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Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measure thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) were compared with thiamin status assessed by the measurement of TDP by HPLC. 55% of 230 included patients were male, and the median age was 10 (range 0.5–73) years. Using α ≥ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%), with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the results of the two tests (Kendall’s tau = −0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall’s tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays.

Vitamin B1 (thiamine) is an essential nutrient that acts as a cofactor for a number of metabolic processes, particularly in energy metabolism. Symptoms of classic thiamine deficiency are recognized as beriberi, although clinical symptoms are nonspecific and recognition of subclinical deficiency is difficult. Therefore, reliable biomarkers of thiamine status are required. Thiamine diphosphate is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay as an indirect, functional marker of thiamine status has been used for over 50 years. The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for deficiency, partly due to a lack of assay harmonization. Here, we provide a step‐by‐step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient, including detailed explanations of equipment and chemicals required and guidance for quality control procedures. Harmonization of the protocol will provide the basis for the development of internationally recognized cutoffs for thiamine insufficiency. The establishment of quality control materials and a quality assurance scheme are recommended to provide reliability. This will ensure that the ETK activity assay remains an important method for the assessment of thiamine status., Reliable biomarkers of thiamine status are required to help diagnose thiamine deficiency. Thiamine diphosphate (ThDP) is a cofactor for transketolase, including erythrocyte transketolase (ETK). The ETK activity assay provides a sensitive and specific biomarker of thiamine status; however, there is a lack of consensus over the cutoffs for thiamine deficiency, partly due to a lack of assay harmonization. Here, we provide a step‐by‐step protocol for the measurement of ETK activity and the calculation of the ETK activity coefficient (ETKAC).

One of the biochemical abnormalities found in diabetic tissues is a decrease in the cytosolic oxidized to reduced forms of the nicotinamide adenine dinucleotide ratio (NAD+/NADH also known as pseudohypoxia) caused by oxidation of excessive substrates (glucose through the polyol pathway, free fatty acids and lactate). Subsequently, a decline in NAD+ levels as a result of the activation of poly adenine nucleotide diphosphate‐ribose polymerase (mainly in type 1 diabetes) or the inhibition of adenine nucleotide monophosphate‐activated protein kinase (in type 2 diabetes). Thus, replenishment of NAD+ levels by nicotinamide‐related compounds could be beneficial. However, these compounds also increase nicotinamide catabolites that cause oxidative stress. This is particularly troublesome for patients with diabetes, because they have impaired nicotinamide salvage pathway reactions at the level of nicotinamide phosphoribosyl transferase and phosphoribosyl pyrophosphate, which occurs by the following mechanisms. First, phosphoribosyl pyrophosphate synthesis from pentose phosphate pathway is compromised by a decrease in plasma thiamine and transketolase activity. Second, nicotinamide phosphoribosyl transferase expression is decreased because of reduced adenosine monophosphate‐activated protein kinase activity, which occurs in type 2 diabetes. The adenosine monophosphate‐activated protein kinase inhibition is caused by an activation of protein kinase C and D1 as a result of enhanced diacylglycerol synthesis caused by pseudohypoxia and increased fatty acids levels. In this regard, nicotinamide‐related compounds should be given with caution to treat diabetes. To minimize the risk and maximize the benefit, nicotinamide‐related compounds should be taken with insulin sensitizers (for type 2 diabetes), polyphenols, benfotiamine, acetyl‐L‐carnitine and aldose reductase inhibitors. The efficacy of these regimens can be monitored by measuring serum NAD+ and urinary nicotinamide catabolites.

The spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter’s role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to ‘virulence priming’. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830. IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects’ fate, the response of Bt to hemolymph and the latter’s role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

Dietary thiamin requirement of fingerling Catla catla (3.5 ± 0.15 g) was evaluated by feeding casein-gelatin-based iso-nitrogenous (350 g/kg crude protein) and iso-caloric (16.72 kJ/g GE) diets containing six graded levels of thiamin (0, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/kg dry diet) for 12 weeks. Significantly (p

Thiamine diphosphate (TDP) and magnesium are co-factors for key enzymes in human intermediary metabolism. However, their role in the systemic inflammatory response (SIR) is not clear. Therefore, the aim of the present study was to examine the relation between acute changes in the SIR and thiamine and magnesium dependent enzyme activity in patients undergoing elective knee arthroplasty (a standard reproducible surgical injury in apparently healthy individuals). Patients (n = 35) who underwent elective total knee arthroplasty had venous blood samples collected pre- and post-operatively for 3 days, for measurement of whole blood TDP, serum and erythrocyte magnesium, erythrocyte transketolase activity (ETKA), lactate dehydrogenase (LDH), glucose and lactate concentrations. Pre-operatively, TDP concentrations, erythrocyte magnesium concentrations, ETKA and plasma glucose were within normal limits for all patients. In contrast, 5 patients (14%) had low serum magnesium concentrations (s = 0.338, p ClinicalTrials.gov: NCT03554668.

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with thiamine transporter-2 (THTR2) primarily involved. Here, we examined the behavior of thiamine transporter-1 (THTR1), THTR2, and their transcription factor Sp1 in response to high glucose and altered thiamine availability in renal cells involved in diabetic nephropathy. Human proximal tubule epithelial cells, podocytes, glomerular endothelial, and mesangial cells were exposed to high glucose and/or thiamine deficiency/oversupplementation. Localization and modulation of THTR1, THTR2, and Sp1, intracellular thiamine, transketolase activity, and permeability to thiamine were examined. Reduced thiamine availability and hyperglycemia impaired thiamine transport and THTR2/Sp1 expression. Intracellular thiamine, transketolase activity, and permeability were strongly dependent on thiamine concentrations and, partly, excess glucose. Glomerular endothelial cells were the most affected by the microenvironmental conditions. Our results confirmed the primary role of THTR2 in altered thiamine transport in cells involved in diabetic microvascular complications. Lack of thiamine concurs with hyperglycemia in impairing thiamine transport. Thiamine supplementation could represent a therapeutic option to prevent or slow the progression of these complications.

International audience; Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.

Nature chemical biology xx, 1-20 (2020). doi:10.1038/s41589-020-0628-4, The natural antivitamin 2′-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2′-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential., Published by Nature Publishing Group, Basingstoke

Objective To determine whether B vitamin treatment was sufficient to reduce cognitive impairment associated with high-fat diets in rats and to modulate transketolase (TK) expression and activity. Methods To test this, we separated 50 rats into five groups that were either fed a standard chow diet (controls) or a high-fat diet (experimental groups H0, H1, H2, and H3). H0 group animals received no additional dietary supplementation, while H1 group animals were administered 100 mg/kg body weight (BW) thiamine, 100 mg/kg BW riboflavin, and 250 mg/kg BW niacin each day, and group H2 animals received daily doses of 100 mg/kg BW pyridoxine, 100 mg/kg BW cobalamin, and 5 mg/kg BW folate. Animals in the H3 group received the B vitamin regimens administered to both H1 and H2 each day. Results Over time, group H0 exhibited greater increases in BW and fat mass relative to other groups. When spatial and memory capabilities in these animals were evaluated via conditioned taste aversion (CTA) and Morris Water Maze (MWM), we found B vitamin treatment was associated with significant improvements relative to untreated H0 controls. Similarly, B vitamin supplementation was associated with elevated TK expression in erythrocytes and hypothalamus of treated animals relative to those in H0 (P Conclusion Together, these findings suggest B vitamin can modulate hypothalamic TK activity to reduce the severity of cognitive deficits in a rat model of obesity. As such, B vitamin supplementation may be a beneficial method for reducing cognitive dysfunction in clinical settings associated with high-fat diets.

Diabetes mellitus-related morbidity and mortality is a rapidly growing healthcare problem, globally. Several nutraceuticals exhibit potency to target the pathogenesis of diabetes mellitus. The antidiabetic effects of compounds of garlic have been extensively studied, however, limited data are available on the biological effects of a certain garlic component, allithiamine. In this study, allithiamine was tested using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The non-cytotoxic concentration of allithiamine was determined based on MTT, apoptosis, and necrosis assays. Subsequently, cells were divided into three groups: incubating with M199 medium as the control, or with 30 mMol/L glucose, or with 30 mMol/L glucose plus allithiamine. The effect of allithiamine on the levels of advanced glycation end-products (AGEs), activation of NF-&kappa, B, release of pro-inflammatory cytokines including IL-6, IL-8, and TNF-&alpha, and H2O2-induced oxidative stress was investigated. We found that in the hyperglycaemia-induced increase in the level of AGEs, pro-inflammatory changes were significantly suppressed by allithiamine. However, allithiamine could not enhance the activity of transketolase, but it exerts a potent antioxidant effect. Collectively, our data suggest that allithiamine could alleviate the hyperglycaemia-induced endothelial dysfunction due to its potent antioxidant and anti-inflammatory effect by a mechanism unrelated to the transketolase activity.

Background Methanol expression regulator 1 (Mxr1p) is a key transcription factor that plays a vital role in the methanol utilization pathway in Pichia pastoris ( P. pastoris ). Most genes referred to the methanol utilization pathway were regulated by Mxr1p. However, some genes did not show a significant difference between methanol and glycerol even though they play an important role in the methanol utilization pathway. So far, the regulation mechanism about these genes and the relationship with Mxr1p are still unknown. Results Methanol metabolic pathway analysis revealed that most of the methanol-induced genes were upregulated in transcriptional level when cultured in methanol. Whereas some genes like tkl1 (transketolase 1) did not show significant up-regulation in methanol even though it plays a very important role in Xu5P recycle, the reason is still not clear. To clarify this point, firstly, pull-down and MS experiments were performed. The result shows that Tkl1p protein combined with Mxr1p in vitro . Subsequently, this result was further confirmed by yeast two-hybrid in vivo , and the binding region mainly located from 150AA to 400AA. Moreover, Ser215 phosphorylation did not affect this interaction. In addition, Mxr1p-400AA integration into Δmxr1 could rescue cell growth in methanol. All the above results proved that Mxr1p played a post-translational role in the methanol utilization pathway and Mxr1p-400AA may achieved most of Mxr1p functions. Secondly, the function of Mxr1p-Tkl1p complex was expounded by detecting formaldehyde consumption and xylulose production in cell-free systems. Results showed that Mxr1p-Tkl1p mixture could promote formaldehyde consumption and xylulose production in vitro . Conclusion Mxr1p promotes methanol utilization via combining with Tkl1p to accelerate Xu5P recycle and this interaction was not affected by Ser215 phosphorylation.

Thiamine prevents high glucose-induced damage in microvasculature, and progression of retinopathy and nephropathy in diabetic animals. Impaired thiamine availability causes renal damage in diabetic patients. Two single-nucleotide polymorphisms in SLC19A3 locus encoding for thiamine transporter 2 are associated with absent/minimal diabetic retinopathy and nephropathy despite long-term type 1 diabetes. We investigated the involvement of thiamine transporter 1 and thiamine transporter 2, and their transcription factor specificity protein 1, in high glucose-induced damage and altered thiamine availability in cells of the inner blood–retinal barrier. Human endothelial cells, pericytes and Müller cells were exposed to hyperglycaemic-like conditions and/or thiamine deficiency/over-supplementation in single/co-cultures. Expression and localization of thiamine transporter 1, thiamine transporter 2 and transcription factor specificity protein 1 were evaluated together with intracellular thiamine concentration, transketolase activity and permeability to thiamine. The effects of thiamine depletion on cell function (viability, apoptosis and migration) were also addressed. Thiamine transporter 2 and transcription factor specificity protein 1 expression were modulated by hyperglycaemic-like conditions. Transketolase activity, intracellular thiamine and permeability to thiamine were decreased in cells cultured in thiamine deficiency, and in pericytes in hyperglycaemic-like conditions. Thiamine depletion reduced cell viability and proliferation, while thiamine over-supplementation compensated for thiamine transporter 2 reduction by restoring thiamine uptake and transketolase activity. High glucose and reduced thiamine determine impairment in thiamine transport inside retinal cells and through the inner blood–retinal barrier. Thiamine transporter 2 modulation in our cell models suggests its major role in thiamine transport in retinal cells and its involvement in high glucose-induced damage and impaired thiamine availability.

The possible roles of selected B vitamins in the development and progression of sarcopenia are reviewed. Age-related declines in muscle mass and function are associated with huge and increasing costs to healthcare providers. Falls and loss of mobility and independence due to declining muscle mass/function are associated with poor clinical outcomes and their prevention and management are attractive research targets. Nutritional status appears a key modifiable and affordable intervention. There is emerging evidence of sarcopenia being the result not only of diminished anabolic activity but also of declining neurological integrity in older age, which is emerging as an important aspect of the development of age-related decline in muscle mass/function. In this connection, several B vitamins can be viewed as not only cofactors in muscle synthetic processes, but also as neurotrophic agents with involvements in both bioenergetic and trophic pathways. The B vitamins thus selected are examined with respect to their relevance to multiple aspects of neuromuscular function and evidence is considered that requirements, intakes or absorption may be altered in the elderly. In addition, the evidence base for recommended intakes (UK recommended daily allowance) is examined with particular reference to original datasets and their relevance to older individuals. It is possible that inconsistencies in the literature with respect to the nutritional management of sarcopenia may, in part at least, be the result of compromised micronutrient status in some study participants. It is suggested that in order, for example, for intervention with amino acids to be successful, underlying micronutrient deficiencies must first be addressed/eliminated.

The biosynthesis of the azinomycins involves the conversion of glutamic acid to an aziridino[1,2-a]pyrrolidine moiety, which together with the epoxide moiety imparts anticancer activity to these agents. The mechanism of azabicycle formation is complex and involves at least 14 enzymatic steps. Previous research has identified N-acetyl-glutamate 5-semialdehyde as a key intermediate, which originates from protection of the amino terminus of glutamic acid and subsequent reduction of the γ-carboxylate. This study reports on the seminal discovery of a thiamin-dependent transketolase responsible for the formation of 2-acetamido-5,6-dihydroxy-6-oxoheptanoic acid, which accounts for the two-carbon extension needed to complete the carbon framework of the azabicycle moiety.

Staphylococcus aureusis a leading cause of both acute and chronic infections in humans. Its ability to persist within host cells is thought to play an important role in chronicity and treatment failures. The importance of the pentose phosphate pathway (PPP) duringS. aureuschronic infection is currently largely unexplored. Here, we focused on one key PPP enzyme, transketolase. We showed that inactivation of the unique gene encoding transketolase activity inS. aureusUSA300 (Δtkt) led to an impaired growth in broth. Using time-lapse video imaging, we correlated this phenotype with a defect in early intracellular proliferation compared to wild-type strain. As determined by metabolomic analysis,tktinactivation also had an important impact onS. aureusmetabolism. We then monitored long-term intracellular persistence over 10 days by counting of viable bacteria. Unexpectedly for such a slow-growing strain, the Δtktmutant was almost completely eliminated by endothelial cells after ten days, as opposed to a prototypical slow-growing ΔhemDBLmutant for which we recovered 1,000 fold more viable bacteria. We found that in infected cells, the transcriptional activity of the two master regulators Sigma B and RpiRc was drastically reduced in the Δtktmutant compared to wild-type strain. Concomitantly, RNAIII transcription was strongly increased. This transcriptional profile is likely to explain the inability of this slow-growing mutant to sustain long-term intracellular survival, suggesting that TKT -or a functional PPP-is required for intracellular bacteria to enable a transcriptional program geared towards persistence.ImportanceStaphylococcus aureusis a leading cause of severe bacterial infections. This bacterium is readily internalized by non-professional phagocytes and infected cells have been proposed to play an important role in chronic infections and treatment failures.Here, we show the importance of the unique transketolase TKT ofS. aureusUSA300 in bacterial adaptation during chronic intracellular infection. We show that TKT is mandatory for the metabolomic homeostasis ofS. aureusduring intracellular persistence. This work unravels the critical role of TKT in the transcriptional regulation of the master regulators Sigma B, RpiRc and RNAIII linking the pentose phosphate pathway to the control of chronicS. aureusinfections.

Background Three transketolase genes have been identified in the human genome to date: transketolase (TKT), transketolase-like 1 (TKTL1) and transketolase-like 2 (TKTL2). Altered TKT functionality is strongly implicated in the development of diabetes and various cancers, thus offering possible therapeutic utility. It will be of great value to know whether TKTL1 and TKTL2 are, similarly, potential therapeutic targets. However, it remains unclear whether TKTL1 and TKTL2 are functional transketolases. Results Homology modelling of TKTL1 and TKTL2 using TKT as template, revealed that both TKTL1 and TKTL2 could assume a folded structure like TKT. TKTL1/2 presented a cleft of suitable dimensions between the homodimer surfaces that could accommodate the co-factor-substrate. An appropriate cavity and a hydrophobic nodule were also present in TKTL1/2, into which the diphosphate group fitted, and that was implicated in aminopyrimidine and thiazole ring binding in TKT, respectively. The presence of several identical residues at structurally equivalent positions in TKTL1/2 and TKT identified a network of interactions between the protein and co-factor-substrate, suggesting the functional fidelity of TKTL1/2 as transketolases. Conclusions Our data support the hypothesis that TKTL1 and TKTL2 are functional transketolases and represent novel therapeutic targets for diabetes and cancer. Electronic supplementary material The online version of this article (10.1186/s12900-018-0099-y) contains supplementary material, which is available to authorized users.

IntroductionThiamine (vitamin B1) deficiency remains a concern in Cambodia where women with low thiamine intake produce thiamine-poor milk, putting their breastfed infants at risk of impaired cognitive development and potentially fatal infantile beriberi. Thiamine fortification of salt is a potentially low-cost, passive means of combating thiamine deficiency; however, both the dose of thiamine required to optimise milk thiamine concentrations as well as usual salt intake of lactating women are unknown.Methods and analysisIn this community-based randomised controlled trial, 320 lactating women from Kampong Thom, Cambodia will be randomised to one of four groups to consume one capsule daily containing 0, 1.2, 2.4 or 10 mg thiamine as thiamine hydrochloride, between 2 and 24 weeks postnatal. The primary objective is to estimate the dose where additional maternal intake of thiamine no longer meaningfully increases infant thiamine diphosphate concentrations 24 weeks postnatally. At 2, 12 and 24 weeks, we will collect sociodemographic, nutrition and health information, a battery of cognitive assessments, maternal (2 and 24 weeks) and infant (24 weeks only) venous blood samples (biomarkers: ThDP and transketolase activity) and human milk samples (also at 4 weeks; biomarker: milk thiamine concentrations). All participants and their families will consume study-provided saltad libitumthroughout the trial, and we will measure salt disappearance each fortnight. Repeat weighed salt intakes and urinary sodium concentrations will be measured among a subset of 100 participants. Parameters of Emaxdose–response curves will be estimated using non-linear least squares models with both ‘intention to treat’ and a secondary ‘per-protocol’ (capsule compliance ≥80%) analyses.Ethics and disseminationEthical approval was obtained in Cambodia (National Ethics Committee for Health Research 112/250NECHR), Canada (Mount Saint Vincent University Research Ethics Board 2017–141) and the USA (University of Oregon Institutional Review Board 07052018.008). Results will be shared with participants’ communities, as well as relevant government and scientific stakeholders via presentations, academic manuscripts and consultations.Trial registration numberNCT03616288.

Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We identified a syndrome due to an autosomal-recessively inherited deficiency of transketolase, encoded by TKT, on chromosome 3p21. Our series includes three families with a total of five affected individuals, ranging in age from 4 to 25 years. Two families of Ashkenazi Jewish ancestry were homozygous for an 18 base pair in-frame insertion in TKT. The third family was compound heterozygous for nonsense and missense variants in TKT. All affected individuals had short stature and were developmentally delayed. Congenital heart defects were noted in four of the five affected individuals, and there was a history of chronic diarrhea and cataracts in the older individuals with the homozygous 18 base pair insertion. Enzymatic testing confirmed significantly reduced transketolase activity. Elevated urinary excretion of erythritol, arabitol, ribitol, and pent(ul)ose-5-phosphates was detected, as well as elevated amounts of erythritol, arabitol, and ribitol in the plasma of affected individuals. Transketolase deficiency reduces NADPH synthesis and nucleic acid synthesis and cell division and could explain the problems with growth. NADPH is also critical for maintaining cerebral glutathione, which might contribute to the neurodevelopmental delays. Transketolase deficiency is one of a growing list of inborn errors of metabolism in the non-oxidative part of the pentose phosphate pathway.

Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter-conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose-5-P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol-3-P synthesis. Using lacZ fusions, we show that ribose-5-P enhances GlpR-mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose-5-P made the DNA-GlpR complex less sensitive to the inducer glycerol-3-P. In addition to inhibition of glycerol assimilation, obstruction of ribose-5-P metabolism retards growth from glycerol-3-P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose-5-P can modulate glycerol-3-P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms.

Thiamine is an essential co-factor for aerobic metabolism. Both thiamine deficiency and sepsis may be associated with hyperlactatemia and hypotension. We assessed the relationship between thiamine compounds, lactate concentrations and clinical outcomes in septic patients. We undertook a prospective observational single-center study. Erythrocyte levels of total thiamine, free thiamine, thiamine mono, di and triphosphate (TMP, TDP, and TTP respectively), the erythrocyte transketolase activity (ETKA) and the effect of thiamine diphosphate on ETKA were measured in septic patients by high performance liquid chromatography and correlated with arterial lactate. Vital status at the end of intensive care unit stay was recorded. Overall, 28 patients suffering from sepsis were included. Median (interquartile range [IQR]) age was 60 [44–77.3] years, 15 (53.6%) patients were male, median [IQR] simplified acute physiology score II was 40 [27–50]. There was no correlation between total thiamine and lactate levels (P = .33). There was no correlation between free thiamine (P = .81), TMP (P = .71), TDP (P = .31), TTP (P = .86), and lactate levels in our population. There was no correlation between ETKA (P = .58) or the effect of TDP on ETKA (P = .40) and lactate concentration. Total thiamine and TDP concentration were significantly higher in intensive care unit (ICU) survivors than in nonsurvivors (P = .03 and P = .03). The effect of TDP on ETKA was significantly higher in nonsurvivors compared to survivors (P = .04). We found no correlation between thiamine compounds and lactate concentration in sepsis. Thiamine deficiency in sepsis may be associated with ICU-mortality.

The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46–47% similarity to the known TktA and TktB isoforms. Assessing the mRNA and protein expression for each of the three transketolases, we determined that all are expressed in WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for V(max) (539–1362 μm NADH consumed/s), K(m) (80–739 μm), and catalytic efficiency (1.02 × 10(8)-1.06 × 10(9) m(−1)/s) for each substrate tested. The recombinant form of TktC had lower K(m) values (23–120 μm), whereas the V(max) (7.8–16 μm NADH consumed/s) and catalytic efficiency (5.58 × 10(6) to 6.07 × 10(8) m(−1)/s) were 10–100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S. Typhimurium possesses three transketolases that contribute to pathogenesis.

Zymomonas mobilis is capable of producing ethanol at high rates and titers from glucose. This bacterium has previously been engineered to consume the pentose sugars xylose and arabinose, but the rate of consumption of these sugars is low. Recent research has utilized adaptive evolution to isolate strains of Z. mobilis capable of rapidly fermenting xylose. In this study, we also used adaptive evolution to isolate strains of Z. mobilis capable of rapidly fermenting xylose and arabinose. To determine the bottlenecks in pentose metabolism, we then used high-throughput sequencing to pinpoint the genetic changes responsible for the phenotypes of the adapted strains. We found that the transport of both xylose and arabinose through the native sugar transporter, Glf, limits pentose fermentations in Z. mobilis. We also found that mutations in the AddB protein increase plasmid stability and can reduce cellular aggregation in these strains. Consistent with previous research, we found that reduced xylitol production improves xylose fermentations in Z. mobilis. We also found that increased transketolase activity and reduced glyceraldehyde-3-phosphate dehydrogenase activity improve arabinose fermentations in Z. mobilis. Biotechnol. Bioeng. 2015;112: 2228–2240. © 2015 Wiley Periodicals, Inc.

It is well known that tumor cells mainly depend on the nonoxidative pathway of the pentose phosphate pathway (PPP), and transketolase-like 1 (TKTL1), a kind of crucial metabolism enzyme, participates in the regulation of PPP; notably, overwhelming evidence has demonstrated that TKTL1 plays pivotal roles in the development and progression of multiple tumors. However, there were no reports about the role of TKTL1 in esophageal squamous cell carcinoma (ESCC). Here, we investigated TKTL1 expression and preliminarily elucidated its underlying biological functions in ESCC. We found that TKTL1 exhibited the high expression in ESCC tissues and cells, and the survival rate of patients with negative TKTL1 expression was significantly higher than that of patients with positive TKTL1 staining (P

This study was conducted to determine dietary thiamine requirement of juvenile Sclizothorax prenanti and evaluate the effect of dietary thiamine levels on growth performance, body composition and haemato-biochemical parameters for this fish species. The seven experimental diets were formulated to contain the graded levels of thiamine (0, 10, 20, 30, 40, 60 and 100 mg kg−1 diet, respectively), providing the actual dietary thiamine values of 0.31 (control), 9.82, 21.49, 29.83, 41.66, 62.24 and 114.58 mg kg−1 diet, respectively. Each diet was assigned to three replicate groups of S. prenanti (initial body weight: 13.46 ± 0.28 g, means ± SD) for 60 days. Increasing dietary thiamine level up to 21.49 mg kg−1 diet increased weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and protein efficiency ratio (PER) (P 0.05). Analysis by the broken-line regression of WGR, SGR, FE, PER, hepatic thiamine concentration and serum transketolase activity indicated that dietary thiamine requirements in juvenile S. prenanti were 18.45–25.91 mg kg−1 diet.

Aim Pentose phosphate pathway (PPP) with key enzyme transketolase (TKT), represents a potentially ‘protective’ mechanism in hyperglycaemia. Diabetic kidney disease (DKD), a common complication of both type 1 and type 2 diabetes associated with significant morbidity and mortality, represents the most common cause of chronic kidney disease (CKD). We hypothesized that protective PPP action in diabetes and eventually even more severely in concomitant DKD might be compromised by limited intracellular availability of an active TKT cofactor thiamine diphosphate (TDP). Methods Effect of hyperglycaemia on gene expression and protein levels of key PPP loci was studied in vitro using human cell lines relevant to diabetes (HUVEC and HRGEC) and (together with measurement of TKT activity, plasma thiamine and erythrocyte TDP concentration) in vivo in diabetic vs. non-diabetic subjects with comparable renal function (n=83 in total). Results Hyperglycaemia significantly decreased protein levels of RFC-1, THTR1, THTR2 and TKT (P Conclusion Both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while TKT activity in parallel with intracellular TDP was increased in CKD patients with or without diabetes. Therefore, lack of adaptive increase of thiamine transmembrane transport allowing further increase of TKT activity might contribute to compromised PPP function in diabetes and CKD and to the development of glycotoxic injury.

Plant phenolics encompass a wide range of aromatic compounds and functions mainly related to abiotic and biotic environmental responses. In calcareous soils, the presence of bicarbonate and a high pH cause a decrease in iron (Fe) bioavailability leading to crop yield losses both qualitatively and quantitatively. High increases in phenolics were reported in roots and root exudates as a consequence of decreased Fe bioavailability suggesting their role in chelation and reduction of inorganic Fe(III) contributing to the mobilization of Fe oxides in soil and plant apoplast. Shikimate pathway represents the main pathway to provide aromatic precursors for the synthesis of phenylpropanoids and constitutes a link between primary and secondary metabolism. Thus the increased level of phenolics suggests a metabolic shift of carbon skeletons from primary to secondary metabolism. Parietaria judaica, a spontaneous plant well adapted to calcareous environments, demonstrates a high metabolic flexibility in response to Fe starvation. Plants grown under low Fe availability conditions showed a strong accumulation of phenolics in roots as well as an improved secretion of root exudates. P. judaica exhibits enhanced enzymatic activities of the shikimate pathway. Furthermore, the non-oxidative pentose phosphate pathway, through the transketolase activity supplies erythrose-4-phosphate, is strongly activated. These data may indicate a metabolic rearrangement modifying the allocation of carbon skeletons between primary and secondary metabolism and the activation of a nonoxidative way to overcome a mitochondrial impairment. We suggest that high content of phenolics in P. judaica play a crucial role in its adaptive strategy to cope with low Fe availability.

Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.

Introduction The activity of erythrocyte transketolase induced by thiamine pyrophosphate and normalized to age of the patient is a marker of thiamine metabolism disturbances with pathological consequences in the central and peripheral nervous system. The measurement of erythrocyte transketolase activity enables evaluation of the thiamine status and therapeutic decisions in the disorders of the nervous system related to its deficiency. The aim of the study was to compare different modes of expression of transketolase activity in the most frequent acquired neuropathies. Material and methods The study included 29 patients with type 2 diabetes mellitus (21 males, 8 females) aged 48.3 years (range: 20-70 years) and 31 subjects with a history of alcohol dependence (23 males, 8 females) aged 54.5 years (range: 28-60 years). All participants of the study showed signs and symptoms of neuropathy. The cases in both groups presented the involvement of either axon, myelin, or both, what was evidenced by electrophysiological tests (electromyography and estimation of nerve conduction velocity). The control group consisted of 20 healthy persons aged 49.1 years (range: 23-70 years). Transketolase activity in erythrocytes (TK) was assessed by means of the spectrophotometric method. Basic TK activity was expressed as units per gram of haemoglobin (g Hb), moreover the normalized transketolase activity ratio (NTKZ) and percentage of activation of thiamine pyrophosphate (TPP) were calculated. Results The basal TK activity was decreased in cases with diabetic neuropathy (0.64 ± 0.342 U/g Hb, p = 0.0131), whereas in patients with alcoholic neuropathy only a trend (p = 0.058) of the decrease was noticed (0.85 ± 0.484 U/g Hb), in relation to the control group (1.005 ± 0.390 U/g Hb). Normalized transketolase activity ratio in erythrocytes has not shown any statistically significant differences. The median TPP did not indicate any thiamine deficiency, both in the group of diabetic and alcoholic neuropathy. Conclusions The decrease in transketolase activity in diabetic neuropathy may be independent of thiamine deficiency. Abnormalities in transketolase activity, when expressed in three modalities: basal activity, normalized transketolase activity ratio and the activity after the stimulation with thiamine pyrophosphate may be differentiated as thiamine-dependent or resulting from posttranslational modification.

Recent clinical trials suggest that patients with myelofibrosis can develop Wernicke's encephalopathy (WE) when treated with fedratinib, a specific Janus kinase-2 (JAK-2) inhibitor. To investigate this issue, we have examined (1) if fedratinib can produce or alter the course of this disorder, (2) its effects on thiamine-dependent enzyme activity and thiamine status, and (3) its influence on the uptake of thiamine. Animals administered fedratinib for 28days at a comparable dose used to treat human cases of myelofibrosis showed no evidence of clinical signs of thiamine deficiency (TD). Rats treated with a combination of fedratinib and TD exhibited no neurological differences in their progress to the symptomatic stage when compared to thiamine-deficient animals only. Treatment with the JAK-2 inhibitor did not compromise erythrocyte transketolase activity, and thiamine status was not affected in a major way unlike animals with TD. In addition, treatment of cultured astrocytes with fedratinib did not diminish the uptake of thiamine into these cells. Our findings suggest that treatment with fedratinib does not lead to or alter the progress of TD, and do not support the notion that administration of this JAK-2 inhibitor directly results in the development of WE due to inhibition of thiamine transport. Known adverse effects of fedratinib involving compromised gastrointestinal function may be an important indirect contributing factor to previously reported cases of WE in patients with myelofibrosis.

Chronic alcoholism and thiamine deficiency are well documented factors in the aetiology of Wernicke-Korsakoff Syndrome. More recently, acetaldehyde (ACH) has been implicated as a possible aetiological factor. In the present investigation the direct effect of ACH was studied on the activity of transketolase, a thiaminedependent enzyme, as well as two non-thiamine-dependent enzymes (aspartate aminotransferase and lactate dehydrogenase), isolated from five control human brains. The concentration of ACH required to inhibit 50% activity of holo- and apo-transketolase was 80 mM and 60 mM, respectively, whereas that for aspartate aminotransferase and lactate dehydrogenase was 14 mM and 10 mM, respectively. None of the enzymes were completely inhibited by the range of ACH concentrations used in the study. It was concluded that the thiamineindependent enzymes were markedly affected by the concentrations of ACH which did not affect the thiaminedependent enzyme, transketolase. In vitro studies with homogenates pre-treated with ACH in the presence of various concentrations of glutathione showed that the latter had a protective effect against loss of transketolase activity.

Eosinophilic esophagitis is an inflammatory condition, causing dysphagia, food impaction and chest pain. A minimum of 15 eosinophils per high power field by esophageal biopsy is necessary for diagnosis. It has become an increasingly important cause of gastrointestinal morbidity in the past two decades. A 14 year old boy had received a diagnosis of eosinophilic esophagitis elsewhere at the age of 8 years. His medical history had been thought for many years to be psychosomatic until endoscopy was performed. The only other positive laboratory study was eosinophilia. In spite of conventional therapy, none of which improved overall symptoms, esophagitis persisted and he had failed to gain weight and stature. Physical examination revealed many indications of autonomic dysfunction and an erythrocyte transketolase test indicated abnormal thiamine homeostasis. Treatment was started with intravenous infusions of water-soluble vitamins that included thiamine hydrochloride (THCL). Because the transketolase test became worse, THCL was replaced with thiamine tetrahydrofurfuryl disulfide (TTFD), with consequent improvement in transketolase and symptomatic response. There was a family history of alcoholism and the patient was addicted to sugar, suggesting a genetic risk to explain the abnormal thiamine homeostasis. Beriberi causes dysautonomia in its early stages. Inflammation is now known to be suppressed reflexly through the vagus nerve, itself dependent on acetylcholine. Abnormal motility of the esophagus has been reported in eosinophilic esophagitis. Failure of THCL to improve transketolase activity suggested a genetic failure in a thiamine transporter and its consequent correction with TTFD that does not require the thiamine transport system.

Background: Cardiac acute beriberi (Shoshin syndrome) is a rare disease that may lead to a fatal outcome if not treated specifically. Objectives: We report a case of Shoshin syndrome with an unusual presentation of cardiogenic shock and an electrocardiographic pattern of severe myocardial ischemia suggesting left main coronary artery obstruction. Case Report: A 35-year-old man presented with chest discomfort, diffuse ST-segment depression in the 12-lead electrocardiogram (ECG) with ST-segment elevation in aVR, and rapidly evolving congestive heart failure leading to cardiogenic shock. Intensive support was required, including mechanical ventilation, high doses of inotropics and vasopressors, intra-aortic balloon counterpulsation, and continuous renal replacement therapy. An emergency coronary angiogram was performed that showed normal coronary arteries. Right heart catheterization showed a high-output state with elevated filling pressures suggesting high-output heart failure. The echocardiography confirmed normal left and right ventricular contraction. Thiamine deficiency was suspected as the cause of the high-output heart failure. After a single dose of intravenous thiamine (100 mg), the patient's hemodynamic status improved dramatically within minutes, allowing a rapid discontinuation of hemodynamic support. Subsequent ECGs showed complete resolution of ST-segment abnormalities. Serial lactate measurements, red blood cell transketolase activity, and the thiamine pyrophosphate response test were concordant with a thiamine deficiency state. Conclusion: Shoshin syndrome may present as cardiogenic shock with an ECG mimicking severe myocardial ischemia, and if suspected, can be rapidly and effectively treated.

This work describes an experimental ‘ toolbox ’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘ mix and match ’ E. coli -based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables effi cient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘ toolbox ’ , one-pot syntheses of the diastereoisomers (2 S ,3 S )-2-aminopentane-1,3-diol (APD) and (2 S ,3 R )-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave fi nal product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specifi c activity and ee ( enantiomeric excess) of 95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘ toolbox ’ provides an effi cient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.

Residual renal function and the integrity of the peritoneal membrane contribute to morbidity and mortality among patients treated with peritoneal dialysis. Glucose and its degradation products likely contribute to the deterioration of the remnant kidney and damage to the peritoneum. Benfotiamine decreases glucose-induced tissue damage, suggesting the potential for benefit in peritoneal dialysis. Here, in a model of peritoneal dialysis in uremic rats, treatment with benfotiamine decreased peritoneal fibrosis, markers of inflammation, and neovascularization, resulting in improved characteristics of peritoneal transport. Furthermore, rats treated with benfotiamine exhibited lower expression of advanced glycation endproducts and their receptor in the peritoneum and the kidney, reduced glomerular and tubulointerstitial damage, and less albuminuria. Increased activity of transketolase in tissue and blood contributed to the protective effects of benfotiamine. In primary human peritoneal mesothelial cells, the addition of benfotiamine led to enhanced transketolase activity and decreased expression of advanced glycation endproducts and their receptor. Taken together, these data suggest that benfotiamine protects the peritoneal membrane and remnant kidney in a rat model of peritoneal dialysis and uremia.

Methylmercury (MeHg) is a global pollutant that causes malformations. There has been no direct evidence for the effect of MeHg on pentose phosphate pathway (PPP). In embryonic development, PPP is much more active. This pathway produces ribose for DNA/RNA production. It is possible that one of teratogenicity mechanisms of MeHg is through PPP. The fetus fibroblast cells were incubated with different concentrations of MeHg (0.1–100 μm). A dose–response dependence was observed in MTT assay. Transketolase activity and DNA content were determined in cell exposed to MeHg. A defect at the level of DNA content was observed. This amount of DNA was highly correlated with transketolase activity (r = 0.76). This study has demonstrated that the potential teratogenic action of MeHg is through PPP. To assess the protective effects of thiamin, the infected cells were incubated with different concentrations of thiamin. The obtained results show that thiamin pyrophosphate supplementation correlated with the toxicity. This finding confirms that thiamin therapy is suitable for the prevention of MeHg toxicity. Our study provides basic data for prevention and treatment of MeHg toxicity via boosting PPP. Copyright © 2011 John Wiley & Sons, Ltd.

The etiology of insulin resistance is complex and multi-faceted.For example, metabolic perturbations associated with obesity and/orincreased consumption of energy-dense foodstuffs, coupled witha sedentary lifestyle may fuel this process [1,2].Nutrientexcess,and more specifically excursions of acute hyperglycemia, can playa significant role in the development of insulin resistance and theassociated onset of cardiovascular diseases [3,4].Recent data from our laboratory show that short-term hypergly-cemia exerts a number of detrimental metabolic alterations contrib-uting to both myocardial insulin resistance and impaired cardiaccontractile function. This includes greater oxidative stress and theinduction of non-oxidative glucose pathways (NOGPs)— i.e.enhanced formation of advanced glycation-end products (AGEs),stimulation of proteinkinase C(PKC)isoforms, andincreased activa-tion of the polyol and hexosamine biosynthetic pathways (HBP);leading to invitrocardiac insulin resistance [5],and increased infarctsizes and apoptosis duringex vivo ischemia/reperfusion (I/R) of rathearts [6]. However, activation of a pentose phosphate pathway(PPP) enzyme (transketolase) by thiamine (derivative of vitaminB1) or its lipophilic analog benfotiamine (BFT) is associated withprotective effects against various diabetic complications. For exam-ple, we demonstrated that BFT treatment augmented transketolaseactivity, thereby reversing cellular insulin resistance and alsoprotecting against I/R-mediated damage[5,6]. Since our earlierwork was generally conducted in cultured heart cells, the currentstudy evaluated myocardial NOGP regulation and thiamine adminis-tration during pre- and overt diabetes within an in vivo context. Wehypothesized that thiamine improves diabetes-induced cardio-metabolic dysfunction by upregulation of transketolase activity,thereby re-directing metabolites into the PPP and subsequentlydecreasing activation of damaging NOGPs.Heart tissues were kindly provided by Dr. Takao Tanaka (OsakaUniversity of Pharmaceutical Sciences, Osaka, Japan). Four-weekold male Otsuka Long-Evans Tokushima Fatty (OLETF) rats wererandomly divided into a) untreated and b) thiamine-treated groupsreceiving thiamine (2 g/L) in their drinking water[7]. Groups werefurther divided according to treatment time, with rats killed at 25 or55 weeks of age, corresponding to 21 and 51 weeks of thiamine treat-ment, respectively (n = 8 per group).Thiamine administration reduced plasma glucose, insulin,triglycerides and HbA1c levels compared to untreated controls[7].Although the levels of plasma glucose and insulin were both higher inthe 25-week-old controls, the 55-week-old rats displayed elevatedglucose but decreased insulin levels. These data therefore indicate ahyperinsulinemic/hyperglycemic (pre-diabetic) state at 25 weeks,and a full blown diabetic condition at 55 weeks [7]. For the purpose ofthisreport,werefertotheexperimentalgroupsas“pre-diabetic”versus“diabetic”. Furthermore, echocardiographic assessment of diabeticOLETF rats revealed impaired cardiac function that could be improvedby thiamine treatment.For this study, we initially investigated Akt kinase activity (asa marker of insulin signaling) using a commercial kit (Enzo LifeSciences, Farmingdale, NY) and found that thiamine treatmentincreased Akt activity by ~2.4 fold in the pre-diabetic group (P b 0.001vs. pre-diabetic control) (Fig. 1A). Conversely, the diabetic ratsdisplayed reduced myocardial Akt activity at baseline and in responseto thiamine treatment (P b 0.05 vs. pre-diabetic control, P b 0.001 vs.pre-diabetic thiamine). The activities of two key regulatory PPPenzymes, glucose-6-phosphate dehydrogenase (G6PD) (BioVision,Mountain View, CA) and transketolase (modified assay from Sigma,St Louis, MO [5,6]) were next evaluated. Here thiamine treatment had

Thiamine (vitamin B-1) is an essential component of a number of metabolic pathways and thiamine deficiency results in a progressive encephalopathy in both humans and animals. Confirming thiamine deficiency is problematic and relies on demonstrating reduced red blood cells transketolase activity, or indirect methods including urinary organic acid analysis and dietary analysis. The characteristic and selective vulnerability of different brain regions in carnivores has been demonstrated by magnetic resonance (MR) imaging in the dog and cat as an aid to diagnosis. A 2-year-old, female, domestic shorthair cat was presented with an acute onset of seizures and ataxia. MR imaging was consistent with thiamine deficiency and supplementation resulted in a progressive clinical improvement. Repeated MR imaging 4 days after starting thiamine supplementation revealed near complete resolution of the MR abnormalities. Repeated MR imaging following appropriate therapy may be useful to further confirm thiamine deficiency.

Summary The levels of transketolase (TK) and the amount of TK apoenzyme measured by thiamine pyrophosphate (TPP) addition in the erythrocytes of dairy cows, beef cattle and sheep are described. The TK levels were lower and the enhancement of the enzyme activity after TPP addition was greater in high yielding cows than in dry cows. A similar pattern was seen in beef cattle and sheep, with low TK levels and clearly enhanced enzyme activity after TPP addition in animals with clinical signs or suspected to have cerebrocortical necrosis (CCN). The possible presence of thiamine antagonists in the blood of these animals is also discussed. Zusammenfassung Transketolaseaktivitat im Blut von Rind und Schaf in Abhangigkeit von der Thiaminversorgung Bei Milchrindern, Fleischrindern und Schafen werden die Transketolaseaktivitaten (TK) und der Gehalt an TK-Apoenzym nach Hinzufugen von Thiaminpyrophosphat (TPP) beschrieben. Bei Kuhen mit hoher Milchleistung waren die TK-Spiegel niedriger und die Steigerung der Enzymaktivitat nach Hinzufugen von TPP ausgepragter als bei trockenstehenden Kuhen. Ein ahnliches Muster wurde bei Fleischrindern und Schafen beobachtet. Erythrozyten von Tieren mit Verdacht oder klinischen Anzeichen einer Cerebrocorticalnekrose wiesen niedrigere TK-Aktivitaten und deutlich erhohte Enzymaktivitaten nach Hinzufugen von TPP auf. Das mogliche Vorkommen eines Thiaminantagonisten im Blut dieser Tiere wird diskutiert. Resume Activite de la transketolase dans le sang de bovin et de mouton en fonction de l'approvisionnement en thiamine Les activites de la transketolase (TK) et le taux de TK-apoenzyme apres adjonction de pyrophosphate de thiamine (TPP) sont decrits chez des vaches laitieres, des bovins d'engrais et des moutons. Le taux TK fut plus bas et l'augmentation de l'activite enzymatique apres adjonction de TPP fut plus marque chez les vaches fortement laitieres que chez les vaches en periode de mise a goutte. On a observe un phenomene semblable chez des bovins d'engrais et des moutons. Des erythrocytes d'animaux suspects ou cliniquement atteints de necrose du cortex cerebral ont presente des activites TK plus basses et des activites enzymatiques nettement augmentees apres adjonction de TPP. On discute la presence possible d'un antagoniste de la thiamine dans le sang de ces animaux. Resumen La actividad transcetolasica en la sangre de vacunos y ovinos en relacion con la deficiencia de tiamina Se describen en vacas lecheras, vacunos destinados a carniceria y en ovinos las actividades de la transcetolasa (TC) y la tasa de apoenzima TC tras agregar pirofosfato de tiamina (PFT). En vacas con produccion elevada de leche eran menores los niveles de TC y mas evidente el incremento de la actividad enzimatica tras anadir PFT que en las vacas secas. Un modelo semejante fue observado en las reses para carne y en las ovejas. Los eritrocitos de animales con sospecha o signos clinicos de una necrosis cerebrocortical (NCC) presentaron actividades TC menores y actividades enzimaticas aumentadas con toda claridad despues de adicionar PFT. Discutese la presencia posible de un antagonista de la tiamina en la sangre de estos animales.

Tropical ataxic neuropathy (TAN) is a syndrome characterized by sensory polyneuropathy, sensory ataxia, bilateral optic atrophy and bilateral sensorineural deafness. The syndrome has occurred in endemic form in several African countries, and in epidemic form in Cuba. In endemic communities, the syndrome has a high prevalence and a demonstrated risk for high mortality. Despite several studies aimed at elucidating the etiological mechanisms of TAN, the etiology has remained unknown more than five decades after its original description. Chronic cyanide intoxication from a monotonous diet of cassava was long thought to be the major etiological factor, but there has been no evidence of a causal association. Vitamin deficiencies were thought to play little or no role in the pathogenesis of TAN. Evidence from the literature implicating chronic thiamine deficiency in the etiology of TAN is presented in this communication. This includes evidence of abnormal pyruvate metabolism reversed by thiamine in patients with TAN, evidence from erythrocyte transketolase activity indicating significant thiamine deficiency in patients with TAN compared to controls, and a placebo-controlled trial of therapeutic doses of thiamine which showed a clinically dramatic and statistically significant improvement in ataxia. A long-term thiamine supplementation program for susceptible individuals in endemic areas may be effective in the control and eventual eradication of the disease.

Thiamine deficiency (TD) impairs hippocampal neurogenesis. However, the mechanisms involved are not identified. In this work, TD mouse model was generated using a thiamine-depleted diet at two time points, TD9 and TD14 for 9 and 14 days of TD respectively. The activities of pyruvate dehydrogenase (PDH), alpha-ketoglutamate dehydrogenase (KGDH), glucose-6-phosphate dehydrogenase (G6PD), and transketolase (TK), as well as on the contents of NADP(+) and NADPH were determined in whole mouse brain, isolated cortex, and hippocampus of TD mice model. The effects of TK silencing on the growth and migratory ability of cultured hippocampal progenitor cells (HPC), as well as on neuritogenesis of hippocampal neurons were explored. The results showed that TD specifically reduced TK activity in both cortex and hippocampus, without significantly affecting the activities of PDH, KGDH, and G6PD in TD9 and TD14 groups. The level of whole brain and hippocampal NADPH in TD14 group were significantly lower than that of control group. TK silencing significantly inhibited the proliferation, growth, and migratory abilities of cultured HPC, without affecting neuritogenesis of cultured hippocampal neurons. Taken together, these results demonstrate that decreased TK activity leads to pentose-phosphate pathway dysfunction and contributes to impaired hippocampal neurogenesis induced by TD. TK and pentose-phosphate pathway may be considered new targets to investigate hippocampal neurogenesis.

The enantiomeric ratio and absolute configuration of products of the transketolase reaction are typically determined by comparison of the specific rotation or derivatisation and HPLC or GC. A Mosher’s ester method has been developed via ester formation at the primary alcohol C-1 which can be used to determine the stereoselectivity of the reaction, as well as the absolute configuration of the product at C-3.