A vesicular Warburg effect: Aerobic glycolysis occurs on axonal vesicles for local NAD+ recycling and transport.

In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.

eIF4EHP promotes Ldh mRNA translation in and fruit fly adaptation to hypoxia.

Hypoxia induces profound modifications in the gene expression program of eukaryotic cells due to lowered ATP supply resulting from the blockade of oxidative phosphorylation. One significant consequence of oxygen deprivation is the massive repression of protein synthesis, leaving a limited set of mRNAs to be translated. Drosophila melanogaster is strongly resistant to oxygen fluctuations; however, the mechanisms allowing specific mRNA to be translated into hypoxia are still unknown. Here, we show that Ldh mRNA encoding lactate dehydrogenase is highly translated into hypoxia by a mechanism involving a CA-rich motif present in its 3' untranslated region. Furthermore, we identified the cap-binding protein eIF4EHP as a main factor involved in 3'UTR-dependent translation under hypoxia. In accordance with this observation, we show that eIF4EHP is necessary for Drosophila development under low oxygen concentrations and contributes to Drosophila mobility after hypoxic challenge. Altogether, our data bring new insight into mechanisms contributing to LDH production and Drosophila adaptation to oxygen variations.

Downregulated expression of lactate dehydrogenase in adult oligodendrocytes and its implication for the transfer of glycolysis products to axons.

Oligodendrocytes and astrocytes are metabolically coupled to neuronal compartments. Pyruvate and lactate can shuttle between glial cells and axons via monocarboxylate transporters. However, lactate can only be synthesized or used in metabolic reactions with the help of lactate dehydrogenase (LDH), a tetramer of LDHA and LDHB subunits in varying compositions. Here we show that mice with a cell type-specific disruption of both Ldha and Ldhb genes in oligodendrocytes lack a pathological phenotype that would be indicative of oligodendroglial dysfunctions or lack of axonal metabolic support. Indeed, when combining immunohistochemical, electron microscopical, and in situ hybridization analyses in adult mice, we found that the vast majority of mature oligodendrocytes lack detectable expression of LDH. Even in neurodegenerative disease models and in mice under metabolic stress LDH was not increased. In contrast, at early development and in the remyelinating brain, LDHA was readily detectable in immature oligodendrocytes. Interestingly, by immunoelectron microscopy LDHA was particularly enriched at gap junctions formed between adjacent astrocytes and at junctions between astrocytes and oligodendrocytes. Our data suggest that oligodendrocytes metabolize lactate during development and remyelination. In contrast, for metabolic support of axons mature oligodendrocytes may export their own glycolysis products as pyruvate rather than lactate. Lacking LDH, these oligodendrocytes can also "funnel" lactate through their "myelinic" channels between gap junction-coupled astrocytes and axons without metabolizing it. We suggest a working model, in which the unequal cellular distribution of LDH in white matter tracts facilitates a rapid and efficient transport of glycolysis products among glial and axonal compartments.

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. merolae L-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.

Principles of Cold Adaptation of Fish Lactate Dehydrogenases Revealed by Computer Simulations of the Catalytic Reaction.

Cold-adapted enzymes from psychrophilic and psychrotolerant species are characterized by a higher catalytic activity at low temperature than their mesophilic orthologs and are also usually found to be more thermolabile. Computer simulations of the catalytic reactions have been shown to be a very powerful tool for analyzing the structural and energetic origins of these effects. Here, we examine the cold adaptation of lactate dehydrogenases from two Antarctic and sub-Antarctic fish species using this approach and compare our results with those obtained for the orthologous dogfish enzyme. Direct calculations of thermodynamic activation parameters show that the cold-adapted fish enzymes are characterized by a lower activation enthalpy and a more negative entropy term. This appears to be a universal feature of psychrophilic enzymes, and it is found to originate from a higher flexibility of certain parts of the protein surface. We also carry out free energy simulations that address the differences in thermal stability and substrate binding affinity between the two cold-adapted enzymes, which only differ by a single mutation. These calculations capture the effects previously seen in in vitro studies and provide straightforward explanations of these experimental results.

Deciphering Evolutionary Trajectories of Lactate Dehydrogenases Provides New Insights into Allostery.

Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to NAD+. LDH is a relevant enzyme to investigate structure-function relationships. The present work provides the missing link in our understanding of the evolution of LDHs. This allows to explain (i) the various evolutionary origins of LDHs in eukaryotic cells and their further diversification and (ii) subtle phenotypic modifications with respect to their regulation capacity. We identified a group of cyanobacterial LDHs displaying eukaryotic-like LDH sequence features. The biochemical and structural characterization of Cyanobacterium aponinum LDH, taken as representative, unexpectedly revealed that it displays homotropic and heterotropic activation, typical of an allosteric enzyme, whereas it harbors a long N-terminal extension, a structural feature considered responsible for the lack of allosteric capacity in eukaryotic LDHs. Its crystallographic structure was solved in 2 different configurations typical of the R-active and T-inactive states encountered in allosteric LDHs. Structural comparisons coupled with our evolutionary analyses helped to identify 2 amino acid positions that could have had a major role in the attenuation and extinction of the allosteric activation in eukaryotic LDHs rather than the presence of the N-terminal extension. We tested this hypothesis by site-directed mutagenesis. The resulting C. aponinum LDH mutants displayed reduced allosteric capacity mimicking those encountered in plants and human LDHs. This study provides a new evolutionary scenario of LDHs that unifies descriptions of regulatory properties with structural and mutational patterns of these important enzymes.

LDHB-deficient brain exhibits resistance to ischemic neuronal cell death due to increased vasodilation.

Ischemic stroke triggers a cascade of metabolic and inflammatory events leading to neuronal death, particularly in the hippocampus. Here, we investigate the role of lactate metabolism in ischemic resistance using LDHB-deficient mice, which exhibit impaired lactate utilization. Contrary to expectations of severe neuronal damage due to metabolic defects, LDHB-deficient mice displayed significantly increased neuronal survival following ischemic insult. Magnetic resonance spectroscopy revealed elevated lactate levels in LDHB-deficient brains, which correlated with enhanced vasodilation of the posterior communicating artery (PComA) and increased extracellular PGE2 levels. These findings suggest that elevated lactate inhibits PGE2 reabsorption, promoting vasodilation and neuronal protection. Our results highlight lactate's potential role in neuroprotection and its therapeutic promise for ischemic stroke.

The four subunits of rabbit skeletal muscle lactate dehydrogenase do not exert their catalytic action additively.

Oligomeric enzymes containing multiple active sites are usually considered to perform their catalytic action at higher rates when compared with their monomeric counterparts. This implies, in turn, that the activity performed by different holoenzyme subunits features additivity. Nevertheless, the extent of this additivity occurring in holoenzymes is far from being adequately understood. To tackle this point, we used tetrameric rabbit lactate dehydrogenase (rbLDH) as a model system to assay the reduction of pyruvate catalysed by this enzyme at the expense of ß-NADH under pre-steady-state conditions. In particular, we observed the kinetics of reactions triggered by concentrations of ß-NADH equimolar to 1, 2, 3, or all 4 subunits of the rbLDH holoenzyme, in the presence of an excess of pyruvate. Surprisingly, when the concentration of the limiting reactant exceeded that of a single holoenzyme subunit, we observed a sharp slowdown of the enzyme conformational rearrangements associated to the generation and the release of l-lactate. Furthermore, using a model to interpret the complex kinetics observed under the highest concentration of the limiting reactant, we estimated the diversity of the rates describing the action of the different rbLDH subunits.

LDHA-mediated M2-type macrophage polarization via tumor-derived exosomal EPHA2 promotes renal cell carcinoma progression.

Lactate dehydrogenase A (LDHA) is known to promote the growth and invasion of various types of tumors, affects tumor resistance, and is associated with tumor immune escape. But how LDHA reshapes the tumor microenvironment and promotes the progression of renal cell carcinoma (RCC) remains unclear. In this study, we found that LDHA was highly expressed in clear cell RCC (ccRCC), and this high expression was associated with macrophage infiltration, while macrophages were highly infiltrated in ccRCC, affecting patient prognosis via M2-type polarization. Our in vivo and in vitro experiments demonstrated that LDHA and M2-type macrophages could enhance the proliferation, invasion, and migration abilities of ccRCC cells. Mechanistically, high expression of LDHA in ccRCC cells upregulated the expression of EPHA2 in exosomes derived from renal cancer. Exosomal EPHA2 promoted M2-type polarization of macrophages by promoting activation of the PI3K/AKT/mTOR pathway in macrophages, thereby promoting the progression of ccRCC. All these findings suggest that EPHA2 may prove to be a potential therapeutic target for advanced RCC.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Lactate dehydrogenase A (LDHA)-mediated lactate generation promotes pulmonary vascular remodeling in pulmonary hypertension.

BACKGROUND: High levels of lactate are positively associated with prognosis and mortality in pulmonary hypertension (PH). Lactate dehydrogenase A (LDHA) is a key enzyme for the production of lactate. This study is undertaken to investigate the role and molecular mechanisms of lactate and LDHA in PH. METHODS: Lactate levels were measured by a lactate assay kit. LDHA expression and localization were detected by western blot and Immunofluorescence. Proliferation and migration were determined by CCK8, western blot, EdU assay and scratch-wound assay. The right heart catheterization and right heart ultrasound were measured to evaluate cardiopulmonary function. RESULTS: In vitro, we found that lactate promoted proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) in an LDHA-dependent manner. In vivo, we found that LDHA knockdown reduced lactate overaccumulation in the lungs of mice exposed to hypoxia. Furthermore, LDHA knockdown ameliorated hypoxia-induced vascular remodeling and right ventricular dysfunction. In addition, the activation of Akt signaling by hypoxia was suppressed by LDHA knockdown both in vivo and in vitro. The overexpression of Akt reversed the inhibitory effect of LDHA knockdown on proliferation in PASMCs under hypoxia. Finally, LDHA inhibitor attenuated vascular remodeling and right ventricular dysfunction in Sugen/hypoxia mouse PH model, Monocrotaline (MCT)-induced rat PH model and chronic hypoxia-induced mouse PH model. CONCLUSIONS: Thus, LDHA-mediated lactate production promotes pulmonary vascular remodeling in PH by activating Akt signaling pathway, suggesting the potential role of LDHA in regulating the metabolic reprogramming and vascular remodeling in PH.

Evaluation of the CCL17/CCL22-CCR4 axis in pediatrics with B-cell acute lymphoblastic leukemia before and after a chemotherapy course.

AIMS: Acute lymphoblastic leukemia (ALL) is the most common type of pediatrics cancer. Chemokines exert different roles in leukemia process through leukocyte recruitment and regulation of disease severity. Due to the prominent roles of chemokine/receptor axes, this study aimed to measure the blood expression levels of CCR4 and their ligands in pediatrics with B-cell ALL (B-ALL). We also evaluated the impact of cytotoxic chemotherapy on this axis. MATERIAL AND METHOD: Thirty children suffering from B-ALL were included in the study and followed up for 30 days after completion of a chemotherapy course. The blood sampling was performed before and after chemotherapy. 30 healthy donors have also entered the study as control subjects. The mRNA expression of CCL17, CCL22 and CCR4 genes was determined by quantitative real-time PCR. The frequency of the peripheral blood mononuclear cells expressing CCR4 (CCR4 + PBMCs) was also evaluated by the flow cytometry method. Moreover, we evaluated the association of the CCL17/CCL22-CCR4 axis with some diagnostic, prognostic and predictive biomarkers in ALL patients. RESULTS: There was overexpression of the CCL17/CCL22-CCR4 axis along with lactate dehydrogenase (LDH) in pediatrics with B-ALL compared to healthy controls. After induction of chemotherapy, the blood expression levels of the CCL17/CCL22-CCR4 axis have reached the levels of healthy controls. The findings for the blood expression levels of CCR4 were also confirmed using flow cytometry. CONCLUSION: The CCL17/CCL22-CCR4 axis can be used as a novel predictive and prognostic biomarker in B-ALL.

Correlation between Lactate Dehydrogenase to Albumin Ratio and the Prognosis of Patients with Cardiac Arrest.

Background: Cardiac arrest (CA) is a common event in the intensive care unit (ICU), which seriously threatens the prognosis of patients. Therefore, it is crucial to determine a simple and effective clinical indicator to judge the prognosis of patients after a CA for later treatments. The purpose of this study was to investigate the relationship between the lactate dehydrogenase to albumin ratio (LAR) and the prognosis of patients after a CA. Methods: The clinical data of participants was obtained from the Medical Information Mart for Intensive Care IV (MIMIC-IV, v2.0; 2008 to 2019). According to the 30-day prognosis, patients were divided into a survivors group (n = 216) and a non-survivors group (n = 304). The optimal LAR threshold was determined using restricted cubic spline (RCS), which divided patients into a high LAR group ( ≥ 15.50, n = 257) and a low LAR group ( < 15.50, n = 263). The ICU hospitalization and 30-day accumulative survival curves of the two groups were plotted following the Kaplan-Meier survival analysis. Multivariate Cox regression was used to analyze the relationship between the LAR and the prognosis of CA patients. Receiver operating characteristic (ROC) curves were drawn to evaluate the predictive efficacy of the LAR on 30-day all-cause mortality, and sensitivity analysis was used to check the reliability of the findings. Results: A total of 520 patients with CA were enrolled and the 30-day mortality was 58.46%. The LAR in the non-survivors group was higher than in the survivors group. The RCS showed a linear trend relationship between the LAR and the mortality risk in patients during their ICU stay and 30 days; moreover, as the LAR increased, so did the risk of mortality. The Kaplan-Meier survival curve showed that compared with the low LAR group, the cumulative survival rates of ICU hospitalization and 30 days were lower in the high LAR group among CA patients (p < 0.001). Multivariate Cox regression analysis showed that an elevated LAR ( ≥ 15.50) was an independent risk factor for mortality during ICU stay and 30 days (p < 0.005). ROC analysis suggested that the LAR was superior to the sequential organ failure assessment (SOFA) score in predicting the 30-day all-cause mortality in CA patients (area under the curve (AUC) = 0.676, 95% confidence interval [CI]: 0.629-0.723). To verify the reliability of our findings, we performed sensitivity analyses and found that the findings were reliable. Conclusions: An elevated LAR might be a predictor of mortality in patients following a CA during ICU hospitalization and 30 days, thereby it can be used to provide a reference for the clinical management of these patients.

Identification of Biomarkers for Assessing Treatment Efficacy of Chemotherapy plus Nivolumab as the First Line in Patients with Unresectable Advanced or Recurrent Gastric Cancer: A Multicenter Study.

INTRODUCTION: In this study, we aimed to identify biomarkers for predicting treatment outcomes and efficacy of chemotherapy plus nivolumab, as well as predict immune-related adverse events (irAEs) characteristics of immune checkpoint inhibitors. METHODS: This multicenter study included 104 patients who received chemotherapy plus nivolumab as the primary treatment for unresectable advanced recurrent gastric cancer. Blood test results were collected before the start and after two courses of treatment. The neutrophil-lymphocyte ratio, prognostic nutritional index, and lactate dehydrogenase/albumin ratio (LAR) were examined after treatment in each case to determine changes compared to values before the start of treatment. RESULTS: A total of 57 (54.8%) patients experienced a complete or partial response. The LAR of the stable disease/progressive disease group significantly increased (p = 0.018). An examination of the presence of grade ≥3 irAEs and changes in related factors showed that the LAR of all patients increased. CONCLUSION: The LAR was correlated with the best therapeutic response; therefore, it may be a potential biomarker of treatment outcomes and efficacy.

The role of LDH and ferritin levels as biomarkers for corticosteroid dosage in children with refractory Mycoplasma pneumoniae pneumonia.

BACKGROUND: This study explored the relationship between inflammatory markers and glucocorticoid dosage upon admission. METHODS: We conducted a retrospective analysis of 206 patients with refractory Mycoplasma pneumoniae pneumonia (RMPP) admitted to a Children's Hospital from November 2017 to January 2022. Patients were categorized into three groups based on their methylprednisolone dosage: low-dose (≤ 2 mg/kg/d), medium-dose (2-10 mg/kg/d), and high-dose (≥ 10 mg/kg/d). We compared demographic data, clinical manifestations, laboratory findings, and radiological outcomes. Spearman's rank correlation coefficient was used to assess relationships between variables. RESULTS: The median age was highest in the low-dose group at 7 years, compared to 5.5 years in the medium-dose group and 6 years in the high-dose group (P < 0.001). The body mass index (BMI) was also highest in the low-dose group at 16.12, followed by 14.86 in the medium-dose group and 14.58 in the high-dose group (P < 0.001). More severe radiographic findings, longer hospital stays, and greater incidence of hypoxia were noted in the high-dose group (P < 0.05). Additionally, significant increases in white blood cells, C-reactive protein, procalcitonin, lactate dehydrogenase (LDH), alanine transaminase, aspartate transaminase, ferritin, erythrocyte sedimentation rate, and D-dimer levels were observed in the high-dose group (P < 0.05). Specifically, LDH and ferritin were markedly higher in the high-dose group, with levels at 660.5 U/L and 475.05 ng/mL, respectively, compared to 450 U/L and 151.4 ng/mL in the medium-dose group, and 316.5 U/L and 120.5 ng/mL in the low-dose group. Correlation analysis indicated that LDH and ferritin levels were significantly and positively correlated with glucocorticoid dose (Spearman ρ = 0.672 and ρ = 0.654, respectively; P < 0.001). CONCLUSIONS: Serum LDH and ferritin levels may be useful biomarkers for determining the appropriate corticosteroid dosage in treating children with RMPP.

Lactate oxidation in Paracoccus denitrificans.

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 µM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

Exploring the anti-protozoal mechanisms of Syzygium aromaticum phytochemicals targeting Cryptosporidium parvum lactate dehydrogenase through molecular dynamics simulations.

Cryptosporidium parvum (C. parvum), a protozoan parasite, is known to induce significant gastrointestinal disease in humans. Lactate dehydrogenase (LDH), a protein of C. parvum, has been identified as a potential therapeutic target for developing effective drugs against infection. This study utilized a computational drug discovery approach to identify potential drug molecules against the LDH protein of C. parvum. In the present investigation, we conducted a structure-based virtual screening of 55 phytochemicals from the Syzygium aromaticum (S. aromaticum). This process identified four phytochemicals, including Gallotannin 23, Eugeniin, Strictinin, and Ellagitannin, that demonstrated significant binding affinity and dynamic stability with LDH protein. Interestingly, these four compounds have been documented to possess antibacterial, antiviral, anti-inflammatory, and antioxidant properties. The docked complexes were simulated for 100 ns using Desmond to check the dynamic stability. Finally, the free binding energy was computed from the last 10ns MD trajectories. Gallotannin 23 and Ellagitannin exhibited considerable binding affinity and stability with the target protein among all four phytochemicals. These findings suggest that these predicted phytochemicals from S. aromaticum could be further explored as potential hit candidates for developing effective drugs against C. parvum infection. The in vitro and in vivo experimental validation is still required to confirm their efficacy and safety as LDH inhibitors.

The catalytic action of human d-lactate dehydrogenase is severely inhibited by oxalate and is impaired by mutations triggering d-lactate acidosis.

d-lactate dehydrogenases are known to be expressed by prokaryotes and by eukaryotic invertebrates, and over the years the functional and structural features of some bacterial representatives of this enzyme ensemble have been investigated quite in detail. Remarkably, a human gene coding for a putative d-lactate dehydrogenase (DLDH) was identified and characterized, disclosing the occurrence of alternative splicing of its primary transcript. This translates into the expression of two human DLDH (hDLDH) isoforms, the molecular mass of which is expected to differ by 2.7 kDa. However, no information on these two hDLDH isoforms is available at the protein level. Here we report on the catalytic action of these enzymes, along with a first analysis of their structural features. In particular, we show that hDLDH is strictly stereospecific, with the larger isoform (hDLDH-1) featuring higher activity at the expense of d-lactate when compared to its smaller counterpart (hDLDH-2). Furthermore, we found that hDLDH is strongly inhibited by oxalate, as indicated by a Ki equal to 1.2 µM for this dicarboxylic acid. Structurally speaking, hDLDH-1 and hDLDH-2 were determined, by means of gel filtration and dynamic light scattering experiments, to be a hexamer and a tetramer, respectively. Moreover, in agreement with previous studies performed with human mitochondria, we identified FAD as the cofactor of hDLDH, and we report here a model of FAD binding by the human d-lactate dehydrogenase. Interestingly, the mutations W323C and T412 M negatively affect the activity of hDLDH, most likely by impairing the enzyme electron-acceptor site.

Efficacy and safety of anti-PD-1 inhibitor versus anti-PD-L1 inhibitor in first-line treatment of extensive-stage small cell lung cancer: a multicenter retrospective study.

BACKGROUND: Immunotherapy targeting PD-1/PD-L1 has revolutionized the treatment of extensive-stage small cell lung cancer (ES-SCLC). However, clinical trials suggest differential efficacy of anti-PD-1 agents and anti-PD-L1 agents in first-line treatment of ES-SCLC. This retrospective multicenter study aimed to compare the efficacy and safety of anti-PD-1 agents versus anti-PD-L1 agents in first-line treatment of ES-SCLC in real-world practice. METHODS: Patients with pathologically or cytologically confirmed ES-SCLC treated with platinum plus etoposide combined with anti-PD-1 or PD-L1 agents as first-line treatment in different centers of PLA General Hospital between January 2017 and October 2021 were included for this study. Survival outcomes and safety were compared between patients receiving anti-PD-1 and PD-L1 agents. RESULTS: Of the total 154 included patients, 68 received anti-PD-1 agents plus chemotherapy (PD-1 group), and 86 received anti-PD-L1 agents plus chemotherapy (PD-L1 group). Progression-free survival (PFS) and overall survival (OS) in the entire cohort were 7.6 months (95% confidence interval [CI]: 6.5-8.2 months) and 17.4 months (95% CI: 15.3-19.3 months), respectively. Median PFS and OS were comparable between the PD-1 group and PD-L1 group (PFS: 7.6 months vs. 8.3 months, HR = 1.13, 95% CI: 0.79-1.62, p = 0.415; OS: 26.9 months vs. 25.6 months, HR = 0.96, 95% CI: 0.63-1.47, p = 0.859. The objective response rate and disease control rate were comparable between the two groups: 79.4% vs. 79.1% and 92.6% vs. 94.2%, respectively. The 6-month, 12-month, and 18-month PFS and OS rates were slightly higher in the PD-L1 group than in the PD-1 group, while the 24-month PFS rate was slightly higher in the PD-1 group than in the PD-L1 group. Stratified analysis showed that locoregional thoracic radiotherapy and normal lactate dehydrogenase level were independent predictors of better OS in ES-SCLC patients treated with first-line chemotherapy plus ICI. Adverse events were not significantly different between the two groups. CONCLUSIONS: Anti-PD-1 agents and anti-PD-L1 agents combined with chemotherapy as first-line treatment for ES-SCLC are comparably effective and well tolerated.

All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(ß) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student's t-test or one-way ANOVA was used to analyze data from replicates. RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(ß) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(ß) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression. CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.