The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O₂ ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.
Animals ; Humans ; L-Lactate Dehydrogenase/genetics* ; Lactic Acid ; Adenoviruses, Human ; Ammonia ; HEK293 Cells ; Glucose/metabolism* ; Adenosine Triphosphate/metabolism* ; Kidney/metabolism* ; Mammals/metabolism*

OBJECTIVE: To analyze the clinical and genetic characteristics of a patient with dihydrolipoamide dehydrogenase deficiency. METHODS: Potential variants of the DLD gene were detected by whole exome sequencing and verified by Sanger sequencing. RESULTS: Compound heterozygous variants, c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr), were detected in the DLD gene. The c.1058T>C (p.Ile353Thr) variant was derived from his mother and known to be pathogenic. The c.704_705delTT (p.Leu235Argfs*8) variant was derived from his father and was unreported previously. CONCLUSION The compound heterozygous variants of c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr) of the DLD gene probably underlay the disease in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for the family.
Acidosis, Lactic/genetics* ; Dihydrolipoamide Dehydrogenase/genetics* ; Female ; Genetic Testing ; Genetic Variation ; Humans ; Male ; Maple Syrup Urine Disease/genetics* ; Pregnancy ; Whole Exome Sequencing

PURPOSE: The incidence and mortality of breast cancer is increasing worldwide. There is a constant quest to understand the underlying molecular biology of breast cancer so as to plan better treatment options. The purpose of the current study was to characterize the expression of histone deacetylases-3 (HDAC3), a member of class I HDACs, and assess the clinical significance of HDAC3 in breast cancer. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry, and western blot analysis were used to examine messenger RNA and protein expression levels. The relationships between HDAC3 expression and clinicopathological variables were analyzed. MTT assays were used to detect cell proliferation. Glucose-uptake, lactate, adenosine triphosphate, and lactate dehydrogenase assays were employed to detect aerobic glycolysis. Chromatin immunoprecipitation was used to detect microRNA-31 (miR-31) promoter binding. RESULTS: Our data revealed that HDAC3 was upregulated in breast cancer tissue compared with matched para-carcinoma tissues, and high levels of HDAC3 were positively correlated with advanced TNM stage and N stage of cancer. Furthermore, overexpression of HDAC3 promoted breast cancer cell-proliferation and aerobic glycolysis. The functional involvement of HDAC3 was related in part to the repression of miR-31 transcription via decreased histone H3 acetylation at lysine K9 levels of the miR-31 promoter. Survival analysis revealed that the level of HDAC3 was an independent prognostic factor for breast cancer patients. CONCLUSION: Our findings revealed that HDAC3 served as an oncogene that could promote cell proliferation and aerobic glycolysis and was predictive of a poor prognosis in breast cancer. HDAC3 participated in the cell proliferation of breast cancer, which may prove to be a pivotal epigenetic target against this devastating disease.
Acetylation ; Adenosine Triphosphate ; Blotting, Western ; Breast Neoplasms* ; Breast* ; Cell Proliferation* ; Chromatin Immunoprecipitation ; Epigenomics ; Glycolysis* ; Histone Code ; Histones* ; Humans ; Immunohistochemistry ; Incidence ; L-Lactate Dehydrogenase ; Lactic Acid ; Lysine ; Molecular Biology ; Mortality ; Oncogenes ; Prognosis* ; Real-Time Polymerase Chain Reaction ; Repression, Psychology ; RNA, Messenger

In 1923, Dr. Warburg had observed that tumors acidified the Ringer solution when 13 mM glucose was added, which was identified as being due to lactate. When glucose is the only source of nutrient, it can serve for both biosynthesis and energy production. However, a series of studies revealed that the cancer cell consumes glucose for biosynthesis through fermentation, not for energy supply, under physiological conditions. Recently, a new observation was made that there is a metabolic symbiosis in which glycolytic and oxidative tumor cells mutually regulate their energy metabolism. Hypoxic cancer cells use glucose for glycolytic metabolism and release lactate which is used by oxygenated cancer cells. This study challenged the Warburg effect, because Warburg claimed that fermentation by irreversible damaging of mitochondria is a fundamental cause of cancer. However, recent studies revealed that mitochondria in cancer cell show active function of oxidative phosphorylation although TCA cycle is stalled. It was also shown that blocking cytosolic NADH production by aldehyde dehydrogenase inhibition, combined with oxidative phosphorylation inhibition, resulted in up to 80% decrease of ATP production, which resulted in a significant regression of tumor growth in the NSCLC model. This suggests a new theory that NADH production in the cytosol plays a key role of ATP production through the mitochondrial electron transport chain in cancer cells, while NADH production is mostly occupied inside mitochondria in normal cells.
Adenosine Triphosphate ; Aldehyde Dehydrogenase ; Cytosol ; Electron Transport ; Energy Metabolism ; Fermentation ; Glucose ; Lactic Acid ; Metabolism ; Mitochondria ; NAD ; Oxidative Phosphorylation ; Oxygen ; Symbiosis

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(<0.05). CONCLUSIONS Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.
Adenosine Triphosphatases ; blood ; Animals ; Creatine Kinase ; blood ; Energy Metabolism ; Isoflavones ; pharmacology ; L-Lactate Dehydrogenase ; blood ; Lactic Acid ; blood ; Male ; Mice ; Random Allocation ; Soybeans ; chemistry ; Succinate Dehydrogenase ; blood ; Swimming

The purpose of this study was to compare body composition, anaerobic capacity, and hematologic parameters after intermittent complex training in high school soccer players. The subjects were 15 high school soccer players (age, 15.60±0.82 years; height, 171.93±5.65 cm; body weight, 61.32±7.43 kg). Clinical variables included body composition, anaerobic capacity, and hematologic parameters. Data were analyzed to compare between trials by paired sample t-test. As a result, there were significant differences in body weight (p<0.05) and muscle mass (p<0.05) of body composition after 4 weeks. There were significant differences in shuttle run (p<0.001) and heart rate in rest (p<0.05) of aerobic performance after 4 weeks. There were significant differences in lactate dehydrogenase (p<0.001), creatine kinase (p<0.001), all out lactic acid (p<0.01), and red blood cell (p<0.001) of hematologic parameters after 4 weeks. In summary, intermittent complex training for 4 weeks showed strong influences on body composition and hematologic parameters variables in high school soccer players.
Body Composition* ; Body Weight ; Creatine Kinase ; Erythrocytes ; Heart Rate ; Humans ; L-Lactate Dehydrogenase ; Lactic Acid ; Soccer*

Nonhuman primates are increasingly used in biomedical research since they are highly homologous to humans compared to other rodent animals. However, there is limited reliable reference data of the clinical pathology parameters in cynomolgus monkeys, and in particular, only some coagulation and urinalysis parameters have been reported. Here, we reported the reference data of clinical chemical, hematological, blood coagulation, and urinalysis parameters in cynomolgus monkeys. The role of sex differences was analyzed and several parameters (including hematocrit, hemoglobin, red blood cell, blood urea nitrogen, total bilirubin, alkaline phosphatase, creatinine kinase, gamma-glutamyl tranferase, and lactate dehydrogenase) significantly differed between male and female subjects. In addition, compared to previous study results, lactate dehydrogenase, creatinine kinase, and aspartate aminotransferase showed significant variation. Interstudy differences could be affected by several factors, including age, sex, geographic origin, presence/absence of anesthetics, fasting state, and the analytical methods used. Therefore, it is important to deliberate with the overall reference indices. In conclusion, the current study provides a comprehensive and updated reference data of the clinical pathology parameters in cynomolgus monkeys and provides improved assessment criteria for evaluating preclinical studies or biomedical research.
Alkaline Phosphatase ; Anesthetics ; Animals ; Aspartate Aminotransferases ; Bilirubin ; Blood Coagulation ; Blood Urea Nitrogen ; Chemistry, Clinical ; Creatinine ; Erythrocytes ; Fasting ; Female ; Hematocrit ; Hematology ; Humans ; L-Lactate Dehydrogenase ; Lactic Acid ; Macaca fascicularis* ; Male ; Pathology, Clinical* ; Phosphotransferases ; Primates ; Reference Values* ; Rodentia ; Sex Characteristics ; Urinalysis

PURPOSE: Skin pH, an indicator of skin health, is maintained by various organic factors, which include lactate, free amino acid (FAA), and free fatty acid (FFA). As skin ages or with illness, skin pH becomes less acidic, and functional food has been developed to maintain the acidic pH of skin. In this study, we determined the dietary effect of green tea extract (GTE) on skin pH of photo-aged mice, as measured by epidermal levels of lactate, FAA, and FFA. The protein expression and activity of lactate dehydrogenase (LDH), an enzyme of pyruvate reduction for lactate generation, was further determined. METHODS: Albino hairless mice were fed a control diet (group UV+) or a diet with 1% GTE (group GTE) in parallel with UV irradiation for 10 weeks. A normal control group was fed a control diet without UV irradiation for 10 weeks (group UV-). RESULTS: Skin pH was higher (less acidic) in group UV+ than in group UV-. In parallel, epidermal levels of lactate and FFA, as well as of LDH protein expression and activity, were reduced in group UV+. Dietary supplementation of GTE (group GTE) reduced skin pH to similar to the level of group UV-, and inversely increased epidermal levels of lactate, LDH protein expression and activity, but not of FFA. Although epidermal levels of FAA were similar in groups UV- and UV+, it was increased in group GTE to a level higher than in group UV-. In further analysis of major FFA, epidermal levels of palmitic acid [16:0], oleic acid [18:1(n-9)], and linoleic acid [18:2(n-6), but not of stearic acid [18:0] in group GTE were similar to or lower than those in group UV+. CONCLUSION: Dietary GTE normalized skin pH with increased levels of lactate and FAA, as well as with increased protein expression and activity of LDH in the epidermis of UVB irradiated hairless mice.
Animals ; Diet ; Dietary Supplements ; Epidermis ; Functional Food ; Hydrogen-Ion Concentration* ; L-Lactate Dehydrogenase* ; Lactic Acid* ; Linoleic Acid ; Mice ; Mice, Hairless* ; Oleic Acid ; Palmitic Acid ; Pyruvic Acid ; Skin* ; Tea*

PURPOSE: Acute kidney injury (AKI) in major burn patients is a common complication with high morbidity and mortality. The mainstream treatment is early diagnosis and rapid termination and prevention of the underlying insult. Therefore, it's essential to identify early biomarkers predicting AKI. METHODS: A total of 85 patients who were admitted to the burn intensive care unit from June 2012 to July 2013 were included in this prospective cohort study. Ten biomarkers (blood urea nitrogen, serum creatinine, urine creatinine, cystatin C, cystatin C glomerular filtration rate, AST, lacate dehydrogenase [LD], creatine kinase, lactic acid, and myoglobin) were obtained at time of admission and evaluated as diagnostic biomarkers to predicting AKI and early AKI. RESULTS: Out of 85 patients, 35 patients were dead and overall mortality was 41.2%. The mean age was 49.4 years and mean percentage of total body surface area was 53.2%. Area under the curve (AUC) of receiver operating characteristic curve of biomarkers on predicting AKI were 0.746, 0.718, and 0.717 in LD, lactic acid, and serum creatinine, respectively. AUC of cystatin C predicting AKI was much lower at 0.555. AUC of biomarkers on predicting early AKI were 0.833, 0.816, 0.790, and 0.759 in LD, serum creatinine, AST, and serum myoglobin. CONCLUSION: LD, lactic acid and serum creatinine were acceptable as diagnostic biomarkers of AKI and LD, serum creatinine, AST, and serum myoglobin were reasonable as diagnostic biomarkers of early AKI. However, cystatin C was an unfavorable biomarker in major burn patients.
Acute Kidney Injury* ; Area Under Curve ; Biomarkers* ; Body Surface Area ; Burns* ; Cohort Studies ; Creatine Kinase ; Creatinine ; Cystatin C ; Early Diagnosis ; Glomerular Filtration Rate ; Humans ; Intensive Care Units ; L-Lactate Dehydrogenase ; Lactic Acid ; Mortality ; Myoglobin ; Nitrogen ; Oxidoreductases ; Prospective Studies ; ROC Curve ; Urea