Roles of heat shock protein A12A in the development of diabetic cardiomyopathy.

Long-term hyperglycemia can lead to diabetic cardiomyopathy (DCM), a main lethal complication of diabetes. However, the mechanisms underlying DCM development have not been fully elucidated. Heat shock protein A12A (HSPA12A) is the atypic member of the Heat shock 70kDa protein family. In the present study, we found that the expression of HSPA12A was upregulated in the hearts of mice with streptozotocin-induced diabetes, while ablation of HSPA12A improved cardiac systolic and diastolic dysfunction and increased cumulative survival of diabetic mice. An increased expression of HSPA12A was also found in H9c2 cardiac cells following treatment with high glucose (HG), while overexpression of HSPA12A-enhanced the HG-induced cardiac cell death, as reflected by higher levels of propidium iodide cells, lactate dehydrogenase leakage, and caspase 3 cleavage. Moreover, the HG-induced increase of oxidative stress, as indicated by dihydroethidium staining, was exaggerated by HSPA12A overexpression. Further studies demonstrated that the HG-induced increases of protein kinase B and forkhead box transcription factors 1 phosphorylation were diminished by HSPA12A overexpression, while pharmacologically inhibition of protein kinase B further enhanced the HG-induced lactate dehydrogenase leakage in HSPA12A overexpressed cardiac cells. Together, the results suggest that hyperglycemia upregulated HSPA12A expression in cardiac cells, by which induced cell death to promote DCM development. Targeting HSPA12A may serve as a potential approach for DCM management.

An engineered poly(A) tail attenuates gut ischemia/reperfusion-induced acute lung injury.

BACKGROUND: Gut ischemia/reperfusion causes the release of damage-associated molecular patterns, leading to acute lung injury and high mortality. Cold-inducible ribonucleic acid-binding protein is a ribonucleic acid chaperon that binds the polyadenylation tail of messenger ribonucleic acid intracellularly. Upon cell stress, cold-inducible ribonucleic acid-binding protein is released, and extracellular cold-inducible ribonucleic acid-binding protein acts as a damage-associated molecular pattern, worsening inflammation. To inhibit extracellular cold-inducible ribonucleic acid-binding protein, we have recently developed an engineered polyadenylation tail named A12. Here, we sought to investigate the therapeutic potential of A12 in gut ischemia/reperfusion-induced acute lung injury. METHODS: Male C57BL6/J mice underwent superior mesenteric artery occlusion and were treated with intraperitoneal A12 (0.5 nmol/g body weight) or vehicle at the time of reperfusion. Blood and lungs were collected 4 hours after gut ischemia/reperfusion. Systemic levels of extracellular cold-inducible ribonucleic acid-binding protein, interleukin-6, aspartate transaminase, alanine transaminase, and lactate dehydrogenase were determined. The pulmonary gene expression of cytokines (interleukin-6, interleukin-1ß) and chemokines (macrophage-inflammatory protein-2, keratinocyte-derived chemokine) was also assessed. In addition, lung myeloperoxidase, injury score, and cell death were determined. Mice were monitored for 48 hours after gut ischemia/reperfusion for survival assessment. RESULTS: Gut ischemia/reperfusion significantly increased the serum extracellular cold-inducible ribonucleic acid-binding protein levels. A12 treatment markedly reduced the elevated serum interleukin-6, alanine transaminase, aspartate transaminase, and lactate dehydrogenase by 53%, 23%, 23%, and 24%, respectively, in gut ischemia/reperfusion mice. A12 also significantly decreased cytokine and chemokine messenger ribonucleic acids and myeloperoxidase activity in the lungs of gut ischemia/reperfusion mice. Histological analysis revealed that A12 attenuated tissue injury and cell death in the lungs of gut ischemia/reperfusion mice. Finally, administration of A12 markedly improved the survival of gut ischemia/reperfusion mice. CONCLUSION: A12, a novel extracellular cold-inducible ribonucleic acid-binding protein inhibitor, diminishes inflammation and mitigates acute lung injury when employed as a treatment during gut ischemia/reperfusion. Hence, the targeted approach toward extracellular cold-inducible ribonucleic acid-binding protein emerges as a promising therapeutic strategy for alleviating gut ischemia/reperfusion-induced acute lung injury.

The impact of MYD88 and PIM1 in mature large B-cell non-Hodgkin lymphomas: Defining element of their evolution and prognosis.

Sequence studies of the entire exome and transcriptome of lymphoma tissues have identified MYD88 and PIM1 as involved in the development and oncogenic signaling. We aimed to determine the frequency of MYD88 and PIM1 mutations, as well as their expressions in conjunction with the clinicopathological parameters identified in mature large B-cell non-Hodgkin lymphomas. The ten-year retrospective study included 50 cases of mature large B-cell lymphoma, diagnosed at the Pathology Department of the Emergency County Hospital of Constanta and Sacele County Hospital of Brasov. They were statistically analyzed by demographic, clinicopathological, and morphogenetic characteristics. We used a real-time polymerase chain reaction technique to identify PIM1 and MYD88 mutations as well as an immunohistochemical technique to evaluate the expressions of the 2 genes. Patients with lymphoma in the small bowel, spleen, brain, and testis had a low-performance status Eastern Cooperative Oncology Group (P = .001). The Eastern Cooperative Oncology Group performance status represented an independent risk factor predicting mortality (HR = 9.372, P < .001). An increased lactate dehydrogenase value was associated with a low survival (P = .002). The international prognostic index score represents a negative risk factor in terms of patient survival (HR = 4.654, P < .001). In cases of diffuse large B-cell lymphoma (DLBCL), immunopositivity of MYD88 is associated with non-germinal center B-cell origin (P < .001). The multivariate analysis observed the association between high lactate dehydrogenase value and the immunohistochemical expression of PIM1 or with the mutant status of the PIM1 gene representing negative prognostic factors (HR = 2.066, P = .042, respectively HR = 3.100, P = .004). In conclusion, our preliminary data suggest that the oncogenic mutations of PIM1 and MYD88 in our DLBCL cohort may improve the diagnosis and prognosis of DLBCL patients in an advanced stage.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

Influences of two transport strategies on AMPK-mediated metabolism and flesh quality of shrimp (Litopenaeus vannamei).

BACKGROUND: Water-free transportation (WFT), as a novel strategy for express delivery of live shrimp (Litopenaeus vannamei), was developed recently. However, air exposure during this transportation arouses a series of abiotic stress to the shrimp. In the present study, the influences of WFT stress on glycolysis and lipolysis metabolism and meat quality (umami flavor and drip loss) were investigated in comparison with conventional water transportation (WT). RESULTS: The results showed that type II muscle fibers with the feature of anaerobic metabolism were dominated in shrimp flesh. In addition, the increments of intracellular Ca2+ was detected in WFT and WT, which then activated the AMP-activated protein kinase pathway and promoted the consumption of glycogen, as well as the accumulation of lactate and lipolysis, under the enzymolysis of hexokinase, pyruvate kinase, lactate dehydrogenase and adipose triglyceride lipase. Glycogen glycolyzed to latate. Meanwhile, ATP degraded along with glycolysis resulting in the generation of ATP-related adenosine phosphates such as inosine monophosphate with umami flavor and phosphoric acid. More remarkable (P < 0.05) physiological changes (except lactate dehydrogenase and lactate) were observed in WFT compared to WT. Additionally, the fatty acid profile also slightly changed. CONCLUSION: The transport stress induced significant energy metabolism changes of shrimp flesh and therefore effected the flesh quality. The intensifications of freshness (K-value) of shrimp flesh were detected as a result of ATP degradation, which were more pronounced after WFT. However, the drip loss of shrimp flesh was more significantly increased (P < 0.05) after WFT compared to WT. © 2023 Society of Chemical Industry.

Supplementation of palmitoleic acid improved piglet growth and reduced body temperature drop upon cold exposure.

Piglet survival is a major challenge in the first few days postpartum and interventions during this period may improve survival and growth. This study investigated the effects of palmitoleic acid (C16:1n-7; PA) supplementation on growth performance, body temperature, fatty acid (FA), and energy metabolism in milk-replacer-fed piglets. Forty-eight piglets were stratified by body weight and randomly assigned to one of four dietary treatments (0%, 1%, 2%, and 3% PA supplementation as a percent of milk replacer) and given the diet through an orogastric tube. They were fed dietary treatments every 2 h for 4 d in the first week postpartum and all were sacrificed at the end of the experiment. The piglets were weighed daily, and half in each dietary treatment group, the same piglets each day, were exposed daily to a lower temperature for 2 h. Plasma samples were collected immediately before sacrifice for analyses of FA and other plasma metabolites. The weight of organs and empty body weight were determined after sacrifice. Liver and semimembranosus muscle tissue samples were collected and analyzed for FA content. Contents of C16:1n-7 and C18:1n-7 in both plasma and liver (P < 0.001), and C16:1n-7 in semimembranosus muscle (P < 0.001) increased linearly as PA supplementation increased. Most plasma FA levels (except C16:1n-7, C16:1n-9, and C22:5n-3) were lower in piglets exposed to lower temperatures than those that were not. Plasma glucose, triglycerides, and lactate dehydrogenase levels increased linearly with PA supplementation (P < 0.001). Piglets' average daily gain, liver glycogen pool, liver weight, and gallbladder weight increased linearly (P < 0.05, P < 0.01, P < 0.05, and P < 0.001, respectively), but lung weight, liver nitrogen content, and body temperature drop decreased linearly (P < 0.01, P < 0.001, and P < 0.05, respectively) with PA supplementation. Piglets exposed to low temperature had greater liver nitrogen (P < 0.05) and lactate dehydrogenase (P < 0.001) contents but had lower liver weight (P < 0.01) and plasma lactate concentration (P < 0.05) than those that were not. In conclusion, this study demonstrated the importance of PA on the growth performance of the piglets by increasing their average daily gain and decreasing a drop in body temperature upon cold exposure, most likely due to a modified energy metabolism.

Reducing piglet mortality in the early days after birth is a significant challenge in the modern pig industry. The focus on achieving larger litter sizes has had a negative impact on piglets' birth weight and their intake of colostrum. Additionally, piglets are born without easily oxidizable brown adipose tissue and have limited body reserves, making them more vulnerable to death due to their lower capacity for thermogenesis. Therefore, it is important to explore dietary strategies that can enhance piglets' thermogenesis capacity. In this study, the role of palmitoleic acid supplementation was investigated in a dose-response design to determine its impact on growth performance, fatty acid composition, and energy metabolism of milk-replacer-fed piglets during their first week of life. The results revealed a linear increase in the average daily gain of the piglets, liver weight, and liver glycogen content with increasing palmitoleic acid supplementation. Moreover, increased palmitoleic acid supplementation was associated with a drop in body temperature when piglets were exposed to a lower temperature during the experimental period. Altogether, the study indicated that palmitoleic acid has a sparing effect on glycogen reserves and that a greater proportion of energy utilized by the piglets to maintain their body temperature was derived from the oxidation of fatty acids. The results indicated a promising approach to improve piglet survival and growth through dietary modifications of fatty acids in the diet.

Evaluation of the effect of Remdesivir on some biomarkers in Iraqi patients with coronavirus 2019 (COVID-19): A cross-sectional study.

COVID-19 is a new virus spreading worldwide that can cause mild to severe illness, multi-organ failure, and even death. Injectable antiviral Remdesivir is effective in treating patients with moderate-to-severe COVID-19. Biomarkers linked to clinical outcomes have been found for COVID-19, although only a few antiviral therapies have been studied. This study aimed to assess how Remdesivir affects several biomarkers in patients with COVID-19 and how those changes impact the severity of the illness. According to Chinese care guidelines for COVID-19, 80 patients with COVID-19 were separated into two groups: group 1 did not receive Remdesivir (RDV) medication and Group 2 received it after 5 days. Injectable antiviral Remdesivir has recently been tested in high-risk, individuals with confirmed SARS-CoV-2 infection who were not hospitalized, and it successfully delayed the onset of the illness. From February 2022 to October 2023, blood samples were taken from study participants to evaluate ferritin, Lactate Dehydrogenase (LDH), and C-reactive protein. The results of this investigation showed that various COVID-19 severity biomarkers, including ferritin, C-reactive protein, and lactate dehydrogenase, may improve more quickly with RDV treatment. These biomarkers are linked to better clinical outcomes during infection. These discoveries enhance the understanding of the COVID-19 antiviral treatment's function. In conclusion, there is a clear association between the levels of biomarkers before and after Remdesivir treatment in COVID-19 cases ranging from moderate to severe. This suggests that the COVID-19 infection might lead to the elevation of several biomarkers.

CEBPD REGULATES OXIDATIVE STRESS AND INFLAMMATORY RESPONSES IN HYPERTENSIVE CARDIAC REMODELING.

ABSTRACT: Hypertension seems to inevitably cause cardiac remodeling, increasing the mortality of patients. This study aimed to explore the molecular mechanism of CCAAT/enhancer-binding protein delta (CEBPD)-mediated oxidative stress and inflammation in hypertensive cardiac remodeling. The hypertensive murine model was established through angiotensin-II injection, and hypertensive mice underwent overexpressed CEBPD vector injection, cardiac function evaluation, and observation of histological changes. The cell model was established by angiotensin-II treatment and transfected with overexpressed CEBPD vector. Cell viability and surface area and oxidative stress (reactive oxygen species/superoxide dismutase/lactate dehydrogenase/malondialdehyde) were assessed, and inflammatory factors (TNF-α/IL-1ß/IL-6/IL-10) were determined both in vivo and in vitro . The levels of CEBPD, miR-96-5p, inositol 1,4,5-trisphosphate receptor 1 (IP3R), natriuretic peptide B, and natriuretic peptide A, collagen I, and collagen III in tissues and cells were determined. The binding relationships of CEBPD/miR-96-5p/IP3R 3' untranslated region were validated. CEBPD was reduced in cardiac tissue of hypertensive mice, and CEBPD upregulation improved cardiac function and attenuated fibrosis and hypertrophy, along with reductions of reactive oxygen species/lactate dehydrogenase/malondialdehyde/TNF-α/IL-1ß/IL-6 and increases in superoxide dismutase/IL-10. CEBPD enriched on the miR-96-5p promoter to promote miR-96-5p expression, whereas CEBPD and miR-96-5p negatively regulated IP3R. miR-96-5p silencing/IP3R overexpression reversed the alleviative role of CEBPD overexpression in hypertensive mice. In summary, CEBPD promoted miR-96-5p to negatively regulate IP3R expression to inhibit oxidative stress and inflammation, thereby alleviating hypertensive cardiac remodeling.

Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.

Propofol Pretreatment Inhibits Liver Damage in Mice with Hepatic Ischemia/Reperfusion Injury and Protects Human Hepatocyte in Hypoxia/Reoxygenation.

BACKGROUND/AIMS: The major complication of liver resection is hepatic ischemia/reperfusion injury. Propofol appears to have organprotective effects. Our study aimed to study the protective role of propofol against hepatic ischemia/reperfusion injury and the potential mechanisms. MATERIALS AND METHODS: Mice and human hepatocytes (LO2) were used to establish 2 models: the ischemia/reperfusion injury model in vivo and the hypoxia/reoxygenation model in vitro, respectively. Alanine and aspartate aminotransferase serum levels were detected to evaluate the extent of hepatic cellular injury. Malondialdehyde, superoxide dismutase, glutathione, and catalase expression levels were measured to evaluate the oxidative damage in mice liver. Lactate dehydrogenase levels were detected for hepatocyte cytotoxicity severity. Nuclear factor, erythroid-like 2 and heme oxygenase 1 expression levels were detected. RESULTS: In the ischemia/reperfusion model, propofol pretreatment significantly reduced the alanine aminotransferase and aspartate aminotransferase expression levels, alleviating the hepatic cellular injury. Propofol also protected the mice liver from oxidative damage. In the hypoxia/reoxygenation model, propofol pretreatment reduced lactate dehydrogenase expression levels, suggesting its protective effects in LO2 cells. Furthermore, propofol increased the nuclear factor, erythroid-like 2 and heme oxygenase 1 expression levels both in vivo and in vitro. CONCLUSION: Propofol acts through the nuclear factor, erythroid-like 2, and heme oxygenase 1 pathway to protect the mice liver against ischemia/reperfusion injury and hepatocytes against hypoxia/reoxygenation injury. Propofol should be used as an effective therapeutic drug for hepatic ischemia/reperfusion injury.

(R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha.

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent L-valine depletion caused by dominant L-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent D-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent L-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. KEY POINTS: (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB.

The implications of exercise in Drosophila melanogaster: insights into Akt/p38 MAPK/Nrf2 pathway associated with Hsp70 regulation in redox balance maintenance.

This study investigated the potential effects of exercise on the responses of energy metabolism, redox balance maintenance, and apoptosis regulation in Drosophila melanogaster to shed more light on the mechanisms underlying the increased performance that this emerging exercise model provides. Three groups were evaluated for seven days: the control (no exercise or locomotor limitations), movement-limited flies (MLF) (no exercise, with locomotor limitations), and EXE (with exercise, no locomotor limitations). The EXE flies demonstrated greater endurance-like tolerance in the swimming test, associated with increased citrate synthase activity, lactate dehydrogenase activity and lactate levels, and metabolic markers in exercise. Notably, the EXE protocol regulated the Akt/p38 MAPK/Nrf2 pathway, which was associated with decreased Hsp70 activation, culminating in glutathione turnover regulation. Moreover, reducing the locomotion environment in the MLF group decreased endurance-like tolerance and did not alter citrate synthase activity, lactate dehydrogenase activity, or lactate levels. The MLF treatment promoted a pro-oxidant effect, altering the Akt/p38 MAPK/Nrf2 pathway and increasing Hsp70 levels, leading to a poorly-regulated glutathione system. Lastly, we demonstrated that exercise could modulate major metabolic responses in Drosophila melanogaster aerobic and anaerobic metabolism, associated with apoptosis and cellular redox balance maintenance in an emergent exercise model.

Correlations between serum leptin levels and classical biomarkers in SARS-CoV-2 infection, in critically ill patients.

BACKGROUND: Altered levels of some blood markers might be linked with the degree of severity and mortality of patients with SARS-CoV-2 infection. This study aimed to find out if there are correlations between serum leptin levels and classical biomarkers. MATERIALS AND METHODS: We present a single-center observational cohort study on SARS-CoV-2 infected patients. The study was conducted at Infectious Diseases Clinic of Academic Emergency Hospital Sibiu, from May through November 2020. In this study, we retrospectively analyzed 54 patients, all with confirmed SARS-CoV-2 infection. RESULTS: Our results revealed that there is a negative correlation between serum leptin and Interleukin-6 levels and a positive correlation between serum leptin and blood glucose levels. A positive correlation between ferritin and lactate dehydrogenase levels was also observed. No correlation was found between leptin and other biomarkers such as ferritin, neutrophil/lymphocyte ratio, lactate dehydrogenase, C-reactive protein, fibrinogen, erythrocyte sedimentation rate, or D-dimer. CONCLUSIONS: Further studies need to be conducted to investigate the role of leptin in SARS-CoV-2 infection. The results of this research could contribute to the introduction of the determination of serum leptin levels in the routine evaluation of patients with critical illness.

Plasma cell myeloma with RAS/BRAF mutations is frequently associated with a complex karyotype, advanced stage disease, and poorer prognosis.

BACKGROUND: Mutations in the RAS-MAPK pathway, such as KRAS, NRAS, and BRAF, are known as high-risk factors associated with poor prognosis in patients with various cancers, but studies in myeloma have yielded mixed results. METHODS: We describe the clinicopathologic, cytogenetic, molecular features, and outcomes of 68 patients with RAS/BRAF-mutated myeloma, and compare with 79 patients without any mutations. RESULTS: We show that KRAS, NRAS, and BRAF were mutated in 16%, 11%, and 5% of cases, respectively. RAS/BRAF-mutated patients had lower hemoglobin and platelet counts, higher levels of serum lactate dehydrogenase and calcium, higher percentage of bone marrow plasma cells, and more advanced R-ISS stage. RAS/BRAF mutations were associated with complex karyotype and gain/amplification of CKS1B. The median overall survival and progression-free survival were significantly shorter for RAS/BRAF-mutated patients (69.0 vs. 220.7 months, p = 0.0023 and 46.0 vs. 60.6 months, p = 0.0311, respectively). Univariate analysis revealed that KRAS mutation, NRAS mutation, lower hemoglobin, elevated lactate dehydrogenase, higher R-ISS stage, complex karyotype, gain/amplification of CKS1B, monosomy 13/RB1 deletion and lack of autologous stem cell transplantation were associated with poorer prognosis. Multivariate analysis showed that KRAS mutation, lower hemoglobin level, higher level of serum calcium, higher ISS stage, and lack of autologous stem cell transplantation predict inferior outcome. CONCLUSIONS: RAS/BRAF mutations occur in 30%-40% of myeloma cases and are associated with higher tumor burden, higher R-ISS stage, complex karyotype, and shorter overall survival and progression-free survival. These findings support testing for RAS/BRAF mutations in myeloma patients and underscore the potential therapeutic benefits of RAS/BRAF inhibitors.

Basal NAD(H) redox state permits hydrogen peroxide-induced mesenteric artery dilatation.

Smooth muscle voltage-gated K+ (Kv) channels in resistance arteries control vascular tone and contribute to the coupling of blood flow with local metabolic activity. Members of the Kv1 family are expressed in vascular smooth muscle and are modulated upon physiological elevation of local metabolites, including the glycolytic end-product l-lactate and superoxide-derived hydrogen peroxide (H2 O2 ). Here, we show that l-lactate elicits vasodilatation of small-diameter mesenteric arteries in a mechanism that requires lactate dehydrogenase (LDH). Using the inside-out configuration of the patch clamp technique, we show that increases in NADH that reflect LDH-mediated conversion of l-lactate to pyruvate directly stimulate the activity of single Kv1 channels and significantly enhance the sensitivity of Kv1 activity to H2 O2 . Consistent with these findings, H2 O2 -evoked vasodilatation was significantly greater in the presence of 10 mM l-lactate relative to lactate-free conditions, yet was abolished in the presence of 10 mM pyruvate, which shifts the LDH reaction towards the generation of NAD+ . Moreover, the enhancement of H2 O2 -induced vasodilatation was abolished in arteries from double transgenic mice with selective overexpression of the intracellular Kvß1.1 subunit in smooth muscle cells. Together, our results indicate that the Kvß complex of native vascular Kv1 channels serves as a nodal effector for multiple redox signals to precisely control channel activity and vascular tone in the face of dynamic tissue-derived metabolic cues. KEY POINTS: Vasodilatation of mesenteric arteries by elevated external l-lactate requires its conversion by lactate dehydrogenase. Application of either NADH or H2 O2 potentiates single Kv channel currents in excised membrane patches from mesenteric artery smooth muscle cells. The binding of NADH enhances the stimulatory effects of H2 O2 on single Kv channel activity. The vasodilatory response to H2 O2 is differentially modified upon elevation of external l-lactate or pyruvate. The presence of l-lactate enhances the vasodilatory response to H2 O2 via the Kvß subunit complex in smooth muscle.

A two-enzyme system in an amorphous metal-organic framework for the synthesis of D-phenyllactic acid.

In this study, we synthesized an amorphous metal-organic framework by adjusting the concentration of precursors, and established a two-enzyme system consisting of lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), which successfully achieved coenzyme recycling, and applied it to the synthesis of D-phenyllactic acid (D-PLA). The prepared two-enzyme-MOF hybrid material was characterized using XRD, SEM/EDS, XPS, FT-IR, TGA, CLSM, etc. In addition, reaction kinetic studies indicated that the MOF-encapsulated two-enzyme system exhibited faster initial reaction velocities than free enzymes due to its amorphous ZIF-generated mesoporous structure. Furthermore, the pH stability and temperature stability of the biocatalyst were evaluated, and the results indicated a significant improvement compared to the free enzymes. Moreover, the amorphous structure of the mesopores still maintained the shielding effect and protected the enzyme structure from damage by proteinase K and organic solvents. Finally, the remaining activity of the biocatalyst for the synthesis of D-PLA reached 77% after 6 cycles of use, and the coenzyme regeneration still maintained at 63%, while the biocatalyst also retained 70% and 68% residual activity for the synthesis of D-PLA after 12 days of storage at 4 °C and 25 °C, respectively. This study provides a reference for the design of MOF-based multi-enzyme biocatalysts.

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and ß cells from humans and mice.

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

Mn(III) Porphyrin, MnTnBuOE-2-PyP5+, Commonly Known as a Mimic of Superoxide Dismutase Enzyme, Protects Cardiomyocytes from Hypoxia/Reoxygenation Induced Injury via Reducing Oxidative Stress.

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes.

Lactate Dehydrogenase Superfamily in Rice and Arabidopsis: Understanding the Molecular Evolution and Structural Diversity.

Lactate/malate dehydrogenases (Ldh/Maldh) are ubiquitous enzymes involved in the central metabolic pathway of plants and animals. The role of malate dehydrogenases in the plant system is very well documented. However, the role of its homolog L-lactate dehydrogenases still remains elusive. Though its occurrence is experimentally proven in a few plant species, not much is known about its role in rice. Therefore, a comprehensive genome-wide in silico investigation was carried out to identify all Ldh genes in model plants, rice and Arabidopsis, which revealed Ldh to be a multigene family encoding multiple proteins. Publicly available data suggest its role in a wide range of abiotic stresses such as anoxia, salinity, heat, submergence, cold and heavy metal stress, as also confirmed by our qRT-PCR analysis, especially in salinity and heavy metal mediated stresses. A detailed protein modelling and docking analysis using Schrodinger Suite reveals the presence of three putatively functional L-lactate dehydrogenases in rice, namely OsLdh3, OsLdh7 and OsLdh9. The analysis also highlights the important role of Ser-219, Gly-220 and His-251 in the active site geometry of OsLdh3, OsLdh7 and OsLdh9, respectively. In fact, these three genes have also been found to be highly upregulated under salinity, hypoxia and heavy metal mediated stresses in rice.

Target integration of an exogenous ß-glucosidase enhances cellulose degradation and ethanol production in Clostridium cellulolyticum.

The bacteria Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing (CBP). However, genetic engineering is necessary to improve this organism's cellulose degradation and bioconversion efficiencies to meet standard industrial requirements. In this study, CRISPR-Cas9n was used to integrate an efficient ß-glucosidase into the genome of C. cellulolyticum, disrupting lactate dehydrogenase (ldh) expression and reducing lactate production. The engineered strain showed a 7.4-fold increase in ß-glucosidase activity, a 70% decrease in ldh expression, a 12% increase in cellulose degradation, and a 32% increase in ethanol production compared to wild type. Additionally, ldh was identified as a potential site for heterologous expression. These results demonstrate that simultaneous ß-glucosidase integration and lactate dehydrogenase disruption is an effective strategy for increasing cellulose to ethanol bioconversion rates in C. cellulolyticum.