Exploring the Regulation of Cytochrome P450 in SH-SY5Y Cells: Implications for the Onset of Neurodegenerative Diseases.

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, ß-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following ß-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases.

Identification of Novel Isatin Derivative Bearing a Nitrofuran Moiety as Potent Multi-Isoform Aldehyde Dehydrogenase Inhibitor.

Aldehyde dehydrogenases (ALDHs) are a family of enzymes that aid in detoxification and are overexpressed in several different malignancies. There is a correlation between increased expression of ALDH and a poor prognosis, stemness, and resistance to several drugs. Several ALDH inhibitors have been generated due to the crucial role that ALDH plays in cancer stem cells. All of these inhibitors, however, are either ineffective, very toxic, or have yet to be subjected to rigorous testing on their effectiveness. Although various drug-like compounds targeting ALDH have been reported in the literature, none have made it to routine use in the oncology clinic. As a result, new potent, non-toxic, bioavailable, and therapeutically effective ALDH inhibitors are still needed. In this study, we designed and synthesized potent multi-ALDH isoform inhibitors based on the isatin and indazole pharmacophore. Molecular docking studies and enzymatic tests revealed that among all of the synthesized analogs, compound 3 is the most potent inhibitor of ALDH1A1, ALDH3A1, and ALDH1A3, exhibiting 51.32%, 51.87%, and 36.65% inhibition, respectively. The ALDEFLUOR assay further revealed that compound 3 acts as an ALDH broad spectrum inhibitor at 500 nM. Compound 3 was also the most cytotoxic to cancer cells, with an IC50 in the range of 2.1 to 3.8 µM for ovarian, colon, and pancreatic cancer cells, compared to normal and embryonic kidney cells (IC50 7.1 to 8.7 µM). Mechanistically, compound 3 increased ROS activity due to potent multi-ALDH isoform inhibition, which increased apoptosis. Taken together, this study identified a potent multi-isoform ALDH inhibitor that could be further developed as a cancer therapeutic.

Regulation of H9C2 cell hypertrophy by 14-3-3η via inhibiting glycolysis.

It has been reported that Ywhah (14-3-3η) reduces glycolysis. However, it remains unclear about the downstream mechanism by which glycolysis is regulated by 14-3-3η in cardiac hypertrophy. As an important regulator, Yes-associated protein (YAP) interacts with 14-3-3η to participate in the initiation and progression of various diseases in vivo. In this study, the model of H9C2 cardiomyocyte hypertrophy was established by triiodothyronine (T3) or rotenone stimulation to probe into the action mechanism of 14-3-3η. Interestingly, the overexpression of 14-3-3η attenuated T3 or rotenone induced cardiomyocyte hypertrophy and decreased glycolysis in H9C2 cardiomyocytes, whereas the knockdown of 14-3-3η had an opposite effect. Mechanistically, 14-3-3η can reduce the expression level of YAP and bind to it to reduce its nuclear translocation. In addition, changing YAP may affect the expression of lactate dehydrogenase A (LDHA), a glycolysis-related protein. Meanwhile, LDHA is also a possible target for 14-3-3η to mediate glycolysis based on changes in pyruvate, a substrate of LDHA. Collectively, 14-3-3η can suppress cardiomyocyte hypertrophy via decreasing the nucleus translocation of YAP and glycolysis, which indicates that 14-3-3η could be a promising target for inhibiting cardiac hypertrophy.

Extraction optimization and reactivity of 7α-acetoxy-6ß-hydroxyroyleanone and ability of its derivatives to modulate PKC isoforms.

Protein kinase C is a family of kinases that play important roles in carcinogenesis. Medicinal plants from Plectranthus spp. (Lamiaceae) are a well-known source of interesting abietanes, such as 7α-acetoxy-6ß-hydroxyroyleanone (Roy). This study aimed to extract and isolate Roy from P. grandidentatus Gürke, comparing two extraction methods (CO2 supercritical and ultrasound-assisted acetonic extraction), and design new royleanone derivatives for PKC modulation focusing on breast cancer therapy. The concentration of Roy in the extracts was determined by HPLC-DAD. The supercritical extraction method yielded 3.6% w/w, with the presence of 42.7 µg mg-1 of Roy (yield of 0.13%), while ultrasound-assisted acetonic extraction yielded 2.3% w/w, with the presence of 55.2 µg mg-1 of Roy (yield of 0.15%). The reactivity of Roy was investigated aiming at synthetizing new ester derivatives through standard benzoylation and esterification reactions. The benzoylated (Roy-12-Bz) and acetylated (Roy-12-Ac) derivatives in the C12 position were consistently prepared with overall good yields (33-86%). These results indicate the 12-OH position as the most reactive for esterification, affording derivatives under mild conditions. The reported di-benzoylated (RoyBz) and di-acetylated (RoyAc) derivatives were also synthesized after increasing the temperature (50 °C), reaction time, and using an excess of reagents. The cytotoxic potential of Roy and its derivatives was assessed against breast cancer cell lines, with RoyBz emerging as the most promising compound. Derivatization at position C-12 did not offer advantages over di-esterification at positions C-12 and C-6 or over the parent compound Roy and the presence of aromatic groups favored cytotoxicity. Evaluation of royleanones as PKC-α, ßI, δ, ε, and ζ activators revealed DeRoy's efficacy across all isoforms, while RoyPr showed promising activation of PKC-δ but not PKC-ζ, highlighting the influence of slight structural changes on isoform selectivity. Molecular docking analysis emphasized the importance of microenvironmental factors in isoform specificity, underscoring the complexity of PKC modulation and the need for further exploration.

From X-ray crystallographic structure to intrinsic thermodynamics of protein-ligand binding using carbonic anhydrase isozymes as a model system.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.

Phospholipid Scramblase 1 Localizes Proximal to Sphingomyelin Synthase Isoforms but Is Not Involved in Sphingomyelin Synthesis.

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.

Exploring variations in glycolytic and gluconeogenic enzymes and isoforms across breast cancer cell lines and tissues.

It is well established that tumour cells undergo metabolic changes to acquire biological advantage over normal cells with activation of the glycolytic pathway, a process termed "Warburg effect". Enzyme isoforms are alternative enzymatic forms with the same function but with different biochemical or epigenetic features. Moreover, isoforms may have varying impacts on different metabolic pathways. We challenge ourselves to analyse the glycolytic and gluconeogenic enzymes and isoforms in breast cancer, a complex and heterogeneous pathology, associated with high incidence and mortality rates especially among women. We analysed epithelial and tumour cell lines by RT-PCR and compared values to a publicly available database for the expression profile of normal and tumour tissues (Gepia) of enzymes and enzymatic isoforms from glycolytic and gluconeogenic pathways. Additionally, GeneMANIA was used to evaluate interactions, pathways, and attributes of each glycolytic/gluconeogenic steps. The findings reveal that the enzymes and enzymatic isoforms expressed in cell culture were somewhat different from those in breast tissue. We propose that the tumor microenvironment plays a crucial role in the expression of glycolytic and gluconeogenic enzymes and isoforms in tumour cells. Nonetheless, they not only participate in glycolytic and gluconeogenic enzymatic activities but may also influence other pathways, such as the Pentose-Phosphate-Pathway, TCA cycle, as well as other carbohydrate, lipid, and amino acid metabolism.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

NAT1 inhibits liver metastasis of colorectal cancer by regulating EMT and glycolysis.

Metastasis is the primary cause of cancer-related deaths, and colorectal cancer (CRC) liver metastasis is a major poor prognostic factor in CRC. NAT1 (N-acetyltransferase 1) plays a crucial role in the invasive and metastatic processes of colorectal cancer. The role and molecular mechanism of NAT1 on tumor cells were verified by establishing a cell model of overexpression and knockdown of NAT1, and further verified by establishing a liver metastasis model of colorectal cancer for animal experiments. In vivo and in vitro experiments have demonstrated that overexpression of NAT1 reduces the ability of metastasis and invasion of colorectal cancer cells. NAT1 overexpression inhibits the PI3K/AKT/mTOR signaling pathway, thereby suppressing the EMT (epithelial-mesenchymal transition) process and glycolytic ability of tumor cells. Additionally, decreased glycolytic ability results in reduced VEGF (Vascular endothelial growth factor) expression in colorectal cancer cells. The decreased VEGF expression leads to decreased angiogenesis and vascular permeability in liver metastases, ultimately reducing the occurrence of liver metastasis. Our findings highlight that overexpression of NAT1 significantly inhibits the PI3K/AKT/mTOR signaling pathway, thereby suppressing EMT, glycolytic ability, and VEGF expression in colorectal cancer cells, collectively preventing the development of liver metastasis.

Exploring the role of casein kinase 1α splice variants across cancer cell lines.

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.

Isoform-specific sequestration of protein kinase A fine-tunes intracellular signaling during heat stress.

Protein kinase A (PKA) is a conserved kinase crucial for fundamental biological processes linked to growth, development, and metabolism. The PKA catalytic subunit is expressed as multiple isoforms in diverse eukaryotes; however, their contribution to ensuring signaling specificity in response to environmental cues remains poorly defined. Catalytic subunit activity is classically moderated via interaction with an inhibitory regulatory subunit. Here, a quantitative mass spectrometry approach is used to examine heat-stress-induced changes in the binding of yeast Tpk1-3 catalytic subunits to the Bcy1 regulatory subunit. We show that Tpk3 is not regulated by Bcy1 binding but, instead, is deactivated upon heat stress via reversible sequestration into cytoplasmic granules. These "Tpk3 granules" are enriched for multiple PKA substrates involved in various metabolic processes, with the Hsp42 sequestrase required for their formation. Hence, regulated sequestration of Tpk3 provides a mechanism to control isoform-specific kinase signaling activity during stress conditions.

HDAC3 inhibitors: a patent review of their broad-spectrum applications as therapeutic agents.

INTRODUCTION: Histone deacetylases (HDACs) are a class of zinc-dependent enzymes. They maintain acetylation homeostasis, with numerous biological functions and are associated with many diseases. HDAC3 strictly requires multi-subunit complex formation for activity. It is associated with the progression of numerous non-communicable diseases. Its widespread involvement in diseases makes it an epigenetic drug target. Preexisting HDAC3 inhibitors have many uses, highlighting the need for continued research in the discovery of HDAC3-selective inhibitors. AREA COVERED: This review provides an overview of 24 patents published from 2010 to 2023, focusing on compounds that inhibit the HDAC3 isoenzyme. EXPERT OPINION: HDAC3-selective inhibitors - pivotal for pharmacological applications, as single or combination therapies - are gaining traction as a strategy to move away from complications laden pan-HDAC inhibitors. Moreover, there is an unmet need for HDAC3 inhibitors with alternative zinc-binding groups (ZBGs) because some preexisting ZBGs have limitations related to toxicity and side effects. Difficulties in achieving HDAC3 selectivity may be due to isoform selectivity. However, advancements in computer-aided drug design and experimental data of HDAC3 3D co-crystallized models could lead to the discovery of novel HDAC3-selective inhibitors, which bear alternative ZBGs with balanced selectivity for HDAC3 and potency.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Arogenate dehydratase isoforms strategically deregulate phenylalanine biosynthesis in Akebia trifoliata.

Arogenate dehydratase (ADT) is key for phenylalanine (Phe) biosynthesis in plants. To examine ADT components and function in Akebia trifoliata, a representative of Ranunculaceae, we first identified eight ADTs (AktADT1-8, encoding sequences varying from 1032 to 1962 bp) in the A. trifoliata reference genome and five proteins (AktADT1, AktADT4, AktADT7, AktADT8 and AktADT8s) with moonlighting prephenate dehydratase (PDT) activity and Km values varying from 0.43 to 2.17 mM. Structurally, two basic residue combinations (Val314/Ala317 and Ala314/Val317) in the PAC domain are essential for the moonlighting PDT activity of ADTs. Functionally, AktADT4 and AktADT8 successfully restored the wild-type phenotype of pha2, a knockout mutant of Saccharomyces cerevisiae. In addition, AktADTs are ubiquitously expressed, but their expression levels are tissue specific, and the half maximal inhibitory concentration (IC50) of Phe for AktADTs ranged from 49.81 to 331.17 µM. Both AktADT4 and AktADT8 and AktADT8s localized to chloroplast stromules and the cytosol, respectively, while the remaining AktADTs localized to the chloroplast stroma. These findings suggest that various strategies exist for regulating Phe biosynthesis in A. trifoliata. This provides a reasonable explanation for the high Phe content and insights for further genetic improvement of the edible fruits of A. trifoliata.

Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of Caenorhabditis elegans.

Multiple isozymes are encoded in the Caenorhabditis elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM) from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are both required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing GlcCers. Deletion of sms-5 hardly affected SM levels. RNAi of sms-1, sms-2, and sms-3 all lowered the abundance of certain SMs with an odd-numbered N-acyl chains (mostly C21 and C23, with or without hydroxylation). Unexpectedly, sms-2 RNAi and sms-3 RNAi elevated a subset of SM species containing even-numbered N-acyls. This suggests that sphingolipids containing even-numbered N-acyls could be regulated separately, sometimes in opposite directions, from those containing odd-numbered N-acyls, which are presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of GlcCers and SMs. These findings underscore the effectiveness of this RPLC-MS/MS method in studies of C. elegans sphingolipid biology.

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs).

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs' mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Integrated analysis of single cell-RNA sequencing and Mendelian randomization identifies lactate dehydrogenase B as a target of melatonin in ischemic stroke.

AIMS: Despite the success of single-cell RNA sequencing in identifying cellular heterogeneity in ischemic stroke, clarifying the mechanisms underlying these associations of differently expressed genes remains challenging. Several studies that integrate gene expression and gene expression quantitative trait loci (eQTLs) with genome wide-association study (GWAS) data to determine their causal role have been proposed. METHODS: Here, we combined Mendelian randomization (MR) framework and single cell (sc) RNA sequencing to study how differently expressed genes (DEGs) mediating the effect of gene expression on ischemic stroke. The hub gene was further validated in the in vitro model. RESULTS: We identified 2339 DEGs in 10 cell clusters. Among these DEGs, 58 genes were associated with the risk of ischemic stroke. After external validation with eQTL dataset, lactate dehydrogenase B (LDHB) is identified to be positively associated with ischemic stroke. The expression of LDHB has also been validated in sc RNA-seq with dominant expression in microglia and astrocytes, and melatonin is able to reduce the LDHB expression and activity in vitro ischemic models. CONCLUSION: Our study identifies LDHB as a novel biomarker for ischemic stroke via combining the sc RNA-seq and MR analysis.

LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.