G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), cause of the life-threatening atypical pneumonia, infects many organs, such as lung, liver and immune organ, and induces parenchyma cells apoptosis and necrosis. The genome of SARS-CoV, not closely related to any of the previously characterized coronavirus, encodes replicase and four major structural proteins and a number of non-structural proteins. Published studies suggest that some non-structural proteins may play important roles in the replication, virulence and pathogenesis of viruses. Among the potential SARS-CoV non-structural proteins, 3b protein (ORF4) is predicted encoding 154 amino acids, lacking significant similarities to any known proteins. Till now, there is no report about the function of 3b protein. In this study, 3b gene was linked with the EGFP tag at the C- terminus. Through cell cycle analysis, it was found that over-expression of 3b-EGFP protein in Vero, 293 and COS-7 cells could induce cell cycle arrest at G0/G1 phase, and that especially in COS-7 cells, expression of 3b-EGFP was able to induce the increase of sub-G1 phase from 24 h after transfection, which was most obvious at 48 h. The apoptosis induction of 3b fusion protein in COS-7 cells was further confirmed by double cell labeling with 7-AAD and Annexin V, the function of 3b protein inducing cell G0/G1 arrest and apoptosis may provide a new insight for further study on the mechanism of SARS pathogenesis

SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway

AbstractThe genome of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains four structural genes that are homologous to genes found in other coronaviruses, and also contains six subgroup-specific open reading frames (ORFs). Expression of one of these subgroup-specific genes, ORF7a, resulted in apoptosis via a caspase-dependent pathway. Here, we observed that transient expression of ORF7a protein fused with myc or GFP tags at its N or C terminus inhibited cell growth and prevented BrdU incorporation in different cultural cells, suggesting that ORF7a expression may regulate cell cycle progression. Analysis by flow cytometry demonstrated that ORF7a expression was associated with blockage of cell cycle progression at G0/G1 phase in HEK 293 cells after 24 to 60 h post-transfection. Similar results were observed in COS-7 and Vero cells. Mutation analysis of ORF7a revealed that the domain spanning aa 44–82 of 7a protein was essential for its cytoplasmic localization and for induction of the cell cycle arrest. After analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that ORF7a expression was correlated with a significant reduction of cyclin D3 level of mRNA transcription and expression, and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in HEK 293 cells. These results suggest that the insufficient expression of cyclin D3 may cause a decreased activity of cyclin D/cdk4/6, resulting in the inhibition of Rb phosphorylation. Accumulation of hypo- or non-phosphorylated pRb thus prevents cell cycle progression at G0/G1 phase

A developmental analysis of juxtavascular microglia dynamics and interactions with the vasculature [preprint]

Microglia, the resident macrophages of the central nervous system (CNS), are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared to older ages and the timing of these associations are dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the prenatal human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages contact vascular areas void of astrocyte endfeet and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted upon the arrival of astrocyte endfeet when juxtavascular microglia then establish a long-term, stable contact with the vasculature

A Developmental Analysis of Juxtavascular Microglia Dynamics and Interactions with the Vasculature

Microglia, a resident CNS macrophage, are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared with older ages and that the timing of these associations is dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages associate with vascular areas void of astrocyte endfeet, and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted on the arrival of astrocyte endfeet such that juxtavascular microglia become highly stationary and stable in the mature cortex. SIGNIFICANCE STATEMENT We report the first extensive analysis of juxtavascular microglia in the healthy, developing, and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte cross talk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential mechanism by which vascular interactions facilitate microglial colonization of the brain to later regulate neural circuit development

Prenatal diagnosis of fetuses with ultrasound anomalies by whole-exome sequencing in Luoyang city, China

Background: There is a great obstacle in prenatal diagnosis of fetal anomalies due to their considerable genetic and clinical heterogeneity. Whole-exome sequencing (WES) has been confirmed as a successful option for genetic diagnosis in pediatrics, but its clinical utility for prenatal diagnosis remains to be limited.Methods: A total of 60 fetuses with abnormal ultrasound findings underwent karyotyping or chromosomal microarray analysis (CMA), and those with negative results were further subjected to WES. The identified variants were classified as pathogenic or likely pathogenic (P/LP) and the variant of uncertain significance (VUS). Pregnancy outcomes were obtained through a telephone follow-up.Results: Twelve (20%, 12/60) fetuses were diagnosed to have chromosomal abnormalities using karyotyping or CMA. Of the remaining 48 cases that underwent WES, P/LP variants were identified in 14 cases (29.2%), giving an additional diagnostic yield of 23.3% (14/60). The most frequently affected organ referred for prenatal WES was the head or neck system (40%), followed by the skeletal system (39.1%). In terms of pathogenic genes, FGFR3 was the most common diagnostic gene in this cohort. For the first time, we discovered five P/LP variants involved in SEC24D, FIG4, CTNNA3, EPG5, and PKD2. In addition, we identified three VUSes that had been reported previously. Outcomes of pregnancy were available for 54 cases, of which 24 cases were terminated.Conclusion: The results confirmed that WES is a powerful tool in prenatal diagnosis, especially for fetuses with ultrasonographic anomalies that cannot be diagnosed using conventional prenatal methods. Additionally, newly identified variants will expand the phenotypic spectrum of monogenic disorders and greatly enrich the prenatal diagnostic database

Glycyrrhizin Protects Mice Against Experimental Autoimmune Encephalomyelitis by Inhibiting High-Mobility Group Box 1 (HMGB1) Expression and Neuronal HMGB1 Release

The inflammatory mediator high-mobility group box 1 (HMGB1) plays a critical role in the pathogenesis of human multiple sclerosis (MS) and mouse experimental autoimmune encephalomyelitis (EAE). Glycyrrhizin (GL), a glycoconjugated triterpene extracted from licorice root, has the ability to inhibit the functions of HMGB1; however, GL’s function against EAE has not been thoroughly characterized to date. To determine the benefit of GL as a modulator of neuroinflammation, we used an in vivo study to examine GL’s effect on EAE along with primary cultured cortical neurons to study the GL effect on HMGB1 release. Treatment of EAE mice with GL from onset to the peak stage of disease resulted in marked attenuation of EAE severity, reduced inflammatory cell infiltration and demyelination, decreased tumor necrosis factor-alpha (TNF-α), IFN-γ, IL-17A, IL-6, and transforming growth factor-beta 1, and increased IL-4 both in serum and spinal cord homogenate. Moreover, HMGB1 levels in different body fluids were reduced, accompanied by a decrease in neuronal damage, activated astrocytes and microglia, as well as HMGB1-positive astrocytes and microglia. GL significantly reversed HMGB1 release into the medium induced by TNF-α stimulation in primary cultured cortical neurons. Taken together, the results indicate that GL has a strong neuroprotective effect on EAE mice by reducing HMGB1 expression and release and thus can be used to treat central nervous system inflammatory diseases, such as MS

Metabolic features of orbital adipose tissue in patients with thyroid eye disease

BackgroundThyroid eye disease (TED) is the most frequent orbital disease in adults and is characterized by the accumulation of orbital adipose tissue (OAT). It can lead to eyelid retraction or even vision loss. Orbital decompression surgery serves as the primary treatment for inactive TED by removing the excess OAT. However, there is a lack of alternative treatments to surgery due to the unclear understanding of the pathogenesis, particularly the metabolic features. Accordingly, our study was implemented to explore the content and features of metabolites of OATs from TED patients.MethodThe OATs used in the current study were obtained from the orbital decompression surgery of seven patients with inactive TED. We also collected control OATs from eye surgical samples of five individuals with no history of autoimmune thyroid diseases, TED, or under non-inflammatory conditions. The liquid chromatography mass spectrometer was used for the measurements of the targeted metabolites. Afterwards, we performed differential metabolite assay analysis and related pathway enrichment analysis.ResultsIn our study, a total of 149 metabolite profiles were detected in all participants. There were significant differences in several metabolite profiles between the TED group and the control group, mainly including uric acid, oxidized glutathione, taurine, dGMP, oxidized glutathione 2, uracil, hexose-phosphate, 1-methylnicotinamide, D-sedoheptulose 1,7-bisphosphate, and uridine 5′-monophosphate (all p-value < 0.05). The TED-related pathways identified included purine metabolism, beta-alanine metabolism, glutathione metabolism (p-values < 0.05). Our study found overlaps and differences including uric acid and uracil, which are in accordance with metabolites found in blood of patients with TED from previous study and several newly discovered metabolite by our study such as hexose-phosphate, 1-methylnicotinamide, D-sedoheptulose 1,7-bisphosphate, compared to those tested from blood, OAT, or urine samples reported in previous studies.ConclusionThe findings of our study shed light on the metabolic features of OAT in individuals with TED. These results may help identify new treatment targets for TED, providing potential avenues for developing alternative treatments beyond ophthalmic surgery

CYP2C19 genotype and platelet aggregation test-guided dual antiplatelet therapy after off-pump coronary artery bypass grafting: A retrospective cohort study

BackgroundDual antiplatelet therapy (DAPT) is recommended in patients undergoing off-pump coronary artery bypass graft surgery (OPCAB). Clopidogrel is less effective among patients with loss-of-function (LoF) of CYP2C19 alleles, while ticagrelor has direct effects on P2Y12 receptor. Whether a CYP2C19 genotype plus platelet aggregation test (PAgT)-guided DAPT after CABG could improve clinical outcomes remain uncertain.Materials and methodsFrom August 2019 to December 2020, 1,134 consecutive patients who underwent OPCAB received DAPT for 1 year after surgery in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. According to the actual treatment they received in real-world, 382 (33.7%) of them received a traditional DAPT: aspirin 100 mg qd + clopidogrel 75 mg qd, no matter the CYP2C19 genotype and response in platelet aggregation test (PAgT). The other 752 (66.3%) patients received an individual DAPT based on CYP2C19 genotype and PAgT: aspirin 100 mg qd + clopidogrel 75 mg qd if CYP2C19 was extensive metabolizer, or moderate metabolizer but normal response in PAgT; aspirin 100 mg qd + ticagrelor 90 mg bid if CYP2C19 was poor metabolizer, or moderate metabolizer but no or low response in PAgT. One-year follow-up was achieved for all patients. The primary outcome was major adverse cardiovascular events (MACE), a composite of cardiovascular death, myocardial infarction, and stroke. The safety outcome was thrombolysis in myocardial infarction (TIMI) criteria major bleeding.ResultsCompared with the traditional DAPT group, the risk of MACE in the individual DAPT group was significantly lower (5.5 vs. 9.2%, HR 0.583; 95% CI, 0.371–0.915; P = 0.019), mainly due to the decreased risk of MI (1.7 vs. 4.2%, HR 0.407; 95% CI, 0.196–0.846; P = 0.016). The risk of TIMI major bleeding events was similar between the two groups (5.3 vs. 6.0%, RR 0.883; 95% CI, 0.537–1.453; P = 0.626).ConclusionFor patients who underwent OPCAB, individual DAPT (CYP2C19 genotype plus PAgT-guided strategy) was associated with a lower risk of MACE and a similar risk of major bleeding

The landscape of RNA polymerase II-associated chromatin interactions in prostate cancer

Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA polymerase II-associated (RNA Pol II-associated) chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins - CTCF and cohesins - and are regulated by the cooperative action of master transcription factors, such as the androgen receptor (AR) and FOXA1. By combining analyses from metastatic castration-resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional upregulation of the AR gene by increasing the total number of chromatin interaction modules comprising the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes - VPS53, FAM57A, and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa