Cloning and expression of cDNAs from hepatitis E virus structural gene

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5'-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E. coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

Micropeptides: origins, identification, and potential role in metabolism-related diseases

With the development of modern sequencing techniques and bioinformatics, genomes that were once thought to be noncoding have been found to encode abundant functional micropeptides(miPs), a kind of small polypeptides. Although miPs are difficult to analyze and identify, a number of studies have begun to focus on them. More and more miPs have been revealed as essential for energy metabolism homeostasis, immune regulation, and tumor growth and development. Many reports have shown that miPs are especially essential for regulating glucose and lipid metabolism and regulating mitochondrial function.MiPs are also involved in the progression of related diseases. This paper reviews the sources and identification of miPs, as well as the functional significance of miPs for metabolism-related diseases, with the aim of revealing their potential clinical applications.

Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China

OBJECTIVE: To investigate the genotypes of hepatitis E viruses (HEV) detected in sera of patients from different regions of China. METHODS: The partial genome (nt6461-6860, nt5994-6294) of open reading frame 2 (ORF2) of 45 HEV strains detected from 14 cities of China was amplified and sequenced using polymerase chain reaction (PCR) and direct sequencing. RESULTS: Forty-one of 45 strains (91%) share the same genotype with HEV Burma strain (B), with nucleotide identities higher than 98% with the representative HEV Chinese strain. Only 4 HEV strains are significantly divergent from the 3 prototype strains of HEV, with nucleotide identities of 77%-80% with HEV Burmese/Chinese strain, 74%-76% with Mexican strain and 74%-77% with the newly discovered HEV US/swine strain, respectively. Phylogenetic analysis suggests that these 4 strains may represent 2 different subtypes that belong to a novel genotype of HEV, which is significantly divergent from the prototype Mexico, Burmese and US/swine strains. CONCLUSION: Among patients with hepatitis E in China, most are infected by the Chinese prototype HEV, and only a small part by the new genotype HEV.

Large-Scale Discovery of Non-conventional Peptides in Maize and Arabidopsis through an Integrated Peptidogenomic Pipeline

Non-conventional peptides(NCPs),which include small open reading frame-encoded peptides,play critical roles in fundamental biological processes.In this study,we developed an integrated peptidogenomic pipeline using high-throughput mass spectra to probe a customized six-frame translation database and applied it to large-scale identification of NCPs in plants.A total of 1993 and 1860 NCPs were unambiguously identified in maize and Arabidopsis,respectively.These NCPs showed distinct characteristics compared with conventional peptides and were derived from introns,3′UTRs,5′UTRs,junctions,and intergenic regions.Furthermore,our results showed that translation events in unannotated transcripts occur more broadly than previously thought.In addition,we found that dozens of maize NCPs are enriched within regions associated with phenotypic variations and domestication selection,indicating that they potentially are involved in genetic regulation of complex traits and domestication in maize.Taken together,our study developed an integrated peptidogenomic pipeline for large-scale identification of NCPs in plants,which would facilitate global characterization of NCPs from other plants.The identification of large-scale NCPs in both monocot(maize)and dicot(Arabidopsis)plants indicates that a large portion of plant genome can be translated into biologically functional molecules,which has important implications for functional genomic studies.

Association of HLA-DQB1 coding region with unexplained recurrent spontaneous abortion

Background DNA analysis has shown a lack of significant compatibility between couples affected by unexplained recurrent spontaneous abortion (URSA) compared with normal fertile couples, 8 although one study that made use of a PCR-sequence-specific oligonucleotide (SSO) method did observe evidence of significant compatibility in the HLA-DQA1 and DQB1 alleles between patients and aborted fetuses. 9 This study was designed to investigate whether URSA were associated with particular DQ alleles or promoter alleles.Methods Thirty-two patients with URSA and 54 women who had had at least one successful pregnancy were included in this study. HLA-DQ genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The HLA-DQB1 promoter was detected by the SSO and sequence-specific primer (SSP) methods. The DQA1, DQB1, and DQB1 promoter (QBP) gene frequencies in the patients were compared with the gene frequencies in normal controls. The data were analyzed statistically with the χ 2 and Fisher’s exact tests.Results The results showed that the frequency of DQB1 *0604/0605 was significantly higher and the frequency of DQB1 *0501/0502 was significantly lower in the patient group as compared with the normal controls. In addition, the frequencies of the DQA1 *01-DQB1 *0604/0605 and QBP6.2-DQB1 *0604/0605 haplotypes were overrepresented in the patients relative to the controls. Our results did not show any differences between URSA patients and the controls with regard to DQA1 and QBP allele frequencies. Conclusions Our data suggest that URSA is associated with the HLA-DQB1 coding region, and is not associated with its upstream regulatory region. The DQB1 *0604/0605, DQA1 *01-DQB1 *0604/0605, and QBP6.2-DQB1 *0604/0605 haplotypes may confer susceptibility to URSA, while the DQB1 *0501/0502 allele may protect women from URSA.

SmProt: A Reliable Repository with Comprehensive Annotation of Small Proteins Identified from Ribosome Profiling

Small proteins specifically refer to proteins consisting of less than 100 amino acids translated from small open reading frames(s ORFs),which were usually missed in previous genome annotation.The significance of small proteins has been revealed in current years,along with the discovery of their diverse functions.However,systematic annotation of small proteins is still insufficient.Sm Prot was specially developed to provide valuable information on small proteins for scientific community.Here we present the update of Sm Prot,which emphasizes reliability of translated s ORFs,genetic variants in translated s ORFs,disease-specific s ORF translation events or sequences,and remarkably increased data volume.More components such as non-ATG translation initiation,function,and new sources are also included.Sm Prot incorporated638,958 unique small proteins curated from 3,165,229 primary records,which were computationally predicted from 419 ribosome profiling(Ribo-seq)datasets or collected from literature and other sources from 370 cell lines or tissues in 8 species(Homo sapiens,Mus musculus,Rattus norvegicus,Drosophila melanogaster,Danio rerio,Saccharomyces cerevisiae,Caenorhabditis elegans,and Escherichia coli).In addition,small protein families identified from human microbiomes were also collected.All datasets in Sm Prot are free to access,and available for browse,search,and bulk downloads at http://bigdata.ibp.ac.cn/SmProt/.

Development of two TaqMan real-time reverse transcription-PCR assays for the detection of severe acute respiratory syndrome coronavirus-2

The outbreak of coronavirus disease 2019(COVID-19)in Wuhan,China,was caused by a novel coronavirus(CoV),named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The rapid detection of viral nucleic acids is critical for the early identification of infected cases.We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2.The designed primers target the nucleocapsid(N)and open reading frame(ORF)1b gene regions,where the probes discriminate SARS-CoV-2 from other human and animal CoVs.The sensitivities are one genomic copy per reaction for theN gene assay and ten copies for the ORF 1b gene assay.The overall linear detection ranges are 1–10^(6)and 10–10^(6)copies per reaction for the N gene assay and the ORF 1b gene assay,respectively.Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100%(23/23)and 62.5%(16/23)of clinical specimens by the N gene assay and the ORF 1b gene assay,respectively.All of the samples not detected by the ORF 1b gene assay were throat swabs,indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay.The assays developed in the present study offer alternative diagnostic tests for COVID-19.

Optogenetic control of GGGGCC repeat-containing RNA phase transition

The GGGGCC(G_(4)C_(2))hexanucleotide repeat expansion in the C9ORF72 gene is a major cause of both hereditary amyotrophic lateral sclerosis and familial frontotemporal dementia.Recent studies have shown that G_(4)C_(2)hexanucleotide repeat-containing RNA transcripts((G_(4)C_(2))_(n)RNA)could go through liquid-liquid phase separation to form RNA foci,which may elicit neurodegeneration.However,the direct causality between these abnormal RNA foci and neuronal toxicity remains to be demonstrated.Here we introduce an optogenetic control system that can induce the assembly and phase separation of(G_(4)C_(2))_(n)RNA foci with blue light illumination in human cells,by fusing a specific(G_(4)C_(2))_(n)RNA binding protein as the linker domain to Cry2,a protein that oligomerizes in response to blue light.Our results demonstrate that a higher number of G_(4)C_(2)repeats have the potential to be induced into more RNA foci in the cells.Both spontaneous and induced RNA foci display liquid-like properties according to FRAP measurements.Computational simulation shows strong consistency with the experimental results and supports the effect of our system to promote the propensity of(G_(4)C_(2))_(n)RNA towards phase separation.This system can thus be used to investigate whether(G_(4)C_(2))_(n)RNA foci would disrupt normal cellular processes and lead to pathological phenotypes relevant to repeat expansion disorders.