Impact of GAD65 and/or GAD67 deficiency on perinatal development in rats.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.

Prealbumin and D-dimer as Prognostic Indicators for Rebleeding in Patients with Nonvariceal Upper Gastrointestinal Bleeding.

BACKGROUND: Determining the risk stratification of nonvariceal upper gastrointestinal bleeding (NVUGIB) plays a vital role in treating upper gastrointestinal bleeding (UGIB). Traditional scores like Glasgow-Blatchford score (GBS), Rockall score (RS), and AIMS65 score have been widely utilized in UGIB practice, however exhibiting limited practical use due to relative lack of user-friendly characters. Prealbumin as a nutritional indicator and d-dimer as a fibrinolytic activity monitor, are generally used to evaluate the overall nutritional and fibrinolytic condition in UGIB patients. AIMS: Here, we explored the predictive value of these two markers in NVUGIB for evaluating severity and prognosis including rebleeding and surgery intervention. METHODS: One hundred and eighty-five patients suffering NVUGIB were enrolled. Their GBS, RS, and AIMS65 score, routine laboratory test results including prealbumin and d-dimer were determined after admission. Multivariate regression analysis was performed to define the independent predictors of rebleeding. ROC curves were generated to compare the suitability of prealbumin, d-dimer, and scores for rebleeding prediction. RESULTS: The NVUGIB patients with rebleeding exhibited higher scores, white blood cell counts, d-dimer, CRP, proportion of surgery intervention, and longer hospital stay, but lower hematocrit, hemoglobin, calcium, prealbumin, and fibrinogen than those without rebleeding. The multivariate regression analysis demonstrated that prealbumin and d-dimer were independent predictors for rebleeding. Values of prealbumin and d-dimer were correlated with hospital stay, ulcer degrees, and surgery demand. The ROC curve analyses showed that prealbumin and d-dimer exhibited superior prediction value over the scoring systems. CONCLUSIONS: Prealbumin and d-dimer are promising predictors for severity and prognosis in NVUGIB practice.

CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.

Arenobufagin is a novel isoform-specific probe for sensing human sulfotransferase 2A1.

Human cytosolic sulfotransferase 2A1 (SULT2A1) is an important phase II metabolic enzyme. The detection of SULT2A1 is helpful for the functional characterization of SULT2A1 and diagnosis of its related diseases. However, due to the overlapping substrate specificity among members of the sulfotransferase family, it is difficult to develop a probe substrate for selective detection of SULT2A1. In the present study, through characterization of the sulfation of series of bufadienolides, arenobufagin (AB) was proved as a potential probe substrate for SULT2A1 with high sensitivity and specificity. Subsequently, the sulfation of AB was characterized by experimental and molecular docking studies. The sulfate-conjugated metabolite was identified as AB-3-sulfate. The sulfation of AB displayed a high selectivity for SULT2A1 which was confirmed by in vitro reaction phenotyping assays. The sulfation of AB by human liver cytosols and recombinant SULT2A1 both obeyed Michaelis-Menten kinetics, with similar kinetic parameters. Molecular docking was performed to understand the interaction between AB and SULT2A1, in which the lack of interaction with Met-137 and Tyr-238 of SULT2A1 made it possible to eliminate substrate inhibition of AB sulfation. Finally, the probe was successfully used to determine the activity of SULT2A1 and its isoenzymes in tissue preparations of human and laboratory animals.

Overexpression of YTHDF1 is associated with poor prognosis in patients with hepatocellular carcinoma.

In China, hepatocellular carcinoma (HCC) is the most commonly diagnosed cancer and the leading cause of cancer death in men, followed by lung and stomach cancer. There was an urgent need to identify novel prognostic biomarkers for HCC. We explored the expression pattern of m6A related proteins in HCC tissues by using TCGA in this study. We found that the m6A 'reader' YTHDF1 was significantly upregulated in HCC and was positive correlated with pathology stage. Kaplan-Meier analysis showed that Lower YTHDF1 expression level was associated with better survival of HCC patients. Furthermore, we performed GO and KEGG pathway analysis of YTHDF1 co-expressed genes and found YTHDF1 played an important role in regulating HCC cell cycle progression and metabolism. We believed that this study will provide a potential new therapeutic and prognostic target for HCC.

Expression of microRNA let-7a positively correlates with hepatitis B virus replication in hepatocellular carcinoma tissues.

Let-7a miRNA is downregulated in various cancers. However, in hepatocellular carcinoma (HCC) patients infected with hepatitis B virus (HBV), the relationship between let-7a and HBV replication has not been fully elucidated. Liver specimens were collected from 23 HCC patients with chronically active HBV. The serum levels of the HBV antigens hepatitis B surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the HBV antibodies, anti-HBs, anti-HBe and anti-hepatitis B core antigen (anti-HBc) were measured using the microparticle enzyme immunoassay. Let-7a levels and HBV DNA copy numbers were measured by quantitative real-time PCR (qRT-PCR) and analyzed statistically. A let-7a specific antisense oligonucleotide was introduced to the HBV-producing cell line HepG2.2.15 and a change in HBV DNA copy numbers was assessed by qRT-PCR. HCC patients with highly active HBV replication (>106 DNA copies/mL) showed higher levels of serum HBsAg and anti-HBc than patients with less active HBV replication (<103 DNA copies/mL). The level of let-7a was lower in malignant tissues than in adjacent normal tissues. However, patients with highly active HBV replication demonstrated a significantly higher level of let-7a in hepatocarcinoma tissue than patients with less active HBV replication ( P < 0.05). A higher level of let-7a was observed in the HBV-producing cell line HepG2.2.15 than in HepG2 cells ( P < 0.05), and let-7a down-regulation by antisense oligonucleotides led to a reduction in HBV DNA copy numbers ( P < 0.05), indicating a correlation between the let-7a level and HBV replication. Down-regulation of let-7a reduces HBV replication and could prevent the development of HCC, suggesting it could be an effective therapeutic treatment for HBV infection. Impact statement Although interferon and nucleic acid analogues effectively suppress HBV replication in HBV patients, there is no treatment which eradicates the virus. Moreover, the therapeutic effect can be reduced by virus mutations or drug resistance. Let-7a is a miRNA initially found in the nematode as a master regulator of developmental processes, but also exists in humans. It has been reported that the transcription of let-7a was much lower in HCC than in normal liver tissues and specific miRNA could directly promote virus replication. Therefore we hypothesized that transcription of let-7a promotes HBV replication, which might compromise the therapeutic effects of antivirus treatments. In our present study, we demonstrated a correlation between let-7a transcription and HBV replication in surgical specimens obtained from patients with HCC, as well as in HCC cell lines. Our finding might be the base for a new approach to improve HBV infection treatments in the future.

Downregulated miRNA-1269a variant (rs73239138) decreases the susceptibility to gastric cancer via targeting ZNF70.

Although emerging evidence has indicated that single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) are associated with susceptibility to gastric cancer, a limited number of studies have revealed the underlying molecular mechanisms. In the present study, the results suggested that miR-1269a rs73239138 has a role in decreasing the risk of gastric cancer. The level of miR-1269a variant expression was significantly downregulated compared with the wild-type miR-1269a in the gastric cells (Fig. 1). Furthermore, overexpression of miR-1269a inhibited apoptosis of gastric cancer cells. Expression of the miR-1269a variant inhibited the function of miR-1269a by increasing the apoptotic rate and the expression of Bik, Bim and Bak was upregulated consistently. In addition, zinc-finger protein 70 (ZNF70) was identified to be a target gene of miR-1269a, which was downregulated by miR-1269a and upregulated by miR-1269a variant. ZNF70 was indicated to exert a role as a tumor suppressor in gastric cancer. To the best our knowledge, the present study for the first time highlights a critical role of miR-1269a variant rs73239138 in decreasing the susceptibility to gastric cancer by downregulating its expression and targeting ZNF70, which promotes apoptosis of gastric cancer cells. This SNP is indicated to serve as a potential biomarker and therapeutic target for gastric cancer.

The natural anthraquinones from Rheum palmatum induced the metabolic disorder of melatonin by inhibiting human CYP and SULT enzymes.

Melatonin (Mel) as an endogenous hormone, has been widely used in clinic for multiple therapeutic purposes. Further, the natural anthraquinones were widespread in various plants including herbs, foods, and some flavoring agents. The present work aims to evaluate the metabolic disorder of Mel caused by various common herbs and further identify their underlying mechanism. More importantly, the relationships between inhibitory activity and their structures were also investigated. Our results demonstrate that some herbs containing anthraquinone derivatives exhibited strong inhibition on Mel metabolism. Additionally, five anthraquinones from R. palmatum could inhibit phase I and II metabolism of Mel with a mixed inhibition kinetic model based on the mechanism of inhibiting human CYP1A1, 1A2, and SULT1A1. At last, the influence of R. palmatum and its five major components on the Mel metabolism were verified in human primary hepatocytes. In conclusion, our studies elucidated that herbs or foods containing abundant anthraquinones such as R. palmatum will cause a metabolic disorder of Mel, and should be avoided to combined application with Mel in clinic.

Association between a variant in microRNA-646 and the susceptibility to hepatocellular carcinoma in a large-scale population.

BACKGROUND: Single-nucleotide polymorphisms in microRNAs play important roles in oncogenesis and cancer development. Objective. We aim to explore whether miR-646 rs6513497 is associated with the risk of hepatocellular carcinoma. METHODS: Total 997 HCC patients and 993 cancer-free controls were enrolled in this study. Genotyping was performed using MassARRAY method. RESULTS: Compared with the T allele of rs6513497, the G allele was associated with a significantly decreased risk of HCC (OR = 0.788, 95% CI = 0.631-0.985, P = 0.037); moreover, a more protective effect of the G allele was shown in males (OR = 0.695, 95% CI = 0.539-0.897, P = 0.005 in HCC and OR = 0.739, 95% CI = 0.562-0.972, P = 0.030 in HBV-related HCC), basically in a dominant manner (HCC: OR = 0.681, 95% CI = 0.162-0.896, P = 0.006; HBV-related HCC: OR = 0.715, 95% CI = 0.532-0.962, P = 0.027). CONCLUSIONS: Our findings support the view that the miR-646 SNP rs6513497 may contribute to the susceptibility of HCC.