets of $7 million and liabilities of $9 million. Joyce Leslie is just the latest company to struggle in the rapidly changing apparel market. Weakening business at malls, the growth in e-commerce and the rise of "fast fashion" vendors such as Forever 21, Spain's Zara and Sweden's H&M have all been contributing factors in the decline of the company. UK police charge man with terrorism after arrest at Leeds hospital UK police arrest man over 2021 deaths of 27 migrants Greg Laurie: Disney’s ‘Snow White’ Remake Abandons Original Themes of Love and Redemption Pastor Greg Laurie, founder of the renowned Harvest Christian Fellowship, recently criticized Disney’s live-action remake of Snow White, saying it strays far from the gospel-inspired message of love and redemption found in the original. Netflix has come under fire from Christian audiences after viewers noticed that a significant Bible verse—Isaiah 53:5—appears to be missing from the opening scene of The Passion of the Christ on the platform. Liverpool FC forward Cody Gakpo is set to receive a formal warning from the Football Association (FA) after displaying a Christian message during a recent Premier League match against Tottenham Hotspur. Following a goal, Gakpo lifted his jersey to reveal an undershirt bearing the message “I Belong To Jesus,” raising both hands toward the sky in a clear act of religious expression. The King of Kings, the latest animated depiction of the life of Jesus Christ, is receiving strong reviews from major critics for its creative framing and compelling voice acting. In a development that has stirred both public interest and internal division, the elders of Fellowship Church in Huntersville, North Carolina, have publicly rejected the findings of a months-long investigation that cleared Dr. Michael Brown of any legal or ethical misconduct. Dutch city Aalten name climb after home cyclist Dutch city Aalten officially re-named a climb Saturday after its cyclist Robert Gesink (Rabobank). "It's been permanently named after me, which is obviously very nice," Gesink told De Gelderlander. The short climb "Robert Gesinkbult" is on the N318 ring road, which passes the north side of Aalten. City officials posted a road sign and unveiled it Saturday. Rabobank director sportif Harold Knebel and ex-professional Tristan Hoffman attended the ceremony. Gesink, 23, finished his best season since turning professional in 2007. He won the Giro dell'Emilia and finished seventh at País-Vasco, third at Amstel Gold, fourth at Dauphiné Libéré and sixth at Vuelta a España.  The latest race content, interviews, features, reviews and expert buying guides, direct to your inbox! The number of infections with the bluetongue virus has risen again, but the increase is becoming slower. The virus has been detected in 1722 locations in the country, reports the Netherlands Food and Consumer Product Safety Authority (NVWA). Previously, the number of cases doubled per week, and sometimes the increase was even faster. The NVWA reported 94 cases On Monday, 15 July; a week later, it was 503, and last Monday, the counter was at 1094 cases. Most infections are still in the Achterhoek. For example, 60 cases have been detected in the municipality of Berkelland, and the same number has been detected in Oude IJsselstreek. This is followed by the municipalities of Bronckhorst (46) and Aalten (44). Overijssel is the only province where every municipality has detected the virus. The east of North Brabant and the north of Limburg also have many infections. Bluetongue has now also been detected in Flevoland, which was the only province without infections until last week. The virus has hardly been detected in North Holland, South Holland, and Zeeland. Bluetongue is a disease that mainly affects sheep, goats, cattle, and deer. Other animals can also become infected, such as camels, llamas, giraffes, and okapis, but it is unknown whether this has happened. The virus causes, among other things, a blue tongue, high fever, and swelling in cattle. They can die as a result. Infected midges, the tiny biting flies spread bluetongue. The virus is not contagious to humans. Many animals were vaccinated against bluetongue in the spring. According to an initial analysis by the Royal GD Health Service, the infections are now somewhat milder than last year. The Dutch Sheep and Goat Breeders' Organisation (NSFO) surveyed its members at the end of July. The number of infected animals on affected farms has increased in recent weeks, with some livestock farmers having almost all their animals sick. Approximately one in ten sick animals die. This means that mortality is considerably lower than during the outbreak at the end of last year. At that time, almost all infected animals on an affected farm usually died. © 2012-2025, NL Times, All rights reserved. This Page Has Washed AwayLooks like this page has disappeared with the tide — perhaps it’s buried in the sand at Vazon or drifting off Petit Port If you want to make sure that your meal deliverer can pocket their entire tip you shouldn't use the tip option on the Thuisbezorgt.nl site or app The largest meal delivery service in the Netherlands only pays out tips after deducting taxes and premiums RTL Nieuws reported after speaking to JustEat Takeaway The tipping option was added to Thuisbezorgd.nl's app as a way to reduce physical contact between meal deliverer and customer about 10 percent of orders now include a digital tip But this may not be so great for the delivery person Because while Thuisbezorgd.nl says on its website that 'the whole tip' goes to the delivery person it also says that 'taxes may apply to tips' Thuisbezorgd.nl told RTL Nieuws that it is not obliged to pay tax and premiums on digital tips but chose to do so "to unburden the deliverers and to ensure that they are not faced with unexpected surprises" Spokesperson Linda van Aalten said that the amount deducted depends on the delivery worker's income the delivery person themself is obliged to report that income to the tax authority Ministry of Finance spokesperson Jessica van Wegen said to the broadcaster "But the question is whether everyone actually declares those tips." Deliveroo and UberEats also offer the option for adding a tip to your order Deliveroo spokesperson Eva Huis in 't Veld told RTL that the entire tip is paid to delivery workers the deliverers themselves are responsible for reporting their tips to the tax authorities." UberEats was not available for comment The family of murdered Birmingham businessman Akhtar Javeed have urged anyone harbouring his alleged killer to turn him in Tahir Zarif - who prosecutors claim shot Mr Javeed in the legs throat and mouth - is at the centre of an international manhunt after flying to Pakistan following the killing last February A six-week trial was shown distressing CCTV footage of Mr Javeed being gunned down at point-blank range as he tried to escape from his catering supplies firm in Digbeth Speaking after three other men were convicted of conspiracy to commit robbery was thought to have passed through Heathrow Airport with his mother five days after the murder The trial at Birmingham Crown Court was told Mr Javeed was killed after being asked to take a masked gunman to a safe Zarif's business partner Suraj Mistry was acquitted of murdering Mr Javeed but convicted of manslaughter conspiracy to commit robbery and possession of firearms with intent to cause fear of violence manslaughter and firearms charges but convicted him of conspiracy to rob after both men were driven to the scene by Wali told the trial he took Mistry and Zarif to the warehouse in Rea Street South but did not know they were armed The raid was launched after sacked delivery driver Sander Van Aalten including a plan of the warehouse drawn on the back of a water bill admitted conspiracy to rob before the start of the trial Commenting after the trial on the ongoing operation to locate Zarif cold-blooded murder - there is no other way of describing it "The offenders planned this offence over a number of weeks and days - they brought firearms to the offence location "They brought cable ties with which to incapacitate all of the staff and this was only made possible by the information provided by an inside man." The officer added: "We would urge anybody who has information where Tahir Zarif is currently living or being harboured to contact West Midlands Police contact your embassy and pass that information on so that we can attempt to bring him back to the UK to face trial for this offence." backed Mr Slevin's plea for information about Zarif The 30-year-old counsellor said: "There is no real closure when it's murder "There has been three people brought to justice It's three less criminals roaming the streets but of course it's very important for us that the other person is brought back "I have faith that the relevant authorities will work together to bring him back and face trial so that we can get justice." Wali and Van Aalten are due to be sentenced on September 27.