Porcine epidemic diarrhea virus uses cell-surface heparan sulfate as an attachment factor.

It is well known that many viruses use heparan sulfate as the initial attachment factor. In the present study, we determined whether porcine epidemic diarrhea virus (PEDV), an emerging veterinary virus, infects Vero cells by attaching to heparan sulfate. Western blot analysis, real-time PCR, and plaque formation assay revealed that PEDV infection was inhibited when the virus was pretreated with heparin (an analogue of heparan sulfate). There was no inhibitory effect when the cells were pre-incubated with heparin. We next demonstrated that enzymatic removal of the highly sulfated domain of heparan sulfate by heparinase I treatment inhibited PEDV infection. We also confirmed that sodium chlorate, which interferes with heparan sulfate biosynthesis, also inhibited PEDV infection. Furthermore, we examined the effect of two heparin derivatives with different types of sulfation on PEDV infection. The data suggested de-N-sulfated heparin, but not N-acetyl-de-O-sulfated heparin, inhibits PEDV infection. In summary, our studies revealed that heparan sulfate acts as the attachment factor of PEDV in Vero cells.

Abnormality of anxious behaviors and functional connectivity between the amygdala and the frontal lobe in maternally deprived monkeys.

OBJECTIVE: Anxious behaviors often occur in individuals who have experienced early adversity. Anxious behaviors can bring many hazards, such as social withdrawal, eating disorders, negative self-efficacy, self-injurious thoughts and behaviors, anxiety disorders, and even depression. Abnormal behavior are is closely related to changes in corresponding circuit functions in the brain. This study investigated the relationship between brain circuits and anxious behaviors in maternal-deprived rhesus monkey animal model, which mimic early adversity in human. METHODS: Twenty-five rhesus monkeys (Macaca mulatta) were grouped by two different rearing conditions: 11 normal control and mother-reared (MR) monkeys and 14 maternally deprived and peer-reared (MD) monkeys. After obtaining images of the brain areas with significant differences in maternal separation and normal control macaque function, the relationship between functional junction intensity and stereotypical behaviors was determined by correlation analysis. RESULTS: The correlation analysis revealed that stereotypical behaviors were negatively correlated with the coupling between the left lateral amygdala subregion and the left inferior frontal gyrus in both MD and MR macaques. CONCLUSION: This study suggests that early adversity-induced anxious behaviors are associated with changes in the strength of the amygdala-prefrontal connection. The normalization of the regions involved in the functional connection might reverse the behavioral abnormality. It provides a solid foundation for effective intervention in human early adversity. SIGNIFICANCE STATEMENT: This study suggests that early adversity-induced anxious behaviors are associated with changes in the strength of the amygdala-prefrontal connection. The higher the amygdala-prefrontal connection strength, the less stereotyped behaviors exhibited by monkeys experiencing early adversity. Thus, in the future, changing the strength of the amygdala-prefrontal connection may reverse the behavioral abnormalities of individuals who experience early adversity. This study provides a solid foundation for effective intervention in humans' early adversity.

Developmental patterns and characteristics of epicardial cell markers Tbx18 and Wt1 in murine embryonic heart.

BACKGROUND: Although recent studies have highlighted the role of epicardial cells during cardiac development and regeneration, their cardiomyogenic potential is still controversial due to the question of lineage tracing of epicardial cells. The present study therefore aimed to examine the the expression of Tbx18 and Wt1 in embryonic heart and to identify whether Tbx18 and Wt1 themselves expressed in the cardiomyocyte. METHODS: Mouse embryonic hearts were collected at different stages for immunofluorescence costaining with either Tbx18 and the cardiac transcription factor Nkx2.5 or Wilms tumor 1 (Wt1) and Nkx2.5. RESULTS: Tbx18 and Wt1, but not Nkx2.5, were expressed in the proepicardium and epicardium. Tbx18 was expressed in cells within the heart from E10.5 to at least E14.5; these Tbx18-expressing cells were Nkx2.5 positive, except for a few cells that were Nkx2.5 negative at E14.5. Wt1 was expressed in cells within the heart from E12.5 to at least E14.5, but these Wt1-expressing cells were Nkx2.5 negative. CONCLUSION: The data obtained in this study demonstrate that Tbx18 is expressed in the cardiomyocytes from E10.5 to at least E14.5, and Wt1 is expressed within the heart from E12.5 to at least E14.5, but not in the cardiomyocyte. These findings may provide new insights on the role of the epicardial cells in cardiac regeneration.

Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys.

Although CRISPR/Cas9-mediated gene editing is widely applied to mimic human disorders, whether acute manipulation of disease-causing genes in the brain leads to behavioral abnormalities in non-human primates remains to be determined. Here we induced genetic mutations in MECP2, a critical gene linked to Rett syndrome (RTT) and autism spectrum disorders (ASD), in the hippocampus (DG and CA1-4) of adolescent rhesus monkeys (Macaca mulatta) in vivo via adeno-associated virus (AAV)-delivered Staphylococcus aureus Cas9 with small guide RNAs (sgRNAs) targeting MECP2. In comparison to monkeys injected with AAV-SaCas9 alone (n = 4), numerous autistic-like behavioral abnormalities were identified in the AAV-SaCas9-sgMECP2-injected monkeys (n = 7), including social interaction deficits, abnormal sleep patterns, insensitivity to aversive stimuli, abnormal hand motions, and defective social reward behaviors. Furthermore, some aspects of ASD and RTT, such as stereotypic behaviors, did not appear in the MECP2 gene-edited monkeys, suggesting that different brain areas likely contribute to distinct ASD symptoms. This study showed that acute manipulation of disease-causing genes via in vivo gene editing directly led to behavioral changes in adolescent primates, paving the way for the rapid generation of genetically engineered non-human primate models for neurobiological studies and therapeutic development.

Direct sunlight exposure reduces hair cortisol levels in rhesus monkeys (Macaca mulatta).

Major depressive disorder (MDD), commonly known as depression, is a mental disease characterized by a core symptom of low mood. It lasts at least two weeks (Badamasi et al., 2019; Wang et al., 2019) and is frequently accompanied by low self-esteem, loss of interest in routinely enjoyable activities, low energy, and unexplained pain (Huey et al., 2018; Park et al., 2012; Post & Warden, 2018; Rice et al., 2019; Xiao et al., 2018). Approximately 2%-8% of adults with MDD commit suicide (Richards & O'Hara, 2014; Strakowski & Nelson, 2015), and around half of suicidal individuals suffer depression or other mood disorders (Bachmann, 2018).

An overview of veterinary medical education in China: current status, deficiencies, and strategy for improvement.

Especially in developing countries, the profession of veterinary medicine is closely tied with agriculture and government economic development, the national structure of education, and national public health. Currently, the Chinese veterinary medical educational system and accreditation standards are distinctly different from those of some more developed countries, such as the United States, Japan, or the countries of the European Union. Chinese veterinary education is still closely based on traditional Chinese education approaches and standards, which has led to some deficiencies in the Chinese system. With the development of a stronger economy in China and the growing trend toward globalization, and particularly since China joined the World Trade Organization (WTO), some important questions about China's system of veterinary education are being raised: How can veterinary science develop more rapidly in China? How can it meet the needs of the growing Chinese society? How can China bring its veterinary medical practice more in line with that of other, more advanced countries? This article describes some of the realities of veterinary medical education in China, discusses several existing problems, and puts forward some ideas for possible reforms. It is hoped that by this means those outside China may gain insight into our veterinary education program and that this, in turn, will lead to helpful input from international educators and other professionals to help improve our programs.

[Response and Control Factors of Groundwater to Extreme Weather, Jiguan Cave, Henan Province, China].

Geochemical dynamics of cave water were monitored to unveil its variation and controlling factors from October 2009 to December 2013 in Jiguan Cave,west of Henan province,southeastern coast of the loess plateau. The results showed that: (1) the hydrochemical types of the cave water are HCO(3-)-Ca(2+)-Mg2+ and HCO(3-)-Mg(2+)-Ca2+. HCO(3-) are over 80% of the anions, Ca2+ and Mg2+ are the dominate cations, and ground river keeping in erosion and pool water drips in deposition all the year. (2) Dripping water and pool water in Ji guan cave can respond perfectly to the change of external climate environment, which geochemistry indexes possess the extraordinary seasonal effects. (3) The concentration changes of the Ca2+, Mg2+ , SO4(2-) responded sensitively to annual precipitation change. Ca2+, Mg2+, SO4(2-) rise in waterlogging year and fall in drought year. Because HCO(3-) controlled by CO2 concentration. HCO(3-) concentration showed a unconspicuous response to the change of external climate environment. (4) The concentration changes of Ca2+, Mg2+, SO4(2-) have no obvious seasonal variation and showed a unconspicuous response to the change of external climate environment.

Screening of susceptibility genes and multi-gene risk analysis in gastric cancer.

The aim of the study was to explore the relations between the genetic polymorphism and the susceptibility to the gastric cancer in Chinese Han population, and to analyze the multi-genes risk in the development of gastric carcinoma. A case-control study of 1:1 matching was performed on 564 individuals with primary gastric carcinoma in Nanjing, China. The genotypes of CYP2E1, GSTMl, GSTTl, NAT2, ALDH2, MTHFR, XRCCl, IL-1ß, VDR, and TNF were detected by molecular biological techniques (PCR-RFLP and AS-PCR). Sole gene and gene-gene interactions were analyzed using Logistic regression model. The effect of multi-genes on gastric carcinoma was analyzed using multi-gene risk analysis model, which focused on the effect of multi-gene interaction on the development of gastric carcinoma. The genotypes involved in the susceptibility of gastric carcinoma were CYP2E1(c1/c1), NAT2M1(T/T), NAT2M2(A/A), XRCC1194(T/T), NAT2 phenotype (slow acetylator), MTHFR1298(A/C), and VDR TaqI(T/T), respectively. Multi-gene risk analysis model was introduced to analyze the effect of these genes on the gastric carcinoma. The results showed that there was a strong relation between odds ratio (OR) value of polygene combination and the gene frequency. With the increase of susceptibility gene frequency, the risk distribution curve of gastric carcinoma would shift to a more dangerous phase and exhibit a quantitative relation. Our results demonstrated that the OR of each gene can be utilized as an index to assess the effect of multiple susceptible genes on the occurrence of gastric carcinoma.

Triggering unfolded protein response by 2-Deoxy-D-glucose inhibits porcine epidemic diarrhea virus propagation.

The unfolded protein response (UPR) is cyto-protective machinery elicited towards an influx of large amount of protein synthesis in the endoplasmic reticulum (ER). Extensive studies suggest that the UPR can also be activated during virus infection. In the present studies, we first evaluated if porcine epidemic diarrhea virus (PEDV) infection activated the UPR pathways. Electron microscopy analysis demonstrated the morphology changes of ER post-PEDV infection. Western blot and real-time PCR identified the differences of UPR genes in response to PEDV infection. The results suggested that PEDV infection induced UPR in Vero cells. Meanwhile, we silenced the expression of PKR-like ER kinase (PERK) by shRNA, we found that the knockdown of PERK increased virus loads in the cells, which was consistent with the result on 4-phenylbutyrate (4-PBA) treatment. We next determined whether 2-Deoxy-d-glucose (2-DG), an ER stress inducer, possessed antiviral activity against PEDV infection. Plaque formation assay, RT-PCR and Western blot analysis suggested that 2-DG might inhibit virus infection by affecting viral protein translation during the early stage of virus infection. Interestingly, we also found that 2-DG treatment could affect virus assembly, which is similar to previous studies on influenza virus. All these results support the therapeutic potential of using 2-DG or glucose/mannose analogs to induce the UPR to block virus replication.

[Cell autophagy and virus infection].

Autophagy is a catabolic process including self-degradation of intracellular components via the lysosomal machinery. The biological behavior can be regarded as defense mechanism, maintaining the cell growth, metabolism and homeostasis etc. To date, plenty of autophagy related genes have been identified. In addition, it has been recognized that autophagy plays important roles in the context of virus infection: it can transport viruses from cytoplasm to lysosome to degrade viruses; it can transfer viral nucleic acid to intracellular sensors to activate innate immunity; it can also present viral antigens to MHC class II molecules to activate adaptive immune responses. Autophagy may serve as a double-edged sword to intracellular pathogens. On one side, autophagy may degrade and clear invading microorganisms by xenophagy; on the other side, some microorganisms may develop mechanisms to escape from autophagy for their survival. In this paper, the notion of autophay and the function of autophagy related genes are reviewed. Furthermore, the association of autophagy with a variety of viruses is discussed.

Molecular cloning and phylogenetic analysis of ORF7 region of chinese isolate TH-98 from transmissible gastroenteritis virus.

Genomic RNA was extracted from a Chinese isolate of porcine transmissible gastroenteritis virus (TGEV) designated TH-98. Employing RT-PCR technique to amplify ORF7 sequence of TGEV, which located at the 3' end of TGEV genome and is poorly understood functionally so far. A recombinant named pPROEX HTc-hp was constructed via inserting ORF7 gene into prokaryotic expression vector pPROEX HTc. The recombinant was sequenced and compared the DNA and its deduced amino acid (aa) sequences with that of some reference strains after restriction endonuclease and PCR analysis. The ORF7 gene named hp gene (Genbank accession number: AY337931) consists of 237 bp in length encoding a hydrophobic protein (HP) of 78 aa with a molecular weight of 9.1 kDa. The sequences of hp gene and Hp protein share 89%-97% and 87%-96% homologous identities compared with 11 TGEV reference strains derived from other regions or countries respectively, which revealed that there are significant variation within-strains, even though the ORF7 region is relatively conservative. In addition, a phylogenetic tree based on these ORF7 DNA sequences was generated, and the tree topology suggests that possible recombination events happened in the evolutionary history of TGEV.