L-lactate Dehydrogenase Research Articles

Hesperidin Suppressed Colorectal Cancer through Inhibition of Glycolysis.

To explore the role of the natural compound hesperidin in glycolysis, the key ratelimiting enzyme, in colorectal cancer (CRC) cell lines. In vitro, HCT116 and SW620 were treated with different doses of hesperidin (0-500 µmol/L), cell counting kit-8 and colone formation assays were utilized to detected inhibition effect of hesperidin on CRC cell lines. Transwell and wound healing assays were performed to detect the ability of hesperidin (0, 25, 50 and 75 µmol/L) to migrate CRC cells. To confirm the apoptotic-inducing effect of hesperidin, apoptosis and cycle assays were employed. Western blot, glucose uptake, and lactate production determination measurements were applied to determine inhibitory effects of hesperidin (0, 25 and 50 µmol/L) on glycolysis. In vivo, according to the random number table method, nude mice with successful tumor loading were randomly divided into vehicle, low-dose hesperidin (20 mg/kg) and high-dose hesperidin (60 mg/kg) groups, with 6 mice in each group. The body weights and tumor volumes of mice were recorded during 4-week treatment. The expression of key glycolysis rate-limiting enzymes was determined using Western blot, and glucose uptake and lactate production were assessed. Finally, protein interactions were probed with DirectDIA Quantitative Proteomics, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Hesperidin could inhibit CRC cell line growth (P<0.05 or P<0.01). Moreover, hesperidin presented an inhibitory effect on the migrating abilities of CRC cells. Hesperidin also promoted apoptosis and cell cycle alterations (P<0.05). The immunoblotting results manifested that hesperidin decreased the levels of hexokinase 2, glucose transporter protein 1 (GLUT1), GLUT3, L-lactate dehydrogenase A, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), PFKFB3, and pyruvate kinase isozymes M2 (P<0.01). It remarkably suppressed tumor xenograft growth in nude mice. GO and KEGG analyses showed that hesperidin treatment altered metabolic function. Hesperidin inhibits glycolysis and is a potential therapeutic choice for CRC treatment.

Validation of Enzytec™ Liquid Citric Acid for Enzymatic Determination of Citric Acid in Selected Foods and Beverages: First Action 2024.02.

Due to its excellent acidifier, antioxidant, and preservative properties, citric acid is added to or is a constituent in a broad range of foods and beverages. Its measurement should be performed to assure the food quality specifications are met. To validate the performance of the Enzytec™ Liquid Citric acid test kit for the determination in food and beverages such as wines, juices, and tomato products. The kit contains two ready-to-use components which makes handling very easy and suitable for automation. Citrate is cleaved into oxaloacetate and acetate by citrate lyase. Oxaloacetate reacts to L-malate by L-malate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH). Pyruvate, spontaneously formed from oxaloacetate, is converted by L-lactate dehydrogenase to L-lactate. The NADH consumed is equivalent to the amount of citric acid converted and is measured at a wavelength of 340 nm within 20 min. The test is specific to citric acid and shows no relevant interferences. Limit of detection and limit of quantification are 15 and 40 mg/L, respectively when using a test volume of 100 µL. The linear measurement range is from 40 to 1000 mg/L citric acid. Trueness was evaluated using materials from FAPAS, NIST, LGC, and two control wines from the German Wine Analysts. Matrix interference was evaluated by spiking tomato ketchup, tomato paste, orange juice and carbonated beverages and resulted in recoveries around 100%. Intermediate precision is between 6.2% and 8.5% for matrixes with extraction and below 4% for matrixes measured directly.For automation, three applications with different test volumes and different measurement ranges were validated. Linearity is given from 8 mg/L up to 5 g/L (depending on the test volume). The method is robust and accurate for manual and automated applications. The method was approved as AOAC Official Method of Analysis℠. The ready-to-use components of the test kit have a shelf life of at least 24 months from date of manufacture.

Functionalized Metal-Organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

From agricultural biomass to D form lactic acid in ton scale via strain engineering of Lactiplantibacillus pentosus

Worsening environmental conditions make lactic acid a sustainable alternative to petroleum-based plastics. This study created a genetically-engineered strain Lactiplantibacillus pentosus PeL containing a disrupted L-lactate dehydrogenase gene to produce high yield and optically pure D-lactic acid. Cellobiose was identified as the optimal sugar in the single carbon source test, yielding the highest lactic acid. In 5-L fermentation tests, pretreated wood chips hydrolysate was the best lignocellulosic substrate for PeL, resulting in a D-lactic acid yield of 900.7 ± 141.4 mg/g of consumed sugars with an optical purity of 99.8 ± 0.0 %. Gradually scaled-up fermentations using this substrate were achieved in 100-, and 9,000-L fermenters; PeL produced remarkably high D-lactic acid yields of 836.3 ± 11.9 and 915.9 ± 4.4 mg/g of consumed sugars, with optical purities of 95.0 ± 0.0 % and 93.8 ± 0.2 %, respectively. This study is the pioneer in demonstrating economical and sustainable ton-scale production of D-lactic acid.

Peptides inhibiting the assembly of monomeric human l-lactate dehydrogenase into catalytically active homotetramer decrease the synthesis of lactate in cultured cells.

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.

Protein biomarkers in assessing kidney quality before transplantation‑current status and future perspectives (Review).

To meet the demand for kidney transplants (KTx), organs are frequently retrieved not only from standard criteria donors (SCD; a donor who is aged <50 years and suffered brain death from any number of causes, such as traumatic injuries or a stroke) but also from expanded criteria donors (any donor aged >60 years or donors aged >50 years with two of the following: A history of high blood pressure, a creatinine serum level ≥1.5 mg/dl or death resulting from a stroke). This comes at the cost of a higher risk of primary non‑function (the permanent hyperkalemia, hyperuremia and fluid overload that result in the need for continuous dialysis after KTx), delayed graft function (the need for dialysis session at least once during the first week after KTx), earlier graft loss and urinary complications (vesico‑ureteral reflux, obstruction of the vesico‑ureteral anastomosis, urine leakage). At present, there are no commercially available diagnostic tools for assessing kidney quality prior to KTx. Currently available predictive models based on clinical data, such as the Kidney Donor Profile Index, are insufficient. One promising option is the application of perfusion solutions for protein biomarkers of kidney quality and predictors of short‑ and long‑term outcomes. However, to date, protein markers that can be detected with ELISA, western blotting and cytotoxic assays have not been identified to be a beneficial predictors of kidney quality. These include lactate dehydrogenases, glutathione S‑transferases, fatty acid binding proteins, extracellular histones, IL‑18, neutrophil gelatinase‑associated lipocalin, MMPs and kidney injury molecule‑1. However, novel methods, including liquid chromatography‑mass spectrometry (LC‑MS) and microarrays, allow the analysis of all renal proteins suspended/dissolved in the acellular preservation solution used for kidney storage before KTx (including hypothermic machine perfusion as one of kidney storage methods) e.g. Belzer University of Wisconsin. Recent proteomic studies utilizing LC‑MS have identified complement pathway elements (C3, C1QB, C4BPA, C1S, C1R and C1RL), desmoplakin, blood coagulation pathway elements and immunoglobulin heavy variable 2‑26 to be novel predictors of kidney quality before transplantation. This was because they were found to correlate with estimated glomerular filtration rate at 3 and 12 months after kidney transplantation. However, further proteomic studies focusing on distinct markers obtained from hypothermic and normothermic machine perfusion are needed to confirm their predictive value and to improve kidney storage methods. Therefore, the present literature review from PubMed, Scopus, Embase and Web of Science was performed with the aims of summarizing the current knowledge on the most frequently studied single protein biomarkers. In addition, novel analytical methods and insights into organ injury during preservation were documented, where future directions in assessing organ quality before kidney transplantation were also discussed.

Two new oligomers of E. coli small heat-shock protein IbpB identified under heat stress exhibit maximum holding chaperone activity.

Escherichia coli small heat-shock protein IbpB (MW: 16 KDa) has holding chaperone activity and is present in cells at 30 °C as two large oligomers of MW 2.0-3.0 MDa and 600-700 KDa. We report here about the presence of two additional oligomers of MW around 400 and 130 KDa in cells under heat-stress at 50 °C. These two smaller oligomers possess the most chaperone activity, as observed from the extent of inhibition of inactivation and aggregation separately, of L-Lactate dehydrogenase in the presence of the individual oligomers at 52 and 60 °C, respectively. It is suggested here that the two larger oligomers act as poorly active storage forms, which under heat stress dissociate partially into smaller oligomers with high holdase activity.

A novel approach to enhance high optically active L-lactate production from food waste by landfill leachate

The recycling of food waste (FW) through anaerobic fermentation into lactic acid (LA), with two isomers L-LA and D-LA, aligns with the principles of a bio-based circular economy. However, FW fermentation is often limited by competing pathways, acidification inhibition, and trace metals deficiency. This study investigates the introduction of landfill leachate, containing buffering agents (ammonia) and trace metals, into FW fermentation. Various dosages of landfill leachate, ranging from 90 (LN-90) to 450 mg/L (LN-450) based on inclusive ammonia calculation, were employed. Results showed that LA production peaked at 43.65 ± 0.57 g COD/L in LN-180 on day 6, with a high optical activity of L-LA at 92.40 ± 1.15 %. Fermentation pathway analysis revealed that landfill leachate amendment enhances hydrolysis (as evidenced by increased activity of amylase, α-glucosidase, and protease) and glycolysis (resulting in enhanced utilization of carbohydrates and glucose). The inclusive ammonia in leachate plays a crucial role as a buffer, maintaining optimal pH conditions (5–7), thereby reducing volatile fatty acid production and thus intensifying LA orientations. The increased activity of L-lactate dehydrogenase (L-LA generation) and decreased NAD-independent lactate dehydrogenase (LA consumption) in properly dosed leachate further explained the high accumulation of L-LA. Dominance of lactic acid bacteria, including Streptococcus, Enterococcus, Klebsiella, Bifidobacterium, Bavariicoccus, and Lacticaseibacillus, accounted for 91.08% (LN-90), while inhibitory effects were observed in LN-450 (4.45%). Functional gene analysis further supported the enhanced glycolysis, L-lactate dehydrogenase, and nitrogen assimilation. Finally, a network analysis indicates a beneficial effect on the genus Enterococcus and Klebsiella by landfill leachate addition. This study demonstrates the efficiency of utilizing landfill leachate to enhance LA recycling from FW fermentation, aligning with the concept of circular economy by transforming waste into valuable resources.

L-Lactate dehydrogenase from Cyanidioschyzon merolae shows high catalytic efficiency for pyruvate reduction and is inhibited by ATP

l-Lactate is a commodity chemical used in various fields. Microorganisms have produced l-lactate via lactic fermentation using saccharides derived from crops as carbon sources. Recently, l-lactate production using microalgae, whose carbon source is carbon dioxide, has been spotlighted because the prices of the crops have increased. A red alga Cyanidioschyzon merolae produce l-lactate via lactic fermentation under dark anaerobic conditions. The l-lactate titer of C. merolae is higher than those of other microalgae but lower than those of heterotrophic bacteria. Therefore, an increase in the l-lactate titer is required in C. merolae. l-Lactate dehydrogenase (l-LDH) catalyzes the reduction of pyruvate to l-lactate during lactic fermentation. C. merolae possesses five isozymes of l-LDH. The results of previous transcriptome analysis suggested that l-LDHs are the key enzymes in the lactic fermentation of C. merolae. However, their biochemical characteristics, such as catalytic efficiency and tolerance for metabolites, have not been revealed. We compared the amino acid sequences of C. merolael-LDHs (CmLDHs) and characterized one of the isozymes, CmLDH1. BLAST analysis revealed that the sequence similarities of CmLDH1 and the other isozymes were above 99%. The catalytic efficiency of CmLDH1 under its optimum conditions was higher than those of l-LDHs of other organisms. ATP decreased the affinity and turnover number of CmLDH1 for NADH. These findings contribute to understanding the characteristics of l-LDHs of microalgae and the regulatory mechanisms of lactic fermentation in C. merolae.

Formate-Mediated Electroenzymatic Synthesis via Biological Cofactor NADH.

Synthetic biohybrid systems by coupling artificial system with nature's machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate-driven NADH regeneration. Utilizing a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90 % Faraday efficiency at a current density of 150 mA cm-2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilized redox enzymes-alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and L-glutamate dehydrogenase (GDH)-to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Effects of stannous fluoride dentifrice on gingival health and oxidative stress markers: a prospective clinical trial

BackgroundPeriodontal disease results in oral dysbiosis, increasing plaque virulence and oxidative stress. Stannous fluoride (SnF2) binds lipopolysaccharides to reduce plaque virulence. This study prospectively assessed SnF2 effects on oxidative stress in adults with gingivitis.MethodsThis was a 2-month, single-center, single-treatment clinical trial. Twenty “disease” (> 20 bleeding sites with ≥ 3 pockets 3 mm-4 mm deep) and 20 “healthy” (≤ 3 bleeding sites with pockets ≤ 2 mm deep) adults were enrolled. All participants were instructed to use SnF2 dentifrice twice daily for 2 months. An oral examination, Modified Gingival Index (MGI) examination and Gingival Bleeding Index (GBI) examination were conducted at baseline, 1 month and 2 months. Gingival crevicular fluid (GCF), saliva, oral lavage and supragingival plaque were collected at each visit to evaluate: Endotoxins, Protein Carbonyls, L-lactate dehydrogenase (LDH), Ferric reducing antioxidant power (FRAP), Oxidized low density lipoproteins (oxi-LDL), IL-6 and C-reactive protein (CRP). A subset-analysis examined participants considered at higher risk of cardiovascular disease. Change-from-baseline analyses within each group were of primary interest.ResultsThe disease group showed statistically significant reductions in GBI at Month 1 (67%) and Month 2 (85%) and in MGI at Month 1 (36%) and Month 2 (51%) versus baseline (p < 0.001). At baseline, the disease group showed greater LDH in GCF and oxi-LDL levels in saliva versus the healthy group (p ≤ 0.01). Total antioxidant capacity (FRAP) in saliva increased versus baseline for the disease group at Months 1 and 2 (p < 0.05), and levels for the disease group were greater than the healthy group at both timepoints (p < 0.05). SnF2 treatment reduced endotoxins (lavage) for both disease and healthy groups at Month 2 (p ≤ 0.021) versus baseline. There was a reduction in oxidative stress markers, namely protein carbonyl in saliva, at Months 1 and 2 (p < 0.001) for both groups and a reduction in cytokine IL-6 (lavage) in the disease group at Month 2 (p = 0.005). A subset analysis of participants at higher coronary disease risk showed reductions in endotoxins in lavage, oxi-LDL, and CRP in saliva at Month 2 (p ≤ 0.04).ConclusionSnF2 dentifrice use reversed gingival inflammation, suppressed endotoxins and reduced some harmful oxidant products in saliva and gingiva.Clinical trial registrationClinicaltrials.gov NCT05326373, registered on 13/04/2022.

One-pot enantioselective synthesis of chiral phenyllactic acids by combining stereocomplementary d- and l-lactate dehydrogenases with multi-enzyme expression fine-tuning

Chiral phenyllactic acid (PLA) is a new type of antiseptic agent and a valuable precursor for active ingredients in pharmaceuticals and agrochemicals. In this study, we designed a multi-enzyme cascade that combined stereocomplementary d- and l-lactate dehydrogenases with threonine aldolase, phenylserine dehydratase, and formate dehydrogenase for the one-pot conversion of achiral glycine and benzaldehyde to synthesize d-PLA and l-PLA. To overcome the imbalance of multi-enzymes in a single cell, two enzyme modules, overexpressing four enzymes, were assembled in Escherichia coli cells to construct whole-cell catalysis systems (WCCSs). Furthermore, by optimizing reaction conditions and components, recombinant E. coli (WCCS 26) was able to produce 100 mM d-PLA with >99 % ee using a fed-batch strategy, while E. coli (WCCS 60) produced 47.2 mM l-PLA with >99 % ee. This study presents a sustainable and efficient method for synthesizing chiral PLAs from food-grade achiral starting materials.

The L-lactate dehydrogenases of Pseudomonas aeruginosa are conditionally regulated but both contribute to survival during macrophage infection.

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in environments associated with human activity, including soil and water altered by agriculture or pollution. Because L-lactate is a significant product of plant and animal metabolism, it can serve as a carbon source for P. aeruginosa in the diverse settings that it inhabits. In this study, we evaluate the production and use of two redundant P. aeruginosa L-lactate dehydrogenases, termed LldD and LldA. We confirm that the protein LldR represses lldD and identify a new transcription factor, called LldS, that activates lldA; these distinct regulators and the genomic contexts of lldD and lldA contribute to their differential expression. We demonstrate that the lldD and lldA genes are conditionally controlled in response to lactate isomers as well as to glycolate and ɑ-hydroxybutyrate, which, like lactate, are ɑ-hydroxycarboxylates. We also show that lldA is induced when iron availability is low. Our examination of lldD and lldA expression across depth in biofilms indicates a complex pattern that is consistent with the effects of glycolate production, iron availability, and cross-regulation on enzyme preference. Finally, macrophage infection assays reveal that both lldD and lldA contribute to persistence within host cells, underscoring the potential role of L-lactate as a carbon source during P. aeruginosa-eukaryote interactions. Together, these findings help us understand the metabolism of a key resource that may promote P. aeruginosa's success as a resident of contaminated environments and animal hosts.IMPORTANCEPseudomonas aeruginosa is a major cause of lung infections in people with cystic fibrosis, of hospital-acquired infections, and of wound infections. It consumes L-lactate, which is found at substantial levels in human blood and tissues. In this study, we investigated the spatial regulation of two redundant enzymes, called LldD and LldA, which enable L-lactate metabolism in P. aeruginosa biofilms. We uncovered mechanisms and identified compounds that control the preference of P. aeruginosa for LldD versus LldA. We also showed that both enzymes contribute to its ability to survive within macrophages, a behavior that is thought to augment the chronicity and recalcitrance of infections. Our findings shed light on a key metabolic strategy used by P. aeruginosa and have the potential to inform the development of therapies targeting bacterial metabolism during infection.

Formate‐Mediated Electroenzymatic Synthesis via Biological Cofactor NADH

Synthetic biohybrid systems by coupling artificial system with nature’s machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate‐driven NADH regeneration. Utilising a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90% Faraday efficiency at a current density of 150 mA cm−2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilised redox enzymes—alcohol dehydrogenase (ADH), L‐lactate dehydrogenase (LDH), and L‐glutamate dehydrogenase (GDH)—to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.

Decoding halal and jhatka slaughter: novel insights into welfare and protein biomarkers in slow-growing broiler chicken.

The first evidence of blood biochemical and proteomic changes in slow-growing broiler chicken subjected to ritual slaughter like halal (HS) and jhatka (JS) without stunning and commercial slaughter with electrical stunning (ES) was decoded. Significant stress indicators like cortisol and triiodothyronine were markedly elevated in JS birds, whereas increased (P < 0.05) levels of lactate dehydrogenase and creatine kinase were observed in JS and ES birds. Two-dimensional gel electrophoresis coupled to MALDI-TOF MS elucidated the overabundance of glyceraldehyde-3-phosphate dehydrogenase and l-lactate dehydrogenase that are positively correlated with stress in JS broilers. Bioinformatic analysis explored the multifaceted landscape of molecular functions. The study has uncovered that ritual slaughter performed without stunning against commercial slaughter with ES practices elicit varying levels of stress as evident from blood biochemistry and novel protein markers. © 2024 Society of Chemical Industry.

Integrating bioinformatics and multiple machine learning to identify mitophagy-related targets for the diagnosis and treatment of diabetic foot ulcers: evidence from transcriptome analysis and drug docking.

Diabetic foot ulcers are the most common and serious complication of diabetes mellitus, the high morbidity, mortality, and disability of which greatly diminish the quality of life of patients and impose a heavy socioeconomic burden. Thus, it is urgent to identify potential biomarkers and targeted drugs for diabetic foot ulcers. In this study, we downloaded datasets related to diabetic foot ulcers from gene expression omnibus. Dysregulation of mitophagy-related genes was identified by differential analysis and weighted gene co-expression network analysis. Multiple machine algorithms were utilized to identify hub mitophagy-related genes, and a novel artificial neural network model for assisting in the diagnosis of diabetic foot ulcers was constructed based on their transcriptome expression patterns. Finally, potential drugs that can target hub mitophagy-related genes were identified using the Enrichr platform and molecular docking methods. In this study, we identified 702 differentially expressed genes related to diabetic foot ulcers, and enrichment analysis showed that these genes were associated with mitochondria and energy metabolism. Subsequently, we identified hexokinase-2, small ribosomal subunit protein us3, and l-lactate dehydrogenase A chain as hub mitophagy-related genes of diabetic foot ulcers using multiple machine learning algorithms and validated their diagnostic performance in a validation cohort independent of the present study (The areas under roc curve of hexokinase-2, small ribosomal subunit protein us3, and l-lactate dehydrogenase A chain are 0.671, 0.870, and 0.739, respectively). Next, we constructed a novel artificial neural network model for the molecular diagnosis of diabetic foot ulcers, and the diagnostic performance of the training cohort and validation cohort was good, with areas under roc curve of 0.924 and 0.840, respectively. Finally, we identified retinoic acid and estradiol as promising anti-diabetic foot ulcers by targeting hexokinase-2 (-6.6 and -7.2kcal/mol), small ribosomal subunit protein us3 (-7.5 and -8.3kcal/mol), and l-lactate dehydrogenase A chain (-7.6 and -8.5kcal/mol). The present study identified hexokinase-2, small ribosomal subunit protein us3 and l-lactate dehydrogenase A chain, and emphasized their critical roles in the diagnosis and treatment of diabetic foot ulcers through multiple dimensions, providing promising diagnostic biomarkers and targeted drugs for diabetic foot ulcers.

Real-life efficacy of immunotherapy for Sézary syndrome: a multicenter observational cohort study

Sézary syndrome is an extremely rare and fatal cutaneous T-cell lymphoma (CTCL). Mogamulizumab, an anti-CCR4 monoclonal antibody, has recently been associated with increased progression-free survival in a randomized clinical trial in CTCL. We aimed to evaluate OS and prognostic factors in Sézary syndrome, including treatment with mogamulizumab, in a real-life setting. Data from patients with Sézary (ISCL/EORTC stage IV) and pre-Sézary (stage IIIB) syndrome diagnosed from 2000 to 2020 were obtained from 24 centers in Europe. Age, disease stage, plasma lactate dehydrogenases levels, blood eosinophilia at diagnosis, large-cell transformation and treatment received were analyzed in a multivariable Cox proportional hazard ratio model. This study has been registered in ClinicalTrials (SURPASSe01 study: NCT05206045). Three hundred and thirty-nine patients were included (58% men, median age at diagnosis of 70 years, Q1-Q3, 61-79): 33 pre-Sézary (9.7% of 339), 296Sézary syndrome (87.3%), of whom 10 (2.9%) had large-cell transformation. One hundred and ten patients received mogamulizumab. Median follow-up was 58 months (95% confidence interval [CI], 53-68). OS was 46.5% (95% CI, 40.6%-53.3%) at 5 years. Multivariable analysis showed that age ≥ 80 versus <50 (HR: 4.9, 95% CI, 2.1-11.2, p=0.001), and large-cell transformation (HR: 2.8, 95% CI, 1.6-5.1, p=0.001) were independent and significant factors associated with reduced OS. Mogamulizumab treatment was significantly associated with decreased mortality (HR: 0.34, 95% CI, 0.15-0.80, p=0.013). Treatment with mogamulizumab was significantly and independently associated with decreased mortality in Sézary syndrome. French Society of Dermatology, Swiss National Science Foundation (IZLIZ3_200253/1) and SKINTEGRITY.CH collaborative research program.

Immunohistochemical localization of HCA1 receptor in placenta in presence of fetal growth restriction

IntroductionGlucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Fetuses with intrauterine growth restriction are considered to become progressively lactacidemic as well as hypoxic. Roles of lactate in the placenta in the presence of fetal growth restriction (FGR) remain to be clarified. MethodsImmunohistochemical localization of lactate-related substances, such as a receptor for lactate (hydroxy-carboxylic acid 1 receptor (HCA1 receptor/GPR81)), monocarboxylate transporters (MCTs) for lactate, lactate dehydrogenases (LDHs), and proteins expressed in syncytiotrophoblasts or cytotrophoblasts was examined in placentas of appropriate weight for gestational age (AGA) fetus and those showing FGR. ResultsImmunoreactivity for the HCA1 receptor was present in the cytoplasm of some trophoblasts, predominantly localized to their basal (fetus-facing) side, and was frequently colocalized with that for E-cadherin or serine peptidase inhibitor, Kunitz type 1 (SPINT1), a marker protein of cytotrophoblasts. Immunoreactivity for MCT1 and MCT4 was present on the basal and the microvillous (maternal-facing) membranes of trophoblasts in both groups, respectively. Clear immunoreactivity for LDHA and LDHB was also observed in the cytoplasm of trophoblasts, mainly localized to their basal side. However, there were no significant differences in immunohistochemically stained areas of lactate-related substances between AGA and late-onset FGR groups. On the other hand, there were correlations between coefficients of the presence of chorioamnionitis and the values of LDHB and E-cadherin. DiscussionImmunohistochemical localization of the HCA1 receptor was predominantly observed in the cytoplasm located on the basal side of trophoblasts, suggesting a role of lactate in human placental development, including syncytialization.

Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.