Background5-methylcytosine (mC) can be oxidized by the tet methylcytosine dioxygenase (Tet) family of enzymes to 5-hydroxymethylcytosine (hmC), which is an intermediate of mC demethylation and may also be a stable epigenetic modification that influences chromatin structure. hmC is particularly abundant in mammalian brains but its function is currently unknown. A high-resolution hydroxymethylome map is required to fully understand the function of hmC in the human brain.ResultsWe present genome-wide and single-base resolution maps of hmC and mC in the human brain by combined application of Tet-assisted bisulfite sequencing and bisulfite sequencing. We demonstrate that hmCs increase markedly from the fetal to the adult stage, and in the adult brain, 13% of all CpGs are highly hydroxymethylated with strong enrichment at genic regions and distal regulatory elements. Notably, hmC peaks are identified at the 5′splicing sites at the exon-intron boundary, suggesting a mechanistic link between hmC and splicing. We report a surprising transcription-correlated hmC bias toward the sense strand and an mC bias toward the antisense strand of gene bodies. Furthermore, hmC is negatively correlated with H3K27me3-marked and H3K9me3-marked repressive genomic regions, and is more enriched at poised enhancers than active enhancers.ConclusionsWe provide single-base resolution hmC and mC maps in the human brain and our data imply novel roles of hmC in regulating splicing and gene expression. Hydroxymethylation is the main modification status for a large portion of CpGs situated at poised enhancers and actively transcribed regions, suggesting its roles in epigenetic tuning at these regions.

Central to the role of p53 in cell regulation are its sequence-specific interactions with genes that control the cell cycle and apoptosis. p53 response elements contain two or more copies of a somewhat promiscuous consensus sequence: 5'-XXXC(A,T)(T,A)GYY-3' (where X is a purine and Y is a pyrimidine) (ref. 3). The sequence-specific DNA-binding region of p53 resides in its central conserved region. Although this region itself is not known to be phosphorylated, the amino and carboxy termini of human p53 contain sites for phosphorylation by several protein kinases. We have examined the role of cyclin-dependent kinase (Cdk) shown previously to phosphorylate human p53 at serine 315 (ref. 5). We report here that p53 is efficiently and selectively phosphorylated by S and G2/M Cdks. Such phosphorylation markedly stimulates sequence-specific DNA binding by p53 and also causes a distinctive conformational change in p53 as revealed by partial protease analysis. Strikingly, Cdk phosphorylation also confers binding-site preference on p53. These data suggest a potential regulatory mechanism of p53 activity.

Surveys were carried out to better understand the tick vector ecology and genetic diversity of Huaiyangshan virus (HYSV) in both regions of endemicity and regions of nonendemicity. Haemaphysalis longicornis ticks were dominant in regions of endemicity, while Rhipicephalus microplus is more abundant in regions of nonendemicity. HYSV RNA was found in human and both tick species, with greater prevalence in H. longicornis and lesser prevalence in R. microplus. Phylogenetic analyses indicate that HYSV is a novel species of the genus Phlebovirus. Recently, a hemorrhagic fever-like disease caused by a novel bunyavirus occurred in China (14, 16). Yu et al. reported the disease as severe fever with thrombocytopenia syndrome (SFTS) (14). As thrombocytopenia is not specific for this disease and is present in nearly all hemorrhagic fevers caused by viruses (11) or Rickettsia (15), we previously proposed naming the syndrome Huaiyangshan hemorrhagic fever (HYSHF) and the virus Huaiyangshan virus (HYSV) (16). Haemaphysalis longicornis ticks might be the vector of HYSV (14, 16). However, less is known about the arthropod vector ecology, the genetic diversity, and the phylogeny of HYSV. Thus, we performed an investigation in regions of endemicity and nonendemicity in Henan and Hubei provinces ( Fig. 1).A total of 17,731 adult ticks were collected (Table 1). After morphological examination and sequence analysis of mitochondrial 12S ribosomal DNA (rDNA) as described previously (2, 16), only H. longicornis and Rhipicephalus microplus were found. In the regions of endemicity, 4,501 ticks (3,498 H. longicornis and 1,003 R. microplus) were collected from 15 counties of Henan and Hubei. In the regions of nonendemicity, 13,230 ticks (400 H. longicornis and 12,830 R. microplus) were collected from 23 counties of Hubei. These data suggested that H. longicornis and R. microplus were the dominant species in regions of endemicity and regions of nonendemicity, respectively.All ticks were grouped into 1,180 pools (450 pools from a region of endemicity and 730 pools from a region of nonendemicity) according to species, host, and geographic origin. H. longicornis and R. microplus represented 365 (30.93%) and 815 (69.07%) pools, respectively. For screening HYSV and sequencing the partial S segment (nucleotides [nt] 63 to 663) or L segment (nt 2208 to 3121) and whole-genome sequences of HYSV, total RNA was extracted from ticks and human sera and was then subjected to reverse transcription-PCR (RT-PCR) as described previously (16). As a result, HYSV RNA was identified in 18 (4.93%) H. longicornis pools and in 5 (0.613%) R. microplus pools, suggesting that both species can carry HYSV. Remarkably, the HYSV RNA-positive H. longicornis ticks were found only in the regions of endemicity, whereas HYSV RNA was identified in R. microplus ticks from both the regions of endemicity (2 pools) and neighboring regions of nonendemicity (3 pools) (Fig. 1). Obviously, the prevalence of HYSV was higher in H. longicornis ticks than in R. microplus ticks and higher in...

The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus.

Background & Aims The development of COVID-19 vaccines has progressed with encouraging safety and efficacy data. Concerns have been raised about SARS-CoV-2 vaccine responses in the large population of patients with non-alcoholic fatty liver disease (NAFLD). The study aimed to explore the safety and immunogenicity of COVID-19 vaccination in NAFLD. Methods This multicenter study included patients with NAFLD without a history of SARS-CoV-2 infection. All patients were vaccinated with 2 doses of inactivated vaccine against SARS-CoV-2. The primary safety outcome was the incidence of adverse reactions within 7 days after each injection and overall incidence of adverse reactions within 28 days, and the primary immunogenicity outcome was neutralizing antibody response at least 14 days after the whole-course vaccination. Results A total of 381 patients with pre-existing NAFLD were included from 11 designated centers in China. The median age was 39.0 years (IQR 33.0–48.0 years) and 179 (47.0%) were male. The median BMI was 26.1 kg/m 2 (IQR 23.8–28.1 kg/m 2 ). The number of adverse reactions within 7 days after each injection and adverse reactions within 28 days totaled 95 (24.9%) and 112 (29.4%), respectively. The most common adverse reactions were injection site pain in 70 (18.4%), followed by muscle pain in 21 (5.5%), and headache in 20 (5.2%). All adverse reactions were mild and self-limiting, and no grade 3 adverse reactions were recorded. Notably, neutralizing antibodies against SARS-CoV-2 were detected in 364 (95.5%) patients with NAFLD. The median neutralizing antibody titer was 32 (IQR 8-64), and the neutralizing antibody titers were maintained. Conclusions The inactivated COVID-19 vaccine appears to be safe with good immunogenicity in patients with NAFLD. Lay summary The development of vaccines against coronavirus disease 2019 (COVID-19) has progressed rapidly, with encouraging safety and efficacy data. This study now shows that the inactivated COVID-19 vaccine appears to be safe with good immunogenicity in the large population of patients with non-alcoholic fatty liver disease.

Posttranscriptional status of messenger RNAs (mRNA) can be affected by many factors, most of which are RNA-binding proteins (RBP) that either bind mRNA in a nonspecific manner or through specific motifs, usually located in the 39 untranslated regions. RBPs can also be recruited by small noncoding RNAs (sncRNA), which have been shown to be involved in posttranscriptional regulations and transposon repression (eg, microRNAs or P-element–induced wimpy testis–interacting RNA) as components of the sncRNA effector complex. Non–sncRNA-binding RBPs have much more diverse effects on their target mRNAs. Some can cause degradation of their target transcripts and/or repression of translation, whereas others can stabilize and/or activate translation. The splicing and exportation of transcripts from the nucleus to the cytoplasm are often mediated by sequence-specific RBPs. The mechanisms by which RBPs regulate mRNA transcripts involve manipulating the 39 poly(A) tail, targeting the transcript to polysomes or to other ribonuclear protein particles, recruiting regulatory proteins, or competing with other RBPs. Here, we briefly review the known mechanisms of posttranscriptional regulation mediated by RBPs, with an emphasis on how these mechanisms might control spermatogenesis in general.

Exosomes are carriers of pro-tumorigenic factors that participate in glioblastoma (GBM) progression, and many fusion genes are strong driver mutations in neoplasia and are involved in tumorigenesis. However, the ability of fusion genes to be transduced by exosomes is unknown. We characterized exosomes from GBM cells harbouring and not harbouring PTPRZ1–MET fusion (ZM fusion). We also determined the effect of the exosomes from ZM fusion cells (ZM exosomes) on pro-oncogenic secretions and showed that ZM exosomes are internalized by the recipient cells. In addition, we studied the effect of ZM exosome-mediated intercellular communication in the GBM microenvironment. MET proto-oncogene expression was higher in ZM exosomes. Moreover, phosphorylated MET was detected only in ZM exosomes and not in exosomes released by non-ZM fusion GBM cells. ZM exosomes transferred to non-ZM fusion GBM cells and normal human astrocytes altered gene expression and induced epithelial–mesenchymal transition. The uptake of ZM exosomes also induced an exosome-dependent phenotype defined by GBM cell migration and invasion, neurosphere growth and angiogenesis. In addition, ZM exosomes conferred temozolomide resistance to the GBM cells, and exosome-derived ZM fusion network proteins targeted multiple pro-oncogenic effectors in recipient cells within the GBM microenvironment. Our findings show that exosomes mediate the aggressive character of GBM and demonstrate the role of ZM fusion in the exacerbation of this effect. These findings have possible implications for the foundation of gene fusion-based therapy for managing GBM.

The effect of aggressive driving behavior on driver's injury severity is analyzed by considering a comprehensive set of variables at highway-rail grade crossings in the US. In doing so, we are able to use a mixed logit modelling approach; the study explores the determinants of driver-injury severity with and without aggressive driving behaviors at highway-rail grade crossings. Significant differences exist between drivers' injury severity with and without aggressive driving behaviors at highway-rail grade crossings. The level of injury for younger male drivers increases a lot if they are with aggressive driving behavior. In addition, driving during peak-hour is found to be a statistically significant predictor of high level injury severity with aggressive driving behavior. Moreover, environmental factors are also found to be statistically significant. The increased level of injury severity accidents happened for drivers with aggressive driving behavior in the morning peak (6-9 am), and the probability of fatality increases in both snow and fog condition. Driving in open space area is also found to be a significant factor of high level injury severity with aggressive driving behaviors. Bad weather conditions are found to increase the probability of drivers' high level injury severity for drivers with aggressive driving behaviors.