Excess Lactate Research Articles

Sodium oligomannate disrupts the adherence of Ribhigh bacteria to gut epithelia to block SAA-triggered Th1 inflammation in 5XFAD transgenic mice

Sodium oligomannate (GV-971), an oligosaccharide drug approved in China for treating mild-to-moderate Alzheimer’s disease (AD), was previously found to recondition the gut microbiota and limit altered peripheral Th1 immunity in AD transgenic mice. As a follow-up study, we here made advances by pinpointing a Lactobacillus murinus (L.m.) strain that highly expressed a gene encoding a putative adhesin containing Rib repeats (Ribhigh-L.m.) particularly enriched in 5XFAD transgenic mice. Mechanistically, Ribhigh-L.m. adherence to the gut epithelia upregulated fecal metabolites, among which lactate ranked as the top candidate. Excess lactate stimulated the epithelial production of serum amyloid A (SAA) in the gut via the GPR81-NFκB axis, contributing to peripheral Th1 activation. Moreover, GV-971 disrupted the adherence of Ribhigh-L.m. to gut epithelia via direct binding to Rib, which corrected the excess lactate, reduced SAA, and alleviated Th1-skewed inflammation. Together, we gained further insights into the molecular link between gut bacteria and AD progression and the mechanism of GV-971 in treating AD.

Capillary blood parameters are gestational age, birthweight, delivery mode and gender dependent in healthy preterm and term infants.

The measurement of blood pH and gas analytes (BPGA), soon after birth, constitutes the first-line standard of care procedure in high-risk newborns. However, no data is available in capillary blood on perinatal bias such as gestational age (GA), weight at birth (BW), delivery mode, and gender. The aims of the present study were to investigate whether in a cohort of healthy preterm (PT) and term (T)infants BPGA were GA, BW, delivery mode and gender dependent, thus affecting BPGA reliability as diagnostic test. We performed a prospective case-control study in 560 healthy infants (PT: n=115, T: n=445). BPGA was measured within 24-h from birth. Perinatal characteristics, outcomes, and clinical examination were also recorded. PT infants showed higher (p<0.001) carbon dioxide partial pressure (pCO2), fraction of fetal hemoglobin (HbF), base excess (BE), bicarbonate (HCO3), and lower lactate (Lac) levels. When corrected for delivery mode, higher (p<0.001) HbF, BE, HCO3, and lower Lac levels were found. Similarly, higher (p<0.05, for all) pCO2, HbF, BE, HCO3 and lower Lac levels were found between female and male PT and T infants. Repeated multiple logistic regression analysis showed that BPGA was GA, BW, delivery mode and gender dependent. The present results showing that BPGA can be affected by a series of perinatal outcomes open the way to further investigations providing longitudinal BPGA reference curves in the transitional phase, thus empowering BPGA role as a reliable diagnostic and therapeutic strategies efficacy marker.

The Lactate Receptor GPR81 is a Mechanism of Leukemia-Associated Macrophage Polarization in the Bone Marrow Microenvironment.

Interactions between acute myeloid leukemia (AML) and the bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. Altered metabolite levels in the tumor microenvironment contribute to immunosuppression in solid tumors, while this has not been studied yet in the leukemic BMME. Metabolomics of AML patient bone marrow serum detected elevated metabolites, including lactate, compared to age- and sex-matched controls. Excess lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages (TAMs) and correlates with poor prognosis. We describe the role of lactate in the polarization of leukemia-associated macrophages (LAMs) using a murine model of blast crisis chronic myelogenous leukemia (bcCML) and mice genetically lacking the lactate receptor GPR81. LAMs were CD206hi and suppressive in transcriptomics and cytokine profiling. Yet, LAMs had a largely unique expression profile from other types of TAMs. We demonstrate GPR81 signaling as a mechanism of both LAM polarization and the direct support of leukemia cell growth and self-repopulation. Furthermore, LAMs and elevated lactate diminished the function of hematopoietic progenitors and stromal support, while knockout of GPR81 had modest protective effects on the hematopoietic system. We report microenvironmental lactate as a critical driver of AML-induced immunosuppression and leukemic progression, thus identifying GPR81 signaling as an exciting and novel therapeutic target for treating this devastating disease.

Correlation of cardiotocography with combined APGAR scores and diagnostic performance of umbilical cord parameters in predicting low combined APGAR scores – A prospective Cohort study

ObjectivesCombined Apgar score includes utilization of interventions such as Continuous positive airway pressure, Oxygen, Mask and Bag ventilation, I ntubation and ventilation, Ne onatal chest compression, Drugs, and newborn assessment. It has been proposed as a substitute for conventional Apgar score which is the gold standard for evaluating newborns right after birth but is impacted by medical interventions and preterm. Combined Apgar scores were examined to check for correlation with CTG tracing and umbilical cord blood parameters which gives an objective assessment of fetal hypoxia, in response to the demand for a more accurate tool for evaluating the neonate and to be used for medico-legal purposes. The study's objectives were to (1) determine the association of combined Apgar scores with suspicious and pathological CTG (2) the association of umbilical cord parameters with low combined Apgar scores and the diagnostic performance of these parameters in predicting low combined Apgar scores. Study designA prospective observational cohort study was conducted in a tertiary care center in East India. 2350 consecutive laboring mothers who had completed 34 weeks of gestation underwent cardiotocography according to institutional protocol and those with suspicious and pathological CTG who delivered within 1 h of abnormal CTG were recruited. Arterial blood was analyzed and the newborn was evaluated immediately after delivery with a combined Apgar scoring system ResultsOf the 2350 women, 50.7 % and 49.3 %, respectively, exhibited suspicious and abnormal CTG tracings. CTG was reported to have low diagnostic accuracy and specificity, with a sensitivity of 66.7 % and 88.9 %, respectively, in detecting combined Apgar at 1 and 5 min. The combined Apgar score at five minutes showed a strong association with acidosis. There was a statistically significant correlation between low combined Apgar and excess lactate and base at one and five minutes. With 100 % sensitivity and 95 % specificity, high lactate levels > 4.1 mM/L were found to predict newborn encephalopathy. ConclusionUmbilical cord blood parameters were found to be correlated with low combined Apgar scores. Combined Apgar scores may be a more useful tool for neonatal assessment and long-term morbidity of newborns. Additional research is required to determine whether it can take the role of conventional Apgar scores in clinical practice.

P-190 Impact of low lactate culture media in embryo development and IVF outcomes

Abstract Study question Can low lactate culture media improve embryo utilization rate in artificial reproductive technology (ART) cycles? Summary answer Embryos cultured in low lactate media showed a significant improved blastocyst utilization rate compared to embryos cultured in standard culture media. What is known already Culture media plays a key role in the embryonic development in the IVF lab. Currently, different culture medias are commercially available, yet no consensus on the most suitable media composition has been reached. Recently, minimizing metabolic stress for the embryo to enhance implantation and pregnancy rates has been suggested. Research on the metabolism of mammalian preimplantation embryos revealed that excess lactate in the culture medium, in addition to pyruvate and glucose, can burden metabolic efficiency, as embryos naturally produce lactate during energy production. Study design, size, duration The prospective, monocentric study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). A total of 60 women with an average age of 32.5 undergoing ICSI (intracytoplasmic sperm injection) treatment were included. All of them had at least 6 mature (MII) oocytes at time of insemination. Sibling oocytes were cultured in two different culture medias and outcomes were compared. Patients undergoing preimplantation genetic testing were excluded. Participants/materials, setting, methods Mature (MII) oocytes of each patient (total 621 MII oocytes) were inseminated by ICSI and then equally split into two culture medias: standard (313 oocytes) and low lactate (308 oocytes) covered with mineral-oil overlay and incubated in time-lapse under the same conditions. A total of 428 embryos were obtained. Embryos were evaluated using morphokinetic criteria. The fertilization rate, embryo utilization rate, pregnancy rate and time to Blastocyst were analyzed. Main results and the role of chance No differences were observed in the number of fertilized oocytes (n = 211 and 217 in the standard and low lactate media, respectively). The overall embryo utilization rate was significantly different (p &amp;lt; 0.008) resulting in 52% in standard media and 64.3% using low lactate media. Interestingly, age stratification (18-30; 31-35; 36-41) revealed overall higher embryo utilization rates when using low lactate media in all age groups. Blastocyst utilization rate was significantly different (p &amp;lt; 0.007) 43,1% and 58,05% in the standard and low lactate media, respectively. Regarding the time to blastocyst, it was observed that in the standard culture media 80% of the blastocysts reached blastocyst stage on D + 5 whereas in the low lactate media 92,07% reached blastocyst stage on D + 5, which was also significantly different (p &amp;lt; 0.001). No differences in pregnancy and live birth rates were observed. Limitations, reasons for caution The number of patients may be seen as a study limitation. Results should be confirmed with a bigger sample size. Wider implications of the findings Low lactate media could help improve IVF outcomes due to higher embryo utilization rate leading to better cumulative pregnancy rates. Excessive lactate concentration in culture media could negatively influence embryo metabolism. Trial registration number not applicable

Serial paired arterial and jugular venous point-of-care values in dogs undergoing manual basic life support.

To evaluate differences in point-of-care (POC) variables obtained from arterial and jugular venous blood in dogs undergoing manual basic life support (BLS) and report changes over time. Experimental study. Small animal research facility. Twenty-four purpose-bred research dogs. Dogs were anesthetized, and arterial catheters were placed before euthanasia. One minute after cardiopulmonary arrest, BLS consisting of manual chest compressions and ventilation delivered via endotracheal intubation, face mask, mouth-to-nose, or no ventilation was initiated. Paired arterial and jugular venous blood samples were obtained for POC testing before euthanasia (T0), at 3minutes (T3), and at 6minutes (T6) into BLS. The association of POC variables with arterial or venous sample type while controlling for type of ventilation and sampling timepoint was determined using a generalized linear mixed model. Variables obtained from arterial and venous blood samples were compared over time using repeated measures ANOVA or Friedman test. Pao2, anion gap, potassium, chloride, glucose concentration, and PCV were significantly higher in arterial blood samples compared with venous samples (P<0.03). By T6, arterial glucose concentration, arterial and venous base excess, venous pH, and plasma lactate, potassium, creatinine, bicarbonate, and sodium concentrations were significantly increased, and arterial and venous Po2, ionized calcium concentration, PCV, and total plasma protein concentration were significantly decreased from T0 (P<0.05). Although statistically significant, arteriovenous differences and changes in POC blood variables during BLS were small and not clinically relevant over time. Given the challenges of arterial blood sampling, it may be reasonable to pursue venous blood sampling during CPR. Further studies in dogs undergoing BLS and advanced life support are needed to better understand the potential clinical role of POC testing during CPR.

Enhanced Osteosarcoma Immunotherapy via CaCO3 Nanoparticles: Remodeling Tumor Acidic and Immune Microenvironment for Photodynamic Therapy.

Osteosarcoma (OS) is a "cold" tumor enriched in noninflammatory M2 phenotype tumor-associated macrophages (TAMs), which limits the efficacy of immunotherapy. The acidic tumor microenvironment (TME), generated by factors such as excess hydrogen (H+) ions and high lactate levels, activates immunosuppressive cells, further promoting a suppressive tumor immune microenvironment (TIME). Therefore, a multitarget synergistic combination strategy that neutralizes the acidic TME and reprograms TAMs can be beneficial for OS therapy. Here, a calcium carbonate (CaCO3)/polydopamine (PDA)-based nanosystem (A-NPs@(SHK+Ce6)) is developed. CaCO3 nanoparticles are used to neutralize H+ ions and alleviate the suppressive TIME, and the loaded SHK not only synergizes with photodynamic therapy (PDT) but also inhibits lactate production, further reversing the acidic TME and repolarizing TAMs to consequently lead to enhanced PDT-induced tumor suppression and comprehensive beneficial effects on antitumor immune responses. Importantly, A-NPs@(SHK+Ce6), in combination with programmed cell death protein 1 (PD-1) checkpoint blockade, shows a remarkable ability to eliminate distant tumors and promote long-term immune memory function to protect against rechallenged tumors. This work presents a novel multiple-component combination strategy that coregulates the acidic TME and TAM polarization to reprogram the TIME.

Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.

Synthesis of organic photothermal agents for combined photothermal/enzymatic dynamic therapy of breast cancer

Nanoparticle-based photothermal therapy is an effective treatment for superficial tumors. However, due to the highly heterogeneous nature of breast cancer, photothermal therapy (PTT) used alone was unable to achieve satisfactory therapeutic results. Therefore, combining PTT with other therapies is a promising strategy for tumor treatment. Inspired by the significant elevation of lactate levels at the tumor site, we prepared a novel nanocomposite therapeutic platform (PPy-LOX) for combined PTT and enzyme dynamic treatment (EDT) of breast cancer. In detail, Polypyrrole (PPy) nanoparticles were synthesized using a simple chemical oxygenation method and polyvinylpyrrolidone (PVP) was used as a surface linker to enhance its colloidal stability. Further, the loading of lactate oxidase (LOX) is accomplished by simple physical mixing. The in vivo and in vitro results demonstrated the excellent biocompatibility of the synthesized PPy-LOX NPs. More importantly, PPy-LOX NPs has excellent photothermal conversion (η = 29.9%) and lactic acid catalytic ability. On the one hand, the temperature increase induced by near-infrared light irradiation can cause apoptosis of tumor cells; on the other hand, LOX can catalyze the generation of hydrogen peroxide from excess lactate in the tumor microenvironment to induce oxidative stress to kill tumor cells. In conclusion, the combination of PTT and EDT can effectively kill tumor cells. This work provides new ideas on how to design rational nanotherapeutic systems by exploiting the tumor microenvironment.

Excess post-exercise oxygen consumption after reduced exertion high-intensity interval training on the cycle ergometer and rowing ergometer.

To examine differences in oxygen consumption ([Formula: see text]O2), ventilation ([Formula: see text]E), excess post-exercise oxygen consumption (EPOC), energy expenditure (EE), and blood lactate concentration (BLa) between reduced exertion high-intensity interval training (REHIT) performed on the cycle- and rowing ergometer. Fourteen active participants (age = 27 ± 7yr) initially completed two assessments of maximal oxygen uptake. On two subsequent days, participants completed REHIT requiring three 20s "all-out" sprints on the cycle-(REHIT-CE) and rowing ergometer (REHIT-RE), followed by 60min rest during which gas exchange data and BLa were measured. During exercise, [Formula: see text]O2 increased significantly in response to REHIT-CE (0.21 ± 0.04 L/min vs. 1.34 ± 0.37 L/min, p < 0.001) and REHIT-RE (0.23 ± 0.05 L/min vs. 1.57 ± 0.47 L/min, p < 0.001) compared to rest, and [Formula: see text]O2 remained elevated at 15, 30, and 45min post-exercise in REHIT-CE (p < 0.001). However, [Formula: see text]O2 was only elevated 15min after REHIT-RE (0.23 ± 0.05 L/min vs. 0.40 ± 0.11 L/min, p < 0.001). [Formula: see text]O2 (1.57 ± 0.47 L/min vs. 1.34 ± 0.37 L/min, p = 0.003) and EE (94.98 ± 29.60kcal vs. 82.05 ± 22.85kcal, p < 0.001) were significantly greater during REHIT-RE versus REHIT-CE. EPOC was significantly greater after REHIT-CE versus REHIT-RE (6.69 ± 2.18 L vs. 5.52 ± 1.67 L, p = 0.009). BLa was ~ twofold higher in response to REHIT-CE vs. REHIT-RE (11.11 ± 2.43 vs. 7.0 ± 2.4, p < 0.001). Rowing-based REHIT elicits greater oxygen consumption and EE during exercise, yet lower EPOC and BLa. Whether rowing-based REHIT augments reductions in fat loss remains to be determined.

Oxamate enhances the efficacy of CAR-T therapy against glioblastoma via suppressing ectonucleotidases and CCR8 lactylation

BackgroundChimeric antigen receptor (CAR)-T immunotherapy fails to treat solid tumors due in part to immunosuppressive microenvironment. Excess lactate produced by tumor glycolysis increases CAR-T immunosuppression. The mechanism of lactate inducing the formation of immunosuppressive microenvironment remains to be further explored.MethodsImmunocyte subpopulations and molecular characteristics were analyzed in the orthotopic xenografts of nude mice using flow cytometry assay and immunohistochemical staining after oxamate, a lactate dehydrogenase A (LDHA) inhibitor, and control T or CAR-T cells injection alone or in combination. RT-qPCR, western blot, flow cytometry, immunofluorescence, luciferase reporter assay, chromatin immunoprecipitation and ELISA were performed to measure the effect of lactate on the regulation of CD39, CD73 and CCR8 in cultured glioma stem cells, CD4 + T cells or macrophages.ResultsOxamate promoted immune activation of tumor-infiltrating CAR-T cells through altering the phenotypes of immune molecules and increasing regulatory T (Treg) cells infiltration in a glioblastoma mouse model. Lactate accumulation within cells upregulated CD39, CD73 and CCR8 expressions in both lactate-treated cells and glioma stem cells-co-cultured CD4 + T cells and macrophages, and intracellular lactate directly elevated the activities of these gene promotors through histone H3K18 lactylation.ConclusionsUtilizing lactate generation inhibitor not only reprogramed glucose metabolism of cancer stem cells, but also alleviated immunosuppression of tumor microenvironment and reduced tumor-infiltrating CAR-Treg cells, which may be a potential strategy to enhance CAR-T function in glioblastoma therapy.

Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate.

The accumulation of metabolic wastes in cell cultures can diminish product quality, reduce productivity, and trigger apoptosis. The limitation or removal of unintended waste products from Chinese hamster ovary (CHO) cell cultures has been attempted through multiple process and genetic engineering avenues with varied levels of success. One study demonstrated a simple method to reduce lactate and ammonia production in CHO cells with adaptation to extracellular lactate; however, the mechanism behind adaptation was not certain. To address this profound gap, this study characterizes the phenotype of a recombinant CHO K-1 cell line that was gradually adapted to moderate and high levels of extracellular lactate and examines the genomic content and role of extrachromosomal circular DNA (eccDNA) and gene expression on the adaptation process. More than 500 genes were observed on eccDNAs. Notably, more than 1000 genes were observed to be differentially expressed at different levels of lactate adaptation, while only 137 genes were found to be differentially expressed between unadapted cells and cells adapted to grow in high levels of lactate; this suggests stochastic switching as a potential stress adaptation mechanism in CHO cells. Further, these data suggest alanine biosynthesis as a potential stress-mitigation mechanism for excess lactate in CHO cells.

Altered brain energy metabolism related to astrocytes in tauopathies

AbstractBackgroundAccumulated evidence suggests altered energy metabolism in the brain of tauopathies. In Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP), decreased fluorodeoxyglucose (FDG) uptake in the cingulate cortex is considered a marker of neurodegeneration. However, its underlying pathogenesis remains unknown. It is hypothesized that lactate, a glucose metabolite, is produced in astrocytes and subsequently shuttles to neurons as an energy substrate. The current study aims to examine alterations in lactate and glucose concentrations and their association with astrocytic activities in the cingulate cortex of patients with AD and PSP.MethodThirty AD, 30 PSP, and 30 healthy control (HC) subjects were enrolled. Lactate, glucose, and myoinositol (mI), an astroglial marker, in the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) were determined by magnetic resonance spectroscopy (MRS) (3T scanner, SPECIAL sequence, TR/TE 3000/8.5ms, LCModel analysis) (Figure 1). Tau depositions were assessed using PET imaging with 18F‐PM‐PBB3.ResultAll the patients with AD and PSP were confirmed to have the typical distribution of tau pathology. Lactate, glucose, and mI levels were higher in the ACC in PSP and AD, and PCC in AD compared to HC (p &lt; 0.05) (Figure 2). In these cortical regions, lactate levels showed a positive correlation with mI levels (p &lt; 0.05) (Figure 3). Lactate levels in the PCC in AD patients showed a positive correlation with Clinical Dementia Rating‐Sum of Boxes (CDR‐SB) scores (p &lt; 0.05) (Figure 4). Moreover, mI levels in the ACC in PSP patients were negatively correlated with Frontal Assessment Battery (FAB) scores (p &lt; 0.05) (Figure 4).ConclusionWe found elevated lactate and glucose levels accompanied by an increased astroglial marker mainly in the PCC in AD and ACC in PSP patients. Moreover, elevated lactate and mI levels in the cingulate cortex reflected the cognitive decline of AD and PSP, respectively. The excess lactate and glucose levels in the disease‐specific regions might reflect decreased energy utilization as shown in previous FDG‐PET studies. Given the residual lactate levels were correlated with mI levels, we speculated that the impaired lactate shuttle of reactivated astrocytes disrupts energy metabolism, leading to cognitive dysfunctions.

Urinary Metabolomics in Young Soccer Players after Winter Training Season.

During the off-season, soccer players in Korea attend the winter training season (WTS) to build running stamina for the next season. For young soccer players, proper recovery time is needed to prevent injury or muscle damage. In this study, urinary metabolites in young players after 1, 5, and 10 days of the WTS were analyzed using nuclear magnetic resonance spectroscopy (NMR) combined with multivariate analysis to suggest appropriate recovery times for improving their soccer skills. After NMR analysis of the urine samples obtained from young players, 79 metabolites were identified, and each group (1, 5, or 10 days after WTS) was separated from the before the WTS group in the target profiling analysis using partial least squares-discriminant analysis (PLS-DA). Of these, 15 metabolites, including 1-methylnicotinamide, 3-indoxylsulfate, galactarate, glutamate, glycerol, histamine, methylmalonate, maltose, N-phenylacetylglycine, trimethylamine, urea, 2-hydroxybutyrate, adenine, alanine, and lactate, were significantly different than those from before the WTS and were mainly involved in the urea, purine nucleotide, and glucose-alanine cycles. In this study, most selected metabolites increased 1 day after the WTS and then returned to normal levels. However, 4 metabolites, adenine, 2-hydroxybutyrate, alanine, and lactate, increased during the 5 days of recovery time following the WTS. Based on excess ammonia, adenine, and lactate levels in the urine, at least 5 days of recovery time can be considered appropriate.

On-chip dielectrophoretic recovery and detection of a lactate sensing probiotic from model human saliva.

Early detection has led to increased survival for multiple cancers; however, the 5-year survival rate of oral carcinoma (OC) has remained at 40% for the last several decades. Screening for OC is routinely done via visual examinations, followed by tissue biopsy and laboratory testing. Point-of-care testing would be a more convenient and widely available alternative for at-risk individuals. Increased lactate production is a hallmark of many head-and-neck tumors, due to the Warburg Effect, where tumor cells favor glycolysis in the place of oxidative phosphorylation. To detect excess lactate, we have modified the commensal bacterium Escherichia coli Nissle 1917 to express fluorescent reporter genes in response to extracellular lactate. Administering this commensal as a mouth wash and subsequently collecting saliva for the detection of the reporter may allow for noninvasive, early detection of cancerous lesions in at-risk individuals. Furthermore, we demonstrate a new on-chip electrokinetic technique to recover these probiotic probes from model saliva fluid to improve the detection of reporter gene activation.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

The Triangle Relationship Between Long Noncoding RNA, RIG-I-like Receptor Signaling Pathway, and Glycolysis.

Long noncoding RNA (LncRNA), a noncoding RNA over 200nt in length, can regulate glycolysis through metabolic pathways, glucose metabolizing enzymes, and epigenetic reprogramming. Upon viral infection, increased aerobic glycolysis providzes material and energy for viral replication. Mitochondrial antiviral signaling protein (MAVS) is the only protein-specified downstream of retinoic acid-inducible gene I (RIG-I) that bridges the gap between antiviral immunity and glycolysis. MAVS binding to RIG-I inhibits MAVS binding to Hexokinase (HK2), thereby impairing glycolysis, while excess lactate production inhibits MAVS and the downstream antiviral immune response, facilitating viral replication. LncRNAs can also regulate antiviral innate immunity by interacting with RIG-I and downstream signaling pathways and by regulating the expression of interferons and interferon-stimulated genes (ISGs). Altogether, we summarize the relationship between glycolysis, antiviral immunity, and lncRNAs and propose that lncRNAs interact with glycolysis and antiviral pathways, providing a new perspective for the future treatment against virus infection, including SARS-CoV-2.

Dynamics of cortical oxygenation during immediate adaptation to extrauterine life

The neonatal transition involves physiological modifications as a consequence of the complexity of the perinatal period. Various strategies can be used to attain the same level of postnatal cerebral oxygenation, depending on the status of the infant at birth. We evaluated such strategies by recording 20 full-term newborns by near-infrared spectroscopy during the first 10 min of life. The acid–base status at birth revealed two clustered profiles of cerebral oxygenation dynamics. Lower pH and base excess and higher lactate levels were associated with more rapid attainment of the 95% maximal tissue oxygenation index value. These results suggest that metabolic mechanisms drive initial cerebral oxygenation dynamics during this critical period. These results confirm the capacity of newborns to develop multiple strategies to protect the brain.

Therapeutic Hypothermia Modulates the Relationships Between Indicators of Severity of Neonatal Hypoxic Ischemic Encephalopathy and Serum Biomarkers.

Objective: To determine the changes due to therapeutic hypothermia (TH) exposure in the strength of association between traditional clinical and biochemical indicators of severity of neonatal hypoxic-ischemic encephalopathy (HIE) and serum biomarkers. We hypothesized that culmination of TH changes the strength of the relationships between traditional indicators of severity of HIE and serum biomarkers.Methods: This was a single-center observational cohort study of 178 neonates with HIE treated with TH and followed with serum biomarkers: (i) brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) (neurotrophins); (ii) tau and glial fibrillary acidic protein (GFAP) (neural cell injury); and (iii) interleukin 6 (IL-6), IL-8, and IL-10 (cytokines), during their first week of life. Adjusted mixed-effect models tested associations with HIE indicators in relation to TH exposure.Results: At admission, lower Apgar scores and base excess (BE) and higher lactate and nucleated red blood cell (NRBC) count correlated with higher Sarnat scores. These indicators of worse HIE severity, including higher Sarnat score, correlated with lower VEGF and higher tau, GFAP, and IL-10 levels at different time points. Within the first 24 h of life, patients with a Sarnat score >2 had lower VEGF levels, whereas only those with score of 3 also had higher GFAP and IL-10 levels. Tau levels increased during TH in patients with Sarnat score of 3, whereas tau and GFAP increased after TH in those with scores of 2. After adjustments, lower VEGF levels during TH and higher tau, GFAP, and IL-10 levels during and after TH were associated with worse Sarnat scores. Tau and GFAP relationship with Sarnat score became stronger after TH.Conclusion: Therapeutic hypothermia exerts an independent modulatory effect in the relationships between traditional indicators of severity of HIE and serum biomarkers after adjustments. Thus, the timing of biomarker testing in relation to TH exposure must be carefully considered if biomarkers are proposed for patient stratification in novel clinical trials.

The thermogenic activity of adjacent adipocytes fuels the progression of ccRCC and compromises anti-tumor therapeutic efficacy

Clear cell renal cell carcinoma (ccRCC) preferentially invades into perinephric adipose tissue (PAT), a process associated with poor prognosis. However, the detailed mechanisms underlying this interaction remain elusive. Here, we describe a bi-directional communication between ccRCC cells and the PAT. We found that ccRCC cells secrete parathyroid-hormone-related protein (PTHrP) to promote the browning of PAT by PKA activation, while PAT-mediated thermogenesis results in the release of excess lactate to enhance ccRCC growth, invasion, and metastasis. Further, tyrosine kinase inhibitors (TKIs) extensively used in the treatment of ccRCC enhanced this vicious cycle of ccRCC-PAT communication by promoting the browning of PAT. However, if this cross-communication was short circuited by the pharmacological suppression of adipocyte browning via H89 or KT5720, the anti-tumor efficacy of the TKI, sunitinib, was enhanced. These results suggest that ccRCC-PAT cross-communication has important clinical relevance, and use of combined therapy holds great promise in enhancing the efficacy of TKIs.