Transketolase Activity Research Articles

Pharmacological Doses of Thiamine Benefit Patients with the Charcot-Marie-Tooth Neuropathy by Changing Thiamine Diphosphate Levels and Affecting Regulation of Thiamine-Dependent Enzymes.

Charcot-Marie-Tooth (CMT) neuropathy is a polygenic disorder of peripheral nerves with no effective cure. Thiamine (vitaminB1) is a neurotropic compound that improves neuropathies. Our pilot study characterizes therapeutic potential of daily oral administration of thiamine (100mg) in CMT neuropathy and its molecular mechanisms. The patient hand grip strength was determined before and after thiamine administration along with the blood levels of the thiamine coenzyme form (thiamine diphosphate, ThDP), activities of endogenous holo-transketolase (without ThDP in the assay medium) and total transketolase (with ThDP in the assay medium), and transketolase activation by ThDP [1-(holo-transketolase/total transketolase),%], corresponding to the fraction of ThDP-free apo-transketolase. Single cases of administration of sulbutiamine (200mg) or benfotiamine (150mg) reveal their effects on the assayed parameters within those of thiamine. Administration of thiamine or its pharmacological forms increased the hand grip strength in the CMT patients. Comparison of the thiamin status in patients with different forms of CMT disease to that of control subjects without diagnosed pathologies revealed no significant differences in the average levels of ThDP, holo-transketolase, or relative content of holo and apo forms of transketolase. However, the regulation of transketolase by thiamine/ThDP differed in the control and CMT groups: in the assay, ThDP activated transketolase from the control individuals, but not from CMT patients. Thiamine administration paradoxically decreased endogenous holo-transketolase in CMT patients; this effect was not observed in the control group. Correlation analysis revealed sex-specific differences in the relationship between the parameters of thiamine status in both the control subjects and patients with the CMT disease. Thus, our findings link physiological benefits of thiamine administration in CMT patients to changes in their thiamine status, in particular, the blood levels of ThDP and transketolase regulation.

Exploring the impact of age, and body condition score on erythrocytic B1-Dependent transketolase activity in cats: A comprehensive analysis of thiamine status

One of the key factors influencing aging and morbidity is the overall antioxidant status and regenerative capacity. In examining contributors to the antioxidant status, we analyzed the thiamine status in felines and the influence of age, gender, and body condition score. We measured erythrocytic B1-dependent specific transketolase (STKT) activity, an enzyme in the pentose phosphate pathway, in a group of 60 sexually intact, healthy, and specific pathogen-free felines (44 females, 16 males, aged 1–17 years) with thiamine diphosphate (TDP; 0.3 and 3 mM) and without it. Only two parameters (STKT activity with and without 0.3 mM TDP) decreased with age. After adjusting for age, statistical thresholds were established using these and other age-independent parameters, identifying 15 felines with subclinical thiamine deficiency. The red blood cell proteomics analysis revealed that the pentose phosphate shunt, glycolysis, and oxidative stress response were the most affected pathways in deficient felines, confirming the above diagnosis. Age emerged as the primary factor associated with thiamine deficiency, supported by the enrichment of neurodegenerative diseases with a proteotoxicity component; five young-adult felines showed marginal or acute B1 deficiency, and six were adult-mature with a more chronic deficiency, possibly linked to cognitive decline, all with an underweight to ideal body condition scores. Only three senior-adult felines were deficient and overweight-obese. Detecting thiamine deficiency emphasizes the need for more accurate reference values, the establishment of advanced preventive or therapeutic measures to enhance the well-being of aging companion animals, and potential extensions to human health, particularly concerning cognitive function.

Protocol for a seamless phase 2A-phase 2B randomized double-blind placebo-controlled trial to evaluate the safety and efficacy of benfotiamine in patients with early Alzheimer's disease (BenfoTeam).

Benfotiamine provides an important novel therapeutic direction in Alzheimer's disease (AD) with possible additive or synergistic effects to amyloid targeting therapeutic approaches. To conduct a seamless phase 2A-2B proof of concept trial investigating tolerability, safety, and efficacy of benfotiamine, a prodrug of thiamine, as a first-in-class small molecule oral treatment for early AD. This is the protocol for a randomized, double-blind, placebo-controlled 72-week clinical trial of benfotiamine in 406 participants with early AD. Phase 2A determines the highest safe and well-tolerated dose of benfotiamine to be carried forward to phase 2B. During phase 2A, real-time monitoring of pre-defined safety stopping criteria in the first approximately 150 enrollees will help determine which dose (600 mg or 1200 mg) will be carried forward into phase 2B. The phase 2A primary analysis will test whether the rate of tolerability events (TEs) is unacceptably high in the high-dose arm compared to placebo. The primary safety endpoint in phase 2A is the rate of TEs compared between active and placebo arms, at each dose. The completion of phase 2A will seamlessly transition to phase 2B without pausing or stopping the trial. Phase 2B will assess efficacy and longer-term safety of benfotiamine in a larger group of participants through 72 weeks of treatment, at the selected dose. The co-primary efficacy endpoints in phase 2B are CDR-Sum of Boxes and ADAS-Cog13. Secondary endpoints include safety and tolerability measures; pharmacokinetic measures of thiamine and its esters, erythrocyte transketolase activity as blood markers of efficacy of drug delivery; ADCS-ADL-MCI; and MoCA. The BenfoTeam trial utilizes an innovative seamless phase 2A-2B design to achieve proof of concept. It includes an adaptive dose decision rule, thus optimizing exposure to the highest and best-tolerated dose. ClinicalTrials.gov identifier: NCT06223360, registered on January 25, 2024. https://classic.clinicaltrials.gov/ct2/show/NCT06223360.

Assessment of Erythrocyte Transketolase, Whole Blood Thiamine Diphosphate, and Human Milk Thiamine Concentrations to Identify Infants and Young Children Responding Favorably to Therapeutic Thiamine Administration: Findings from the Lao Thiamine Study, a Prospective Cohort Study

BackgroundThere is limited information on relationships among biomarkers of thiamine status (whole blood thiamine diphosphate [ThDP], erythrocyte transketolase activity coefficient [ETKac], and human milk thiamine [MTh]) and clinical manifestations of thiamine deficiency. ObjectivesThis study aimed to explore correlations among these biomarkers and thiamine responsive disorders (TRDs), a diagnosis based on favorable clinical response to thiamine. MethodsHospitalized infants and young children (aged 21 d to <18 mo) with respiratory, cardiac, and/or neurological symptoms suggestive of thiamine deficiency were treated with parenteral thiamine (100 mg daily) for ≥3 d alongside other treatments and re-examined systematically. Clinical case reports were reviewed by 3 pediatricians, who determined TRD or non-TRD status. Children in a community comparison group were matched by age, sex, and residence. Venous whole blood ThDP and MTh were determined by high-performance liquid chromatography fluorescence detection and ETKac in washed erythrocytes by ultraviolet spectrophotometry. Associations between biomarkers were assessed using Spearman correlations, and biomarker cutoffs predictive of TRD and ETKac >1.25 were explored using area under the receiver operating characteristic curve framework. ResultsThiamine biomarkers were available for 287 hospitalized children and 228 community children (mean age 4.7 mo; 59.4% male). Median (interquartile range [IQR]) ThDP and ETKac were 66.9 nmol/L (IQR: 41.4, 96.9 nmol/L) and 1.25 nmol/L (IQR: 1.11, 1.48 nmol/L), respectively, among hospitalized children, and 64.1 nmol/L (IQR: 50.0, 85.3 nmol/L) and 1.22 nmol/L (IQR: 1.12, 1.37 nmol/L) among 228 community children (P > 0.05 for both). Forty-five percent of breastfeeding mothers of infants <6 mo had MTh <90 μg/L. ThDP and ETKac, but not MTh, were significantly different between 152 children with TRD and 122 without TRD, but overlapping distributions undermined prediction of individual responses to thiamine. ConclusionsAlthough ETKac, ThDP, and MTh are useful biomarkers of population thiamine status, none of the biomarkers reliably identified individual children with TRD. ThDP is more practical for population assessment because preparing washed erythrocytes is not required.This trial was registered at clinicaltrials.gov as NCT03626337.

Race explains substantial variance in whole blood thiamine diphosphate concentrations

Deficiency for thiamine (vitamin B1), traditionally assessed via the activity of the thiamine-dependent enzyme erythrocyte transketolase, has been reported in individuals with alcohol use disorder (AUD) and in people with HIV; concentrations of the metabolically active diphosphate form, however, have yet to be reported in HIV cohorts and results in AUD are equivocal. In this cross-sectional study, samples from 170 AUD, 130 HIV, and 100 healthy control individuals were analyzed to test the hypothesis that AUD and HIV groups relative to healthy controls would show low whole blood thiamine diphosphate (TDP) concentrations related to peripheral neuropathy. TDP concentrations were not different in the 3 study groups (P = .6141) but were lower in Black (n = 172) relative to White (n = 155) individuals (P < .0001) regardless of group. In a multiple regression, race relative to diagnoses explained more than 10 times the variance in whole blood TDP concentrations (F4,395 = 3.5, P = .0086; r2 = 15.1]. Performance on a measure of peripheral neuropathy (2-point discrimination) was worse in the HIV and AUD cohorts relative to the healthy control group (P < .0001) but was not associated with TDP concentrations. These findings suggest that Black individuals carry a heightened vulnerability for low whole blood TDP concentrations, but the clinical significance and mechanisms underlying these results remain to be determined.

Posttranslational Acylations of the Rat Brain Transketolase Discriminate the Enzyme Responses to Inhibitors of ThDP-Dependent Enzymes or Thiamine Transport.

Transketolase (TKT) is an essential thiamine diphosphate (ThDP)-dependent enzyme of the non-oxidative branch of the pentose phosphate pathway, with the glucose-6P flux through the pathway regulated in various medically important conditions. Here, we characterize the brain TKT regulation by acylation in rats with perturbed thiamine-dependent metabolism, known to occur in neurodegenerative diseases. The perturbations are modeled by the administration of oxythiamine inhibiting ThDP-dependent enzymes in vivo or by reduced thiamine availability in the presence of metformin and amprolium, inhibiting intracellular thiamine transporters. Compared to control rats, chronic administration of oxythiamine does not significantly change the modification level of the two detected TKT acetylation sites (K6 and K102) but doubles malonylation of TKT K499, concomitantly decreasing 1.7-fold the level of demalonylase sirtuin 5. The inhibitors of thiamine transporters do not change average levels of TKT acylation or sirtuin 5. TKT structures indicate that the acylated residues are distant from the active sites. The acylations-perturbed electrostatic interactions may be involved in conformational shifts and/or the formation of TKT complexes with other proteins or nucleic acids. Acetylation of K102 may affect the active site entrance/exit and subunit interactions. Correlation analysis reveals that the action of oxythiamine is characterized by significant negative correlations of K499 malonylation or K6 acetylation with TKT activity, not observed upon the action of the inhibitors of thiamine transport. However, the transport inhibitors induce significant negative correlations between the TKT activity and K102 acetylation or TKT expression, absent in the oxythiamine group. Thus, perturbations in the ThDP-dependent catalysis or thiamine transport manifest in the insult-specific patterns of the brain TKT malonylation and acetylations.

Effects of Low Light after Heading on the Yield of Direct Seeding Rice and Its Physiological Response Mechanism.

As a photophilous plant, rice is susceptible to low-light stress during its growth. The Sichuan Basin is a typical low-light rice-producing area. In this study, eight rice varieties with different shade tolerances were studied from 2021 to 2022. The physiological adaptability and yield formation characteristics of rice were studied with respect to photosynthetic physiological characteristics and dry matter accumulation characteristics, and the response mechanism of rice to low light stress was revealed. The results showed that the shading treatment significantly increased the chlorophyll a, chlorophyll b, and total chlorophyll contents in the leaves of direct-seeded rice after heading, and the total chlorophyll content increased by 1.68-29.70%. Nitrate reductase (NR) activity first increased and then decreased under each treatment, and the shading treatment reduced the NR activity of direct-seeded rice. Compared to the control treatment, the peroxidase (POD) activity of each variety increased from 7 to 24 d after the shading treatment. The transketolase (TK) activity in direct-seeded hybrid rice increased under low light stress. Compared with the control, shading treatment significantly reduced the aboveground dry matter, grain number per panicle, and seed setting rate of direct-seeded rice at the full heading stage and maturity stage, thus reducing the yield of direct-seeded rice by 26.10-34.11%. However, under the shading treatment, Zhenliangyou 2018 and Jingliangyou 534 maintained higher chlorophyll content and related enzyme activities, accumulated more photosynthetic products, and reduced yield. In general, Zhenliangyou 2018 and Jingliangyou 534 still had a yield of 7.06-8.33 t·hm-2 under low light. It indicated that Zhenliangyou 2018 and Jingliangyou 534 had better stability and stronger tolerance to weak light stress and had a higher yield potential in weak light areas such as Sichuan.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

Search for Inhibitors of Mycobacterium tuberculosis Transketolase in a Series of Sulfo-Substituted Compounds.

As a result of the computer screening of a library of sulfo-substituted compounds, molecules capable of binding to the active site of transketolase from Mycobacterium tuberculosis were identified. An experimental verification of the inhibitory activity of the most promising compound, STK045765, against a highly purified recombinant enzyme preparation was carried out. It was shown that the STK045765 molecule competes for the binding site of the pyrophosphate group of the thiamine diphosphate cofactor and, at a micromolar concentrations, is able to suppress the activity of mycobacterial transketolase. The discovered furansulfonate scaffold may serve as the basis for the creation of anti-tuberculosis drugs.

Efficacy of dietary thiamine supplementation on growth performance, digestive enzymes, immunological responses, and antioxidant capacity of juvenile beluga (Huso huso)

The aim of the current study was to investigate the beneficial effects of dietary thiamine supplementation on physiological, immunological and antioxidant responses in juvenile beluga, Huso huso. The juvenile fish (∼ 3 g) were fed four times a day with diets containing 0 (control), 5 (5 T), 10 (10 T), and 20 (20 T) mg kg−1 thiamine for 12 weeks. Significant increases were observed in growth performance indicators of the fish fed different levels of thiamine, compared to the control group and the best performance was observed in 10 T treatment. The carcass protein content significantly increased in the thiamine-treated fish and the highest protein content was observed in the 10 T treatment. Digestive enzymes’ activities (pepsin, trypsin, lipase, and amylase) significantly increased in the fish fed the thiamine-supplemented diets. The serum alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase activities significantly decreased in the 10 T and 20 T treatments, compared to the control group. Dietary thiamine supplementation significantly increased the serum lysozyme, alternative complement activities, and IgM level, compared to the control diet and the highest lysozyme activity and IgM level were observed in the 10 T and 20 T treatments. The hepatic superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase, transketolase activities of 10 T and 20 T treatments were significantly higher than those of the control treatment. The hepatic catalase activities in the thiamine-treated fish were significantly higher than that of the control fish. Contrastingly, the hepatic malondialdehyde contents of the 5 T, 10 T, and 20 T treatments were significantly lower than the control treatment. Although all the dietary thiamine levels (5–20 mg kg−1) showed beneficial effects on different tested parameters, 10 mg kg−1 was the most beneficial level and is recommended for dietary inclusion to improve the growth and health of juvenile beluga.

Association between diabetes and thiamine status - A systematic review and meta-analysis

BackgroundThiamine (vitamin B1) is an essential cofactor in glucose metabolism, but it remains unclear whether thiamine status is lower in individuals with diabetes compared to individuals with normal glucose metabolism. AimsWe conducted a systematic review and meta-analysis to study whether the circulating concentrations of various thiamine analytes differ between people with and those without diabetes. MethodsPubMed and the Cochrane Central Register of Controlled Trials were searched according to the study protocol. The standardized mean difference (SMD) and 95 % confidence intervals (CI) of thiamine markers between individuals with and without diabetes were used as effect size (random effects model). Subgroup analysis considered albuminuria as an additional variable. ResultsOut of the 459 articles identified, 24 full-texts were eligible for the study, 20 of which qualified for the data analysis and four were evaluated for coherence. Compared to controls, individuals with diabetes showed lower concentrations of thiamine (pooled estimate SMD [95 % CI]: −0.97 [−1.89, −0.06]), thiamine monophosphate (−1.16 [−1.82, −0.50]), and total thiamine compounds (−1.01 [−1.48, −0.54]). Thiamine diphosphate (−0.72 [−1.54, 0.11] and erythrocyte transketolase activity (−0.42 [−0.90, 0.05]) tended to be lower in persons with diabetes than in controls without reaching statistical significance. Subgroup analysis showed that individuals with diabetes and albuminuria had lower thiamine levels than the controls (−2.68 [−5.34, −0.02]). ConclusionsDiabetes is associated with lower levels of various thiamine markers, suggesting that individuals with diabetes may have higher thiamine requirements than those without diabetes, but well-designed studies are required to confirm these findings.

The Thiamine-Dependent Hysteretic Behavior of Human Transketolase: Implications for Thiamine Deficiency

We have investigated the hysteretic properties of human transketolase with emphasis on its dependency on thiamine pyrophosphate concentration. As demonstrated previously, the reaction progress curves revealed a slow transition from an initial low velocity to a faster final steady-state velocity, characterized by the rate constant τ-1. The rate of the transition was dependent on the concentration of the thiamine pyrophosphate cofactor, with progressively longer transition times found as the concentration of thiamine pyrophosphate was decreased. At physiological thiamine pyrophosphate concentrations, the inverse rate constant was in the range of 10 to 20 min for fibroblast-derived transketolase and increased dramatically with only small decreases from these levels of thiamine pyrophosphate. Variation in the lag was found when transketolase from different individuals was examined. Moreover, at low levels of thiamine, the rate of the transition was different between fibroblast- and lymphoblast-derived transketolase. The substantial lag in formation of active holoenzyme and the findings of interindividual variation and cell type variation in the lag period suggest mechanisms for the loss of transketolase activity during thiamine deficiency and may explain, at least in part, the differential sensitivity to deficiency demonstrated by tissues and individuals.

Thiamine Pyrophosphate-Requiring Enzymes are Altered during Pyrithiamine-Induced Thiamine Deficiency in Cultured Human Lymphoblasts

The most common of the severe complications of thiamine deficiency are beriberi and Wernicke-Korsakoff syndrome. To help clarify the biochemical basis for these disorders, a cell culture system has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both transketolase and α-ketoglutarate dehydrogenase (α-KGDH) decreased at the same rate and to roughly the same levels in response to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzymatic activities, was found when different cell lines were compared. When exogenous thiamine pyrophosphate was added to the activity assays, differences between transketolase and α-KGDH became readily apparent. Only 25% of the lost transketolase activity was present as apo-enzyme, whereas 70% of the lost α-KGDH activity was present in the apo-enzyme form. For transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thiamine has a direct effect on the expression of the transketolase gene and/or protein.

Protocol and application of basal erythrocyte transketolase activity to improve assessment of thiamine status.

Thiamine (vitamin B1) is an essential micronutrient required as a cofactor in many metabolic processes. Clinical symptoms of thiamine deficiency are poorly defined, hence biomarkers of thiamine status are important. The erythrocyte transketolase activity coefficient (ETKac) is a sensitive measure of thiamine status, but its interpretation may be confounded where the availability of the transketolase enzyme is limited. Basal ETK activity per gram of hemoglobin provides a complementary biomarker of thiamine status; however, its measurement and calculation are poorly described. Here, we describe in detail the assessment of basal ETK activity, including the calculation of path length in microplates and the molar absorption coefficient of NADH specific to the assay, and the measurement of hemoglobin in sample hemolysates. To illustrate the application of the methods, we present ETKac and basal ETK activity from women in The Gambia and UK. In conclusion, we present a clear protocol for the measurement of basal ETK activity that will permit the harmonization of methods to improve replication between laboratories.

Transketolase in human Müller cells is critical to resist light stress through the pentose phosphate and NRF2 pathways

The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of “traffic flow” through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.

Randomised trial of intravenous thiamine and/or magnesium sulphate administration on erythrocyte transketolase activity, lactate concentrations and alcohol withdrawal scores

Alcohol withdrawal syndrome (AWS) occurs in 2% of patients admitted to U.K. hospitals. Routine treatment includes thiamine and benzodiazepines. Laboratory studies indicate that thiamine requires magnesium for optimal activity, however this has not translated into clinical practice. Patients experiencing AWS were randomized to three groups: (group 1) thiamine, (group 2) thiamine plus MgSO4 or (group 3) MgSO4. Pre- and 2-h post-treatment blood samples were taken. AWS severity was recorded using the Glasgow Modified Alcohol Withdrawal Score (GMAWS). The primary outcome measure was 15% change in erythrocyte transketolase activity (ETKA) in group 3. Secondary outcome measures were change in plasma lactate concentrations and time to GMAWS = 0. 127 patients were recruited, 115 patients were included in the intention-to-treat analysis. Pre-treatment, the majority of patients had normal or high erythrocyte thiamine diphosphate (TDP) concentrations (≥ 275–675/> 675 ng/gHb respectively) (99%), low serum magnesium concentrations (< 0.75 mmol/L) (59%), and high plasma lactate concentrations (> 2 mmol/L) (67%). Basal ETKA did not change significantly in groups 1, 2 or 3. Magnesium deficient patients (< 0.75 mmol/L) demonstrated less correlation between pre-treatment basal ETKA and TDP concentrations than normomagnesemic patients (R2 = 0.053 and R2 = 0.236). Median plasma lactate concentrations normalized (≤ 2.0 mmol/L) across all three groups (p < 0.001 for all groups), but not among magnesium deficient patients in group 1 (n = 22). The median time to achieve GMAWS = 0 for groups 1, 2 and 3 was 10, 5.5 and 6 h respectively (p < 0.001). No significant difference was found between groups for the primary endpoint of change in ETKA. Co-administration of thiamine and magnesium resulted in more consistent normalization of plasma lactate concentrations and reduced duration to achieve initial resolution of AWS symptoms.ClinicalTrials.gov: NCT03466528.

LncRNA PMAT–PtoMYB46 module represses PtoMATE and PtoARF2 promoting Pb2+ uptake and plant growth in poplar

Lead (Pb2+) is one of the most toxic heavy-metal contaminants. Fast-growing woody plants with substantial biomass are ideal for bioremediation. However, the transcriptional regulation of Pb2+ uptake in woody plants remains unclear. Here, we identified 226 Pb2+-induced, differentially expressed long non-coding RNAs (DELs) in Populus tomentosa. Functional annotation revealed that these DELs mainly regulate carbon metabolism, biosynthesis of secondary metabolites, energy metabolism, and signal transduction through their potential target genes. Association and epistasis analysis showed that the lncRNA PMAT (Pb2+-induced multidrug and toxic compound extrusion (MATE) antisense lncRNA) interacts epistatically with PtoMYB46 to regulate leaf dry weight, photosynthesis rate, and transketolase activity. Genetic transformation and molecular assays showed that PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth. We concluded that the PMAT–PtoMYB46–PtoMATE–PtoARF2 regulatory module control Pb2+ tolerance, uptake, and plant growth. This study demonstrates the involvement of lncRNAs in response to Pb2+ in poplar, yielding new insight into the potential for developing genetically improved woody plant varieties for phytoremediating lead-contaminated soils.

Thiamin and riboflavin status with related enzyme activities in pulmonary tuberculosis with diabetes mellitus in Shandong province of China.

Poor nutritional status is a common finding in pulmonary tuberculosis (TB) patients with and without type 2 diabetes mellitus (T2DM), thiamin (VB-1) and riboflavin (VB-2) are coenzymes important for the activation of many enzymes involved in improving nutritional status. We aimed to investigate enzymatic activities and the associations between VB-1 and VB-2, and their relations to nutritional status in TB and TB+T2DM patients. This was a cross-sectional study that prospectively enrolled TB 40 patients with or without T2DM respectively from the Chest Hospital of Qingdao and 76 healthy controls with similar age and gender distributions were recruited from the medical center of the affiliated hospital of Qingdao Medical College. The erythrocyte transketolase activation coefficient (ETKac, for VB-1 deficiency), the glutathione reductase activation coefficient (EGRac, for VB-2 deficiency), and metabolic enzyme activities were analyzed. VB-1 and VB-2 deficiency rates were higher, and enzyme activities were lower in TB and TB+T2DM relative to control group. ETKac and EGRac were negatively correlated with enzyme activities, either with body mass index (BMI), while enzyme activities were positively associated with BMI. VB-1 and VB-2 concentrations were lower in TB patients with or without T2DM relative to controls, with concomitant reductions in the activity levels of key metabolic enzymes. Significant correlations were observed between VB-1 and VB-2 concentrations and the activity of these metabolic enzymes, they all correlated with nutrition status. VB-1 and VB-2 concentrations may thus impact metabolic enzyme activity and thereby influence nutritional status.

Dietary thiamine requirement and its effects on glycolipid metabolism in oriental river prawn (Macrobrachium nipponense)

A 10-week feeding trial was conducted to investigate the optimal dietary thiamine requirement of oriental river prawn Macrobrachium nipponense with the effects on glycolipid metabolism evaluated. A total of 960 prawns with an average weight of 0.18 g were selected and randomly fed six semi-purified diets containing graded levels of thiamine (14.10, 35.20, 43.30, 61.40, 95.60 and 165.00 mg/kg, respectively) in an indoor water circulation system. Each diet was tested in 4 replicates. Final weight, weight gain, specific growth rate, protein efficiency rate, hemolymph thiamine and triglycerides concentrations all increased significantly (P < 0.05) with increasing dietary thiamine levels up to 61.40 mg/kg, and then decreased. A similar result was also observed in hemolymph transketolase activities except for the fact that the maximum value was observed in the 95.70 mg/kg group (P < 0.05). However, the opposite was true for hemolymph glucose and pyruvate concentrations with no statisticaldifference observed (P > 0.05). As dietary thiamine levels increased, the expressions of hexokinase and acetyl-CoA carboxylase both increased significantly (P < 0.05), and peaked at the 95.70 mg/kg group. Similar results were also observed in the expressions of glucose transporter 2, pyruvate kinase, glycogen synthase and fatty acid synthase except that the maximum values were all observed in the 61.40 mg/kg group. Furthermore, the expressions of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, carnitine palmitoyltransferase I and hormone-sensitive triglayceride lipase all decreased significantly (P < 0.05) with increasing dietary thiamine levels up to 61.40 mg/kg, and then plateaued. Based on the broken-line regression analysis of growth performance and hemolymph thiamine content, the optimal thiamine requirement of M.nipponense was estimated to be 66.80 and 67.57 mg/kg, respectively.

Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects.

ABSTRACTThe 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.