Elucidating the role of liver enzymes as markers and regulators in ovarian cancer: a synergistic approach using Mendelian randomization, single-cell analysis, and clinical evidence.

OBJECTIVE: To investigate the association between liver enzymes and ovarian cancer (OC), and to validate their potential as biomarkers and their mechanisms in OC. Methods Genome-wide association studies for OC and levels of enzymes such as Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase, and gamma-glutamyltransferase were analyzed. Univariate and multivariate Mendelian randomization (MR), complemented by the Steiger test, identified enzymes with a potential causal relationship to OC. Single-cell transcriptomics from the GSE130000 dataset pinpointed pivotal cellular clusters, enabling further examination of enzyme-encoding gene expression. Transcription factors (TFs) governing these genes were predicted to construct TF-mRNA networks. Additionally, liver enzyme levels were retrospectively analyzed in healthy individuals and OC patients, alongside the evaluation of correlations with cancer antigen 125 (CA125) and Human Epididymis Protein 4 (HE4). RESULTS: A total of 283 single nucleotide polymorphisms (SNPs) and 209 SNPs related to ALP and AST, respectively. Using the inverse-variance weighted method, univariate MR (UVMR) analysis revealed that ALP (P = 0.050, OR = 0.938) and AST (P = 0.017, OR = 0.906) were inversely associated with OC risk, suggesting their roles as protective factors. Multivariate MR (MVMR) confirmed the causal effect of ALP (P = 0.005, OR = 0.938) on OC without reverse causality. Key cellular clusters including T cells, ovarian cells, endothelial cells, macrophages, cancer-associated fibroblasts (CAFs), and epithelial cells were identified, with epithelial cells showing high expression of genes encoding AST and ALP. Notably, TFs such as TCE4 were implicated in the regulation of GOT2 and ALPL genes. OC patient samples exhibited decreased ALP levels in both blood and tumor tissues, with a negative correlation between ALP and CA125 levels observed. CONCLUSION: This study has established a causal link between AST and ALP with OC, identifying them as protective factors. The increased expression of the genes encoding these enzymes in epithelial cells provides a theoretical basis for developing novel disease markers and targeted therapies for OC.

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolizes aspartate to enable N2 fixation in pea nodules.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.

The Increased aspartate levels in transgenic cotton (Gossypium hirsutum L.) lead to improved tolerance against whitefly (Bemisia tabaci, Gennadius).

The whitefly, a polyphagous insect pest feeding on nearly 1328 plant species, is a major threat to global cotton production and incurs up to 50% yield losses in cotton production in Pakistan. We investigated whether increased aspartate in phloem sap imparts whitefly toxicity and protects cotton plants from intense damage. The enzymatic step for aspartate production is carried through aspartate aminotransferase (AAT). In this study, we constitutively overexpressed the Oryza sativa cytoplasmic AAT (OsAAT2) under the CaMV35S promoter in Gossypium hirsutum cv. CIM-482. Real-time PCR analysis of the AAT transcripts revealed a 2.85- to 31.7-fold increase in mRNA levels between the different cotton lines. A substantial increase in the free-amino acid content of the major N-assimilation and transport amino acids (aspartate, glutamate, asparagine, and glutamine) was seen in the phloem sap of the transgenic cotton lines. The bioassay revealed that the two transgenic cotton lines with the highest free aspartate content in the phloem sap exhibited 97 and 94% mortality in the adult whitefly population and a 98 and 96% decline in subsequent nymph populations, respectively. There was also a significant change in the physiological behaviour of the transgenic cotton lines, with an increased net assimilation (A), gaseous exchange (Gs) and rate of transpiration (E). Improved morphological characteristics like plant height, total number of bolls and fiber yield were recorded in transgenic cotton lines. The AAT gene shows promise in mitigating whitefly infestations and enhancing the overall health and yield of cotton plants.

The Glucose-Succinate Pathway: A Crucial Anaerobic Metabolic Pathway in the Scallop Chlamys farreri Experiencing Heat Stress.

Recently, the increase in marine temperatures has become an important global marine environmental issue. The ability of energy supply in marine animals plays a crucial role in avoiding the stress of elevated temperatures. The investigation into anaerobic metabolism, an essential mechanism for regulating energy provision under heat stress, is limited in mollusks. In this study, key enzymes of four anaerobic metabolic pathways were identified in the genome of scallop Chlamys farreri, respectively including five opine dehydrogenases (CfOpDHs), two aspartate aminotransferases (CfASTs) divided into cytoplasmic (CfAST1) and mitochondrial subtype (CfAST2), and two phosphoenolpyruvate carboxykinases (CfPEPCKs) divided into a primitive type (CfPEPCK2) and a cytoplasmic subtype (CfPEPCK1). It was surprising that lactate dehydrogenase (LDH), a key enzyme in the anaerobic metabolism of the glucose-lactate pathway in vertebrates, was absent in the genome of scallops. Phylogenetic analysis verified that CfOpDHs clustered according to the phylogenetic relationships of the organisms rather than substrate specificity. Furthermore, CfOpDHs, CfASTs, and CfPEPCKs displayed distinct expression patterns throughout the developmental process and showed a prominent expression in muscle, foot, kidney, male gonad, and ganglia tissues. Notably, CfASTs displayed the highest level of expression among these genes during the developmental process and in adult tissues. Under heat stress, the expression of CfASTs exhibited a general downregulation trend in the six tissues examined. The expression of CfOpDHs also displayed a downregulation trend in most tissues, except CfOpDH1/3 in striated muscle showing significant up-regulation at some time points. Remarkably, CfPEPCK1 was significantly upregulated in all six tested tissues at almost all time points. Therefore, we speculated that the glucose-succinate pathway, catalyzed by CfPEPCK1, serves as the primary anaerobic metabolic pathway in mollusks experiencing heat stress, with CfOpDH3 catalyzing the glucose-opine pathway in striated muscle as supplementary. Additionally, the high and stable expression level of CfASTs is crucial for the maintenance of the essential functions of aspartate aminotransferase (AST). This study provides a comprehensive and systematic analysis of the key enzymes involved in anaerobic metabolism pathways, which holds significant importance in understanding the mechanism of energy supply in mollusks.

[Mechanism of Biejiajian Pills against non-alcoholic steatohepatitis based on lipidomics].

This study aims to explore the potential mechanism of Biejiajian Pills in the treatment of non-alcoholic steatohepatitis(NASH) based on lipidomics. A mouse model of NASH was induced by high-fat/high cholesterol diet, and the mice of the normal group were fed with a normal diet. The therapeutic efficacy of Biejiajian Pills against NASH was evaluated through biochemical indexes in both of serum and liver, as well as the hepatic histopathology. Lipid metabolites in the liver were detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)-based lipidomics. Then the partial least-squares discriminant analysis, t-test and receiver operating characteristic curve analysis were performed to screen the differential lipid metabolites and the main biomarkers. The proteins and genes involved in the lipid metabolism and inflammatory response were detected by Western blot and qPCR. The results demonstrated that Biejiajian Pills notably lowered the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), and alkaline phosphatase(ALP) in the serum and the levels of triglyceride(TG) and total cholesterol(TC) in the liver tissue. In addition, Biejiajian Pills alleviated the lipid accumulation, hepatocyte ballooning, and liver fibrosis. Lipidomics revealed that Biejiajian Pills regulated the content of 11 biomarkers including phosphatidyl choline(PC), phosphatidyl ethanolamine(PE), sphingomyelin(SM), and ceramide(Cer). The results of Western blot and qPCR demonstrated that Biejiajian Pills regulated the expression of sterol regulatory element-binding protein 1(SREBP1), peroxisome proliferator-activated receptor gamma(PPARγ) and phospho-AMP-activated protein kinase(p-AMPK), and the mRNA level of fatty acid translocase 36 gene(Cd36), Pparγ, cardiolipin synthase 1 gene(Crls1), and phospholipase Cß2 gene(Plcß2). Furthermore, Biejiajian Pills displayed inhibitory effects on phospho-p38 MAPK(p-p38 MAPK) and phospho-ERK1/2(p-ERK1/2) and the mRNA levels of interleukin-6 gene(Il-6), interleukin-1ß gene(Il-1ß) and tumor necrosis factor-α gene(Tnf-α). In conclusion, Biejiajian Pills could alleviate the lipid metabolism disorders and regulate the expression of SREBP1, PPARγ, and p-AMPK and the mRNA levels of pro-inflammatory cytokines.

[Mechanism of liver injury induced by alcohol extract of salt-processed Psoraleae Fructus based on chemical modification of protein].

Salt-processed Psoraleae Fructus is a commonly used tonic in clinical practice. However, its usage is restricted due to the inherent toxicity. The covalent modification of proteins by reactive metabolites(RMs) plays a role in the hepatotoxicity of salt-processed Psoraleae Fructus. This study delves into the protein covalent modification by RMs generated from psoralen/isopsoralen, the primary toxic components of salt-processed Psoraleae Fructus, by liquid chromatography-mass spectrometry(LC-MS), aiming to elucidate the mechanism underlying the hepatic injury induced by salt-processed Psoraleae Fructus. Biochemical methods were utilized to measure the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), catalase(CAT), malondialdehyde(MDA), superoxide dismutase(SOD), reduced glutathione(GSH), and glutathione S-transferase(GST) in mice. The pathological changes in the liver were observed by hematoxylin-eosin(HE) staining. Subsequently, ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS) was employed to identify the primary toxic components of psoralen/isopsoralen and the RMs in salt-processed Psoraleae Fructus. Covalent bonding adducts of the toxic components/RMs with GSH and free amino acids were identified to investigate the effects of the toxic components on modification sites and patterns of amino acids. The modifications of RMs were incorporated into the variable modifications of Proteome Discoverer, and the target proteins of psoralen/isopsoralen were detected by liquid chromatography-quadrupole exactive-mass spectrometry. Lastly, Label-free quantitative proteomics was adopted to screen differential proteins, which were further subjected to KEGG and GO enrichment analyses and confirmed by qPCR. The results indicated that compared with the control group, salt-processed Psoraleae Fructus significantly elevated the ALT, AST, and MDA levels and lowered the SOD, CAT, GSH, and GST levels in a dose-dependent manner, while causing obvious vacuolization and inflammatory cell infiltration in mouse hepatocytes. Furthermore, the livers of mice in the salt-processed Psoraleae Fructus group showed the presence of five RMs of psoralen/isopsoralen, two adducts with GSH, and one adduct with cysteine. In addition, 10 proteins modified by the RMs of psoralen/isopsoralen were identified. A total of 133 differential proteins were detected in the livers of mice in the salt-processed Psoraleae Fructus group, including 92 with up-regulated expression and 41 with down-regulated expression. These differential proteins mainly involved ribosomes, rRNAs, and glutathione, affecting the proteasome pathway. The qPCR results were consistent with the differential proteins. These findings suggest that the RMs of psoralen/isopsoralen can covalently bind to GSH and modify cysteine and lysine residues of liver proteins. This covalent modification of proteins by harmful substances can potentially result in liver damage. Therefore, it can be inferred that the oxidative stress damage induced by salt-processed Psoraleae Fructus may be associated with the abnormality of proteasome and its complex, biosynthesis of ribosomes and their nucleoprotein complex, rRNA binding, and glutathione binding.

Genotypic and phenotypic spectrum of cytosolic phosphoenolpyruvate carboxykinase deficiency.

OBJECTIVES: Pathogenic biallelic variants in PCK1 coding for the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) cause PEPCK-C deficiency, a rare disorder of gluconeogenesis presenting with hypoglycemia, lactic acidosis, and hepatopathy. To date, there has been no systematic analysis of its phenotypic, biochemical, and genetic spectrum. METHODS: All currently published individuals and a novel patient with genetically confirmed PEPCK-C deficiency were included. Clinical, biochemical, and genetic findings were analyzed. Protein and in-silico prediction score modeling was applied to analyze potential variant effects. RESULTS: Thirty-two individuals from 25 families were found, including one previously unreported patient. The typical biochemical pattern was hypoglycemia triggered by catabolic situations, elevated urinary concentrations of tricarboxylic acid cycle metabolites, mildly elevated alanine and aspartate aminotransferase and elevated lactate concentrations in serum. Plasma glutamine concentrations were elevated in some patients and may be a suitable marker for newborn screening. With adequate treatment, biochemical abnormalities usually normalized following a hypoglycemic episode. Symptom onset usually occurred in infancy with a broad range from neonatal age to adulthood. Regardless of the genotype, different phenotypes with a broad clinical spectrum were found. To date, eight genotypes with nine different PCK1 variants were identified, of which alleles with the recurrent variant c.925G > A; p.(Gly309Arg) are predominant and appear to be endemic in the Finnish population. Protein modeling suggests altered manganese- and substrate-binding as superordinate pathomechanisms. CONCLUSIONS: Environmental factors appear to be the main determinant for the phenotype in patients with biallelic variants in PCK1. Based on the biochemical pattern, PEPCK-C deficiency is a recognizable cause of childhood hypoglycemia. It is a treatable disease and early diagnosis is important to prevent metabolic derailment and morbidity. Newborn screening can identify at least a sub-cohort of affected individuals through elevated glutamine concentrations in dry blood.

Effects of NR1I2 and ABCB1 Genetic Polymorphisms on Everolimus Pharmacokinetics in Japanese Renal Transplant Patients.

The purpose of this study was to evaluate the effects of NR1I2 (7635G>A and 8055C>T) and ABCB1 (1236C>T, 2677G>T/A, and 3435C>T) genetic polymorphisms on everolimus pharmacokinetics in 98 Japanese renal transplant patients. On day 15 after everolimus administration, blood samples were collected just prior to and 1, 2, 3, 4, 6, 9, and 12 h after administration. The dose-adjusted area under the blood concentration−time curve (AUC0-12) of everolimus was significantly lower in patients with the NR1I2 8055C/C genotype than in those with other genotypes (p = 0.022) and was significantly higher in male patients than female patients (p = 0.045). Significant correlations between the dose-adjusted AUC0-12 of everolimus and age (p = 0.001), aspartate transaminase (p = 0.001), and alanine transaminase (p = 0.005) were found. In multivariate analysis, aging (p = 0.008) and higher alanine transaminase levels (p = 0.032) were independently predictive of a higher dose-adjusted everolimus AUC0-12. Aging and hepatic dysfunction in patients may need to be considered when evaluating dose reductions in everolimus. In renal transplant patients, management using everolimus blood concentrations after administration may be more important than analysis of NR1I2 8055C>T polymorphism before administration.

High protein diet-induced metabolic changes are transcriptionally regulated via KLF15-dependent and independent pathways.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

Inborn disorders of the malate aspartate shuttle.

Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.

Gensenoside Rg1 protects against lipopolysaccharide- and d-galactose-induced acute liver failure via suppressing HMGB1-mediated TLR4-NF-κB pathway.

AIM: Acute liver failure (ALF) is a life-threatening acute liver injury (ALI) with high mortality. Gensenoside Rg1 (G-Rg1) effects on Lipopolysaccharide- (LPS-) and d-galactose-(D-gal-) induced ALI, but its effects on ALF remained unclear. This paper aimed to validate its possible efficacy on ALF prevention. METHODS: For in vivo studies, histological examination was performed using hematoxylin-eosin (H&E) staining, and alanine aminotransferase (ALT), aspartate aminotransminase (AST), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) contents were measured. Levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) were quantified via enzyme-linked immunosorbent assay (ELISA). Human bronchial epithelial cell line BEAS-2B was used for ALF model in vitro and its viability was measured by MTT assay. Expressions of high mobility group box 1 (HMGB1) and toll-like receptor 4-Nuclear Factor-κB (TLR4-NF-κB) pathway-related proteins were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. RESULTS: G-Rg1 relieved LPS- and D-gal-induced hepatic injury, and reduced ALT, AST and MDA levels but upregulated SOD and GSH levels, with downregulation on TNF-α and IL-6 levels. Expressions of HMGB1, TLR4 and NF-κB pathway-related proteins were also down-regulated after G-Rg1 treatment both in vivo and in vitro, while BEAS-2B cell viability was increased. However, overexpressed HMGB1 reversed the effects of G-Rg1 treatment in vitro. CONCLUSION: G-Rg1 had a protective effect against LPS- and D-gal-induced ALF both in vitro and in vivo, which might be related to inhibited HMGB1-mediated TLR4-NF-κB Pathway. These discoveries suggested that G-Rg1 could be a potential agent for prevention against ALF.

The cytosolic form of aspartate aminotransferase is required for full activation of TOR complex 1 in fission yeast.

The evolutionarily conserved TOR complex 1 (TORC1) activates cell growth and proliferation in response to nutritional signals. In the fission yeast Schizosaccharomyces pombe, TORC1 is essential for vegetative growth, and its activity is regulated in response to nitrogen quantity and quality. Yet, how TORC1 senses nitrogen is poorly understood. Rapamycin, a specific TOR inhibitor, inhibits growth in S. pombe only under conditions in which the activity of TORC1 is compromised. In a genetic screen for rapamycin-sensitive mutations, we isolated caa1-1, a loss-of-function mutation of the cytosolic form of aspartate aminotransferase (Caa1). We demonstrate that loss of caa1+ partially mimics loss of TORC1 activity and that Caa1 is required for full TORC1 activity. Disruption of caa1+ resulted in aspartate auxotrophy, a finding that prompted us to assess the role of aspartate in TORC1 activation. We found that the amino acids glutamine, asparagine, arginine, aspartate, and serine activate TORC1 most efficiently following nitrogen starvation. The glutamine synthetase inhibitor l-methionine sulfoximine abolished the ability of asparagine, arginine, aspartate, or serine, but not that of glutamine, to induce TORC1 activity, consistent with a central role for glutamine in activating TORC1. Neither addition of aspartate nor addition of glutamine restored TORC1 activity in caa1-deleted cells or in cells carrying a Caa1 variant with a catalytic site substitution, suggesting that the catalytic activity of Caa1 is required for TORC1 activation. Taken together, our results reveal the contribution of the key metabolic enzyme Caa1 to TORC1 activity in S. pombe.

Identification of a four-gene metabolic signature predicting overall survival for hepatocellular carcinoma.

While hundreds of consistently altered metabolic genes had been identified in hepatocellular carcinoma (HCC), the prognostic role of them remains to be further elucidated. Messenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and GSE14520 data set from the Gene Expression Omnibus database. Univariate Cox regression analysis and lasso Cox regression model established a novel four-gene metabolic signature (including acetyl-CoA acetyltransferase 1, glutamic-oxaloacetic transaminase 2, phosphatidylserine synthase 2, and uridine-cytidine kinase 2) for HCC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was significantly correlated with other negative prognostic factors such as higher α-fetoprotein. The signature was found to be an independent prognostic factor for HCC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. Furthermore, gene set enrichment analyses revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust four-gene metabolic signature for HCC prognosis prediction. The signature might reflect the dysregulated metabolic microenvironment and provided potential biomarkers for metabolic therapy and treatment response prediction in HCC.

Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Macrophage-derived sulfur dioxide is a novel inflammation regulator.

Macrophage-mediated inflammation is a key pathophysiological component of cardiovascular diseases, but the underlying mechanisms by which the macrophage regulates inflammation have been unclear. In our study, we, for the first time, showed an endogenous sulfur dioxide (SO2) production in RAW267.4 macrophages by using HPLC and SO2-specific fluorescent probe assays. Moreover, the endogenous SO2 generating enzyme aspartate aminotransferase (AAT) was found to be expressed by the macrophages. Furthermore, we showed that AAT2 knockdown triggered spontaneous macrophage-mediated inflammation, as represented by the increased TNF-α and IL-6 levels and the enhanced macrophage chemotaxis; these effects could be reversed by the treatment with a SO2 donor. Mechanistically, AAT2 knockdown activated the NF-κB signaling pathway in macrophages, while SO2 successfully rescued NF-κB activation. In contrast, forced AAT2 expression reversed AngII-induced NF-κB activation and subsequent macrophage inflammation. Moreover, treatment with a SO2 donor also alleviated macrophage infiltration in AngII-treated mouse hearts. Collectively, our data suggest that macrophage-derived SO2 is an important regulator of macrophage activation and it acts as an endogenous "on-off switch" in the control of macrophage activation. This knowledge might enable a new therapeutic strategy for cardiovascular diseases.

Rev-erbα regulates hepatic ischemia-reperfusion injury in mice.

Hepatic ischemia-reperfusion (I/R) injury is a complex pathophysiological process that often times occurs in liver transplantation, hepatectomy, and ischemic shock. Aberrant activation of inflammatory responses has been implicated in hepatic I/R injury. In this study, we aimed to investigate the role of circadian clock gene Rev-erbα (a well-known regulator of inflammation) in hepatic I/R injury. We first showed that Rev-erbα ablation sensitized mice to hepatic I/R injury as evidenced by higher levels of plasma alanine aminotransferase and aspartate aminotransferase, an increased histological score, as well as enhanced hepatic myeloperoxidase activity in Rev-erbα-/- mice. More severe hepatic I/R injury in Rev-erbα-/- mice was accompanied by higher expression of pro-inflammatory cytokines, exacerbated activation of Nlrp3 inflammasome, and more extensive infiltration of inflammatory cells. Moreover, pharmacological activation of Rev-erbα by SR9009 significantly alleviated the hepatic damage and inflammatory responses. In addition, I/R operation started at ZT18 (corresponding to low Rev-erbα expression) caused more severe liver damage and inflammatory responses in wild-type mice as compared to operation started at ZT6 (corresponding to high Rev-erbα expression), supporting a protective effect of Rev-erbα on hepatic I/R injury. Collectively, Rev-erbα protects hepatic I/R injury probably via repression of inflammatory responses, and targeting Rev-erbα may be a promising approach for management of hepatic I/R injury.

The malate-aspartate shuttle (Borst cycle): How it started and developed into a major metabolic pathway.

This article presents a personal and critical review of the history of the malate-aspartate shuttle (MAS), starting in 1962 and ending in 2020. The MAS was initially proposed as a route for the oxidation of cytosolic NADH by the mitochondria in Ehrlich ascites cell tumor lacking other routes, and to explain the need for a mitochondrial aspartate aminotransferase (glutamate oxaloacetate transaminase 2 [GOT2]). The MAS was soon adopted in the field as a major pathway for NADH oxidation in mammalian tissues, such as liver and heart, even though the energetics of the MAS remained a mystery. Only in the 1970s, LaNoue and coworkers discovered that the efflux of aspartate from mitochondria, an essential step in the MAS, is dependent on the proton-motive force generated by the respiratory chain: for every aspartate effluxed, mitochondria take up one glutamate and one proton. This makes the MAS in practice uni-directional toward oxidation of cytosolic NADH, and explains why the free NADH/NAD ratio is much higher in the mitochondria than in the cytosol. The MAS is still a very active field of research. Most recently, the focus has been on the role of the MAS in tumors, on cells with defects in mitochondria and on inborn errors in the MAS. The year 2019 saw the discovery of two new inborn errors in the MAS, deficiencies in malate dehydrogenase 1 and in aspartate transaminase 2 (GOT2). This illustrates the vitality of ongoing MAS research.

Heterologous gene expression and characterization of recombinant aspartate aminotransferase from Geobacillus thermopakistaniensis.

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 µmol min-1 mg-1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.

Separation and Characterization of Phenolamines and Flavonoids from Rape Bee Pollen, and Comparison of Their Antioxidant Activities and Protective Effects Against Oxidative Stress.

Phenolamines and flavonoids are two important components in bee pollen. There are many reports on the bioactivity of flavonoids in bee pollen, but few on phenolamines. This study aims to separate and characterize the flavonoids and phenolamines from rape bee pollen, and compare their antioxidant activities and protective effects against oxidative stress. The rape bee pollen was separated to obtain 35% and 50% fractions, which were characterized by HPLC-ESI-QTOF-MS/MS. The results showed that the compounds in 35% fraction were quercetin and kaempferol glycosides, while the compounds in 50% fraction were phenolamines, including di-p-coumaroyl spermidine, p-coumaroyl caffeoyl hydroxyferuloyl spermine, di-p-coumaroyl hydroxyferuloyl spermine, and tri-p-coumaroyl spermidine. The antioxidant activities of phenolamines and flavonoids were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays. It was found that the antioxidant activity of phenolamines was significantly higher than that of flavonoids. Moreover, phenolamines showed better protective effects than flavonoids on HepG2 cells injured by AAPH. Furthermore, phenolamines could significantly reduce the reactive oxygen species (ROS), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and increase the superoxide dismutase (SOD) and glutathione (GSH) levels. This study lays a foundation for the further understanding of phenolamines in rape bee pollen.

Participation of Free-Radical Processes in Structural and Metabolic Disturbances in the Lung Tissues Caused by Exposure to Coal-Rock Dust and their Adaptogenic Correction.

Adaptive correction of structural and metabolic disturbances in the lungs caused by longterm exposure to coal-rock dust were studied in experiments on rats. It was shown that the complex antioxidant preparation containing dihydroquercetin compensated disturbances in the redox balance in the lung tissue, prevented the formation of dust granulomas, and reduced the severity of degenerative changes in the bronchopulmonary system.