Molecular Characteristics and Expression of TKTL1 in Germ Cells: Implications for Nontumour Cell Research.

TKTL1 is a crucial regulatory enzyme in the pentose phosphate pathway (PPP) and plays a significant role in energy synthesis. It is expressed in various tumour tissues, with its expression level closely associated with tumour invasion, metastasis and prognosis. Recent studies utilising proteomic analysis and other methods have highlighted the noteworthy expression of the TKTL1 gene in germ cells, particularly in spermatogonia and ovarian cells. Consequently, this article reviews the molecular characteristics of TKTL1 and its expression in germ cells to provide a reference for research on TKTL1 beyond tumour cells.

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 (vitamin B1) is a neurotropic compound that improves neuropathies. Our pilot study characterizes therapeutic potential of daily oral administration of thiamine (100 mg) 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 (200 mg) or benfotiamine (150 mg) 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.

Association of transketolase polymorphisms with diabetic polyneuropathy in the general population: The KORA F4 study.

AIMS: We recently reported that genetic variability in the TKT gene encoding transketolase, a key enzyme in the pentose phosphate pathway, is associated with measures of diabetic sensorimotor polyneuropathy (DSPN) in recent-onset diabetes. Here, we aimed to substantiate these findings in a population-based KORA F4 study. MATERIALS AND METHODS: In this cross-sectional study, we assessed seven single nucleotide polymorphisms (SNPs) in the transketolase gene in 952 participants from the KORA F4 study with normal glucose tolerance (NGT; n = 394), prediabetes (n = 411), and type 2 diabetes (n = 147). DSPN was defined by the examination part of the Michigan Neuropathy Screening Instrument (MNSI) using the original MNSI > 2 cut-off and two alternative versions extended by touch/pressure perception (TPP) (MNSI > 3) and by TPP plus cold perception (MNSI > 4). RESULTS: After adjustment for sex, age, BMI, and HbA1c, in type 2 diabetes participants, four out of seven transketolase SNPs were associated with DSPN for all three MNSI versions (all p ≤ 0.004). The odds ratios of these associations increased with extending the MNSI score, for example, OR (95% CI) for SNP rs62255988 with MNSI > 2: 1.99 (1.16-3.41), MNSI > 3: 2.27 (1.26-4.09), and MNSI > 4: 4.78 (2.22-10.26); SNP rs9284890 with MNSI > 2: 2.43 (1.42-4.16), MNSI > 3: 3.46 (1.82-6.59), and MNSI > 4: 4.75 (2.15-10.51). In contrast, no associations were found between transketolase SNPs and the three MNSI versions in the NGT and prediabetes groups. CONCLUSIONS: The link of genetic variation in transketolase enzyme to diabetic polyneuropathy corroborated at the population level strengthens the concept suggesting an important role of pathways metabolising glycolytic intermediates in the evolution of diabetic polyneuropathy.

Multilocus sequence typing database for Streptococcus agalactiae contains a spurious allele of the transketolase gene.

The tkt (transketolase) gene is one of the seven gene fragments used in the multilocus sequence typing (MLST) system for Streptococcus agalactiae. We discovered that the tkt_134 allele is derived from a homologous gene (which we designate tktX) that is not present in all S. agalactiae; all known strains that contain a match to the tkt_134 allele also contain a gene sequence that is much closer in sequence identity to the other non-tkt_134 alleles (i.e., the canonical tkt gene) in the database. Based on these data, the tkt_134 allele has been removed from the MLST database as of September 2021, and all sequence types containing tkt_134 have also been removed.IMPORTANCEMultilocus sequence typing (MLST) databases are a common good and remain important for research, medical, and epidemiological purposes. This remains true even in the context of widespread whole-genome sequencing. We discovered a contaminating allele of the tkt gene in the S. agalactiae MLST database that led to unstable, ambiguous, or erroneous MLST assignment. The allele has since been removed from the public database based on the results presented in this manuscript.

HMGA1 promotes the progression of esophageal squamous cell carcinoma by elevating TKT-mediated upregulation of pentose phosphate pathway.

Esophageal squamous cell carcinoma (ESCC) possesses a poor prognosis and treatment outcome. Dysregulated metabolism contributes to unrestricted growth of multiple cancers. However, abnormal metabolism, such as highly activated pentose phosphate pathway (PPP) in the progression of ESCC remains largely unknown. Herein, we report that high-mobility group AT-hook 1 (HMGA1), a structural transcriptional factor involved in chromatin remodeling, promoted the development of ESCC by upregulating the PPP. We found that HMGA1 was highly expressed in ESCC. Elevated HMGA1 promoted the malignant phenotype of ESCC cells. Conditional knockout of HMGA1 markedly reduced 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumorigenesis in mice. Through the metabolomic analysis and the validation assay, we found that HMGA1 upregulated the non-oxidative PPP. With the transcriptome sequencing, we identified that HMGA1 upregulated the expression of transketolase (TKT), which catalyzes the reversible reaction in non-oxidative PPP to exchange metabolites with glycolytic pathway. HMGA1 knockdown suppressed the PPP by downregulating TKT, resulting in the reduction of nucleotides in ESCC cells. Overexpression of HMGA1 upregulated PPP and promoted the survival of ESCC cells by activating TKT. We further characterized that HMGA1 promoted the transcription of TKT by interacting with and enhancing the binding of transcription factor SP1 to the promoter of TKT. Therapeutics targeting TKT with an inhibitor, oxythiamine, reduced HMGA1-induced ESCC cell proliferation and tumor growth. Together, in this study, we identified a new role of HMGA1 in ESCCs by upregulating TKT-mediated activation of PPP. Our results provided a new insight into the role of HMGA1/TKT/PPP in ESCC tumorigenesis and targeted therapy.

Glucose-6-phosphate dehydrogenase and transketolase: Key factors in breast cancer progression and therapy.

Breast cancer is one of the most common malignant tumors in women and is a serious threat to women's health. The pentose phosphate pathway (PPP) is a mode of oxidative breakdown of glucose that can be divided into oxidative (oxPPP) and non-oxidative (non-oxPPP) stages and is necessary for cell and body survival. However, abnormal activation of PPP often leads to proliferation, migration, invasion, and chemotherapy resistance in breast cancer. Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in PPP oxidation. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) produced by G6PD is the raw material for cholesterol and lipid synthesis and can resist the production of oxygen species (ROS) and reduce oxidative stress damage to tumor cells. Transketolase (TKT) is a key enzyme in non-oxPPP. Ribose 5-phosphate (R5P), produced by TKT, is a raw material for DNA and RNA synthesis, and is essential for tumor cell proliferation and DNA damage repair. In this review, we describe the role and specific mechanism of the PPP and the two most important enzymes of the PPP, G6PD and TKT, in the malignant progression of breast cancer, providing strategies for future clinical treatment of breast cancer and a theoretical basis for breast cancer research.

Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability.

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution.

Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559-31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

Mapping Conformational Changes in the Saliva Proteome Potentially Associated with Oral Cancer Aggressiveness.

Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.

Transketolase promotes MAFLD by limiting inosine-induced mitochondrial activity.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has a global prevalence of about 25% and no approved therapy. Using metabolomic and proteomic analyses, we identified high expression of hepatic transketolase (TKT), a metabolic enzyme of the pentose phosphate pathway, in human and mouse MAFLD. Hyperinsulinemia promoted TKT expression through the insulin receptor-CCAAT/enhancer-binding protein alpha axis. Utilizing liver-specific TKT overexpression and knockout mouse models, we demonstrated that TKT was sufficient and required for MAFLD progression. Further metabolic flux analysis revealed that Tkt deletion increased hepatic inosine levels to activate the protein kinase A-cAMP response element binding protein cascade, promote phosphatidylcholine synthesis, and improve mitochondrial function. Moreover, insulin induced hepatic TKT to limit inosine-dependent mitochondrial activity. Importantly, N-acetylgalactosamine (GalNAc)-siRNA conjugates targeting hepatic TKT showed promising therapeutic effects on mouse MAFLD. Our study uncovers how hyperinsulinemia regulates TKT-orchestrated inosine metabolism and mitochondrial function and provides a novel therapeutic strategy for MAFLD prevention and treatment.

Transketolase drives the development of aortic dissection by impairing mitochondrial bioenergetics.

AIM: Aortic dissection (AD) is a disease with rapid onset but with no effective therapeutic drugs yet. Previous studies have suggested that glucose metabolism plays a critical role in the progression of AD. Transketolase (TKT) is an essential bridge between glycolysis and the pentose phosphate pathway. However, its role in the development of AD has not yet been elucidated. In this study, we aimed to explore the role of TKT in AD. METHODS: We collected AD patients' aortic tissues and used high-throughput proteome sequencing to analyze the main factors influencing AD development. We generated an AD model using BAPN in combination with angiotensin II (Ang II) and pharmacological inhibitors to reduce TKT expression. The effects of TKT and its downstream mediators on AD were elucidated using human aortic vascular smooth muscle cells (HAVSMCs). RESULTS: We found that glucose metabolism plays an important role in the development of AD and that TKT is upregulated in patients with AD. Western blot and immunohistochemistry confirmed that TKT expression was upregulated in mice with AD. Reduced TKT expression attenuated AD incidence and mortality, maintained the structural integrity of the aorta, aligned elastic fibers, and reduced collagen deposition. Mechanistically, TKT was positively associated with impaired mitochondrial bioenergetics by upregulating AKT/MDM2 expression, ultimately contributing to NDUFS1 downregulation. CONCLUSION: Our results provide new insights into the role of TKT in mitochondrial bioenergetics and AD progression. These findings provide new intervention options for the treatment of AD.

Design, Synthesis, and Herbicidal Activity of Pyrazole Amide Derivatives as Potential Transketolase Inhibitors.

The design and synthesis of new herbicidal active compounds based on a new target are of great significance for the development of new herbicides. Transketolase (TK) plays a key role in the Calvin cycle of plant photosynthesis and has been confirmed as a potential candidate target to develop and discover new herbicides. To obtain compounds with ultraefficient targeting of TK, a series of pyrazole amide derivatives were designed and synthesized through structural optimization for lead compound 4u based on TK as the new target. The bioassay results showed that compounds 6ba and 6bj displayed a highly inhibitory effect with the root inhibition of about 90% against Digitaria sanguinalis (DS) and 80% against Amaranthus retroflexus (AR) and Setaria viridis (SV) by the small cup method, which was better than the positive control mesotrione and nicosulfuron. Furthermore, compounds 6ba and 6bj exhibited an excellent inhibitory effect with the inhibition of about 80% (against DS) and over 80% (against SV) at the dosage of 150 g of active ingredient/ha by the foliar spray method. The TK enzyme activity inhibition test showed that the inhibition effect of target compounds against TK was consistent with the results of herbicidal activities. Also, molecular docking analysis showed that compounds 6ba and 6bj went deep into the active cavity of TK, bound to TK by a strong interaction, and might act on the enzyme TK. Above of all, compounds 6ba and 6bj are promising herbicide lead compounds targeting TK. Hence, they could be developed into more efficient herbicides by further structural optimization.

TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) stands as the fifth most prevalent malignant tumor on a global scale and presents as the second leading cause of cancer-related mortality. DNA damage-based radiotherapy (RT) plays a pivotal role in the treatment of HCC. Nevertheless, radioresistance remains a primary factor contributing to the failure of radiation therapy in HCC patients. In this study, we investigated the functional role of transketolase (TKT) in the repair of DNA double-strand breaks (DSBs) in HCC. Our research unveiled that TKT is involved in DSB repair, and its depletion significantly reduces both non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair. Mechanistically, TKT interacts with PARP1 in a DNA damage-dependent manner. Furthermore, TKT undergoes PARylation by PARP1, resulting in the inhibition of its enzymatic activity, and TKT can enhance the auto-PARylation of PARP1 in response to DSBs in HCC. The depletion of TKT effectively mitigates the radioresistance of HCC, both in vitro and in mouse xenograft models. Moreover, high TKT expression confers resistance of RT in clinical HCC patients, establishing TKT as a marker for assessing the response of HCC patients who received cancer RT. In summary, our findings reveal a novel mechanism by which TKT contributes to the radioresistance of HCC. Overall, we identify the TKT-PARP1 axis as a promising potential therapeutic target for improving RT outcomes in HCC.

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.

Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Enrichment of grape berries and tomato fruit with health-promoting tartaric acid by expression of the Vitis vinifera transketolase VvTK2 gene.

Tartaric acid (TA) is a major non-fermentable plant soluble acid that abundantly occur in grapes and wines, imparting low pH and tart flavour to berries thereby regulating numerous quality attributes of wine, such as flavour, microbial stability, and aging potential. Evaluation of acidity in mature fruits of 21 wine grape (Vitis vinifera) varieties revealed significant variation between 'Beichun' and 'Gewürztraminer', which was correlated with TA content. RNA-seq analysis of fruits from the two cultivars at different developmental stages revealed that a transketolase gene, VvTK2, was significantly dominantly expressed in the high TA phenotype 'Beichun' variety. Subcellular localization assay showed that VvTK2 protein was located in the chloroplast. Virus-induced VvTK2 gene silencing significantly decreased the expression of 2-keto-L-gulonic acid reductase (Vv2-KGR) as well as L-idonate dehydrogenase (VvL-IdnDH3) and inhibited TA accumulation, while its transient over-expression in grape showed the opposite results. Heterologous VvTK2 over-expression in tomato demonstrated its obvious capacity to induce TA synthesis. Overall, these results highlights a novel role of VvTK2 in modulating TA biosynthesis, which could be an excellent strategy for future genetic improvement of grape flavour.

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.

Structural determination and kinetic analysis of the transketolase from Vibrio vulnificus reveal unexpected cooperative behavior.

Vibrio vulnificus (vv) is a multidrug-resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose-phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose-6-phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady-state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38-49 inhibitor (2-(4-ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3-b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains.

Utility of peripheral blood macrophage factor Apo10 and TKTL1 as markers in distinguishing malignant from benign lung nodules: a protocol for a prospective cohort study in Southern China.

INTRODUCTION: Lung nodules are one of the most prevalent diseases. Medical imaging methods have a high false positive rate for distinguishing malignant nodules from benign nodules. Therefore, developing new technologies with high accuracy for screening malignant nodules is of great importance for lung nodule surveillance. Use of flow cytometry to detect biomarkers in blood macrophages (epitop detect in macrophages/macrophages) has opened a new era for early and noninvasive diagnosis of cancer. This planned study aims to examine whether the peripheral blood macrophage factors Apo10 and TKTL1 accurately distinguish malignant nodules from benign nodules. METHODS AND ANALYSES: We plan to enrol in this study 3825 participants with lung nodules who will attend their annual physical examination at Sun Yat-sen University Cancer Center. Apo10 and TKTL1 levels in all patients will be tested at 60 min after their last meal every 6 months during their 3-year follow-up. Biopsy or surgical pathology results will be collected as the gold standard to assess the accuracy of Apo10 and TKTL1 in distinguishing malignant nodules from benign nodules. The sensitivity, specificity, positive predictive value, negative predictive value and area under the receiving operating characteristic curve will also be evaluated. ETHICS AND DISSEMINATION: The study is approved by the medical ethics committee of Sun Yat-sen University (SL-G2022-005-02). The results of this study will be disseminated in peer-reviewed publications and presentations at international scientific meetings and will also be disseminated to the participants. TRIAL REGISTRATION NUMBER: ChiCTR2300073823; Pre-results.

Antimalarial Activity of Anacardium occidentale Leaf Extracts Against Plasmodium falciparum Transketolase (PfTK).

BACKGROUND: As per estimates by WHO in 2021 almost half of the world's population was at risk of malaria and > 0.6 million deaths were attributed to malaria. Therefore, the present study was aimed to explore the antimalarial activity of extracts derived from the leaves of the plant Anacardium occidentale L., which has been used traditionally for the treatment of malaria. Different extracts of A. occidentale leaves were prepared and tested for their inhibitory activity against recombinant P. falciparum transketolase (rPfTK) enzyme, in vitro. Further, growth inhibitory activity against cultivated blood stage P. falciparum parasites (3D7 strain), was studied using SYBR Green fluorescence-based in vitro assays. Acute toxicity of the hydro alcoholic extracts of leaves of A. occidentale (HELA) at different concentrations was evaluated on mice and Zebra fish embryos. HELA showed 75.45 ± 0.35% inhibitory activity against the recombinant PfTk and 99.31 ± 0.08% growth inhibition against intra-erythrocytic stages of P. falciparum at the maximum concentration (50 µg/ml) with IC50 of 4.17 ± 0.22 µg/ml. The toxicity test results showed that the heartbeat, somite formation, tail detachment and hatching of embryos were not affected when Zebra fish embryos were treated with 0.1 to 10 µg/ml of the extract. However, at higher concentrations of the extract, at 48 h (1000 µg/ml) and 96 h (100 µg/ml and 1000 µg/ml, respectively) there was no heartbeat in the fish embryos. In the acute oral toxicity tests performed on mice, the extract showed no toxicity up to 300 mg/kg body weight in mice. CONCLUSION: The hydro-alcoholic extract of leaves of A. occidentale L. showed potent antimalarial activity against blood stage P. falciparum. Based on the observed inhibitory activity on the transketolase enzyme of P. falciparum it is likely that this enzyme is the target for the development of bioactive molecules present in the plant extracts. The promising anti-malarial activity of purified compounds from leaves of A. occidentale needs to be further explored for development of new anti-malarial therapy.