Research status and progress of laboratory diagnostic tests for intraocular lymphoma / 中华眼底病杂志

Intraocular lymphoma (IOL) is a rare lymphocytic malignancy. The gold standard for the definite diagnosis remains histopathologic examination of the ocular specimen. But cytologic confirmation of malignant lymphoma cells in vitreous or chorioretinal specimens is challenging and dependending on highly skilled cytopathologist, due to the sparse cellularity and specimen degeneration. Consequently, false-negative rates arecommon, which delays diagnosis and treatment seriously. Because of the limited diagnostic capacity of cytology, other adjunct diagnostic tools have been developed. Additional procedures that may support IOL diagnosis include flow cytometry, immunocytochemistry, cytokines study with identification of interleukin (IL)-10 and IL-6 level, and polymerase chain reaction amplification. And more recently, new techniques of mutational analysis have been validated for the diagnosis of vitreoretinal lymphoma (VRL) and may represent a helpful diagnostic tool for the detection of early cases. Metagenomic deep sequencing technology may provide an important basis for IOL diagnosis and personalized treatment. In the future, it is expected to deepen the understanding of IOL disease phenotypes at the molecular level, discover new target therapies, monitor response to treatment, and detect intraocular recurrences. These may offer insights into how we might create a tailored therapeutic approach for each patient's VRL in the future.

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1

Targeted massively parallel sequencing for congenital generalized lipodystrophy

ABSTRACT Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). Subjects and methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. Conclusions: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Discussion on critical points for a tailored therapy to cure hepatitis C virus infection

Hepatitis C virus (HCV) infects around 71 million people worldwide and in 2018 it is still a major health problem. Since 2011, anti-HCV therapy with availability of direct-acting antiviral drugs has revolutionized the clinical response and paved the way to eradication strategies. However, despite the high rate of sustained virological response, treatment failure may occur in a limited percentage of patients, possibly due to resistance-associated substitutions (RASs), either emergent or pre-existent even in minority viral populations. Clearly this problem may impair success of eradication strategies. With this background, several questions marks still exist around HCV treatment, including whether pan-genotypic treatments with complete effectiveness in any clinical conditions really exist outside clinical trials, the actual cost-effectiveness of genotyping testing, and utility of RAS detection in viral quasispecies by next generation sequencing approach. In this review, we describe these critical points by discussing recent literature data and our research experience.

Human sapovirus GI. 2 and GI. 3 from children with acute gastroenteritis in northern Brazil

Human sapoviruses (HSaV) are considered important causative agents of acute gastroenteritis in humans worldwide. However, knowledge of the genetic characteristics of the whole genome of HSaV in Brazil is limited. Here we report the complete genome sequences of six HSaVs GI.2 and two GI.3 strains obtained from children with acute gastroenteritis in the Northern region of Brazil. Next generation sequencing was used to obtain the full genome and molecular characterization of the genome was performed. Phylogenetic analysis of the genome was also performed. Only one complete HSaV GI.2 genome characterization in the country precedes that of the present study. This is the first complete genome sequence of genotype GI.3 in Brazil. The data obtained in this investigation can contribute to the augmentation of the database on the molecular diversity of HSaVs strains circulating in Brazil, and to the improvement of current typing protocols.

Challenges from Health Management and Deep Sequencing Data Parsing / 医学信息学杂志

The paper introduces impact of deep sequencing technology on biomedical research and the society,discusses challenges confronting and opportunities enjoyed by deep sequencing data parsing and its application in health management,including aspects like data access,computing technology,data application,lack of talent and interdisciplinary talent education,etc.

Complete genome analysis of a sapovirus in a mixed infected patient of hand foot and mouth disease / 中华实验和临床病毒学杂志

Objective@#To understand the potential viral pathogens other than enteroviruses existing in samples of hand foot and mouth disease (HFMD) patient and study their molecular feature and genotype.@*Methods@#The deep sequencing analysis of a fecal specimen collected from HFMD patient was conducted by metagenomics and bioinformatics.@*Results@#Enterovirus A71 and sapovirus mixed infection was found in this case. The nucleic acid of sapovirus was confirmed positive by RT-PCR and the 7 429 bp complete genome sequence of sapovirus was obtained by assembling sequencing reads which consisted of 3 open reading frames. Phylogenetic analysis revealed that this strain of virus should belong to the genotype 1 of sapovirus having a homology of 99.4% with sapovirus Hu/G1/Zhejiang1/China/2014 strain, which is a currently predominant genotype circulating in China.@*Conclusions@#The sapovirus, which is a predominant strain circulating in China, was a mixed infected causative agent existing in HFMD sample identified by deep sequencing. This study will serve as a reference for pathogen detection of HFMD and diarrheal related diseases, as well as provide a sequence reference for molecular feature study of sapovirus in China in the future.

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.</p><p><b>METHODS</b>Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.</p><p><b>RESULTS</b>Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.</p><p><b>CONCLUSION</b>Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.</p>

Identification of zebrafish shield organizer-specific genes / 中国实验动物学报

Objective During zebrafish gastrulation, dramatic movements rearrange cells into three germ layers and contribute to the formation of the shield organizer, which acts as a dorsal signal center to pattern the body axis.Global identification of shield organizer-specific genes in early gastrulas will be valuable for studying the regulatory cascades during organizer formation and body axis establishment.Methods Tg( gsc:GFP) transgenic embryos express GFP in the shield organizer, which is controlled by a 1.8 kb gsc promoter.Flow cytometry technology and RNA deep sequencing analysis were applied to isolate GFP positive cells from the Tg( gsc:GFP) transgenic embryos and systematically uncover the genes highly expressed in the dorsal organizer.Subsequently, the expression of shield organizer-specific genes was further con-firmed by quantitative real-time PCR and whole mount in situ hybridization method during zebrafish embryonic develop-ment.Results GFP-positive cells exceeding 96%purity were isolated from shield-stage Tg(gsc:GFP) transgenic embryos and 657 organizer highly expressed genes were identified through RNA deep sequencing analysis.The results of in situ hy-bridization experiments revealed that a number of genes including KIAA1324, ripply1, twist2, isthmin1, nme4, zgc174153 and rrbp1b were expressed in shield organizer during zebrafish gastrulation.Conclusions The identification of these shield organizer-specific genes in the current study provides useful clues to explore the zebrafish developmental functions in further studies.

HCMV-encoded miRNA during latent infection / 军事医学

Objective To explore humam cytomegalovirus(HCMV) encoded microRNAs during latent infection in order to help study HCMV virology and latent infection mechanisms.Methods A model of HCMV latent infection via THP-1 cells infected with HCMV was constructed.Deep-sequencing was performed using high-resolution Solexa sequencing platform.The secondary structure of the newly sequenced miRNA was predicted by RNAFold bioinformatics software. Results HCMV encoded various miRNAs during latent infection, including miR-US25-2-3p, miR-US25-2-5p, miR-UL112, miR-US25-1, miR-UL22A and PC-5p-148467 with a predicted length of 25 bp, named hcmv-miR-US33as-5p.Conclusion HCMV can express many types of miRNAs during latent infection.

Mutational Analysis of Extranodal NK/T-Cell Lymphoma Using Targeted Sequencing with a Comprehensive Cancer Panel

Extranodal natural killer (NK)/T-cell lymphoma, nasal type (NKTCL), is a malignant disorder of cytotoxic lymphocytes of NK or T cells. It is an aggressive neoplasm with a very poor prognosis. Although extranodal NKTCL reportedly has a strong association with Epstein-Barr virus, the molecular pathogenesis of NKTCL has been unexplored. The recent technological advancements in next-generation sequencing (NGS) have made DNA sequencing cost- and time-effective, with more reliable results. Using the Ion Proton Comprehensive Cancer Panel, we sequenced 409 cancer-related genes to identify somatic mutations in five NKTCL tissue samples. The sequencing analysis detected 25 mutations in 21 genes. Among them, KMT2D, a histone modification-related gene, was the most frequently mutated gene (four of the five cases). This result was consistent with recent NGS studies that have suggested KMT2D as a novel driver gene in NKTCL. Mutations were also found in ARID1A, a chromatin remodeling gene, and TP53, which also recurred in recent NGS studies. We also found mutations in 18 novel candidate genes, with molecular functions that were potentially implicated in cancer development. We suggest that these genes may result in multiple oncogenic events and may be used as potential bio-markers of NKTCL in the future.

The use of ultra deep sequencing technique in the screening program on HIV-1 drug resistance mutation among ART-na?ve patients in Hunan province / 中华流行病学杂志

Objective To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) TDRs among HIV-1 ART-na?ve patients in Hunan province using the ultra deep sequencing(UDS) technique. Methods ART-na?ve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques,to the 1%variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. Results UDS method was performed on 90 ART-na?ve subjects that seeking service of care,in Hunan. In total,42.2%(38/90)of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique,at a HIV variant frequency level of≥1%,15.6%(14/90)showed NRTI TDR,16.7%(15/90) showed a major NNRTI TDR and 10%(9/90)were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. Conclusion ART-na?ve subjects from Hunan province, which had been predominately infected by subtype AE,would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region,identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.

The use of ultra deep sequencing technique in the screening program on HIV-1 drug resistance mutation among ART-na?ve patients in Hunan province / 中华流行病学杂志

Objective To determine the prevalence rates of nucleotide reverse-transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) TDRs among HIV-1 ART-na?ve patients in Hunan province using the ultra deep sequencing(UDS) technique. Methods ART-na?ve subjects diagnosed in Hunan between 2010 and 2011 were evaluated by both UDS technique and Sanger sequencing techniques,to the 1%variant level. Mutations were scored using the Stanford HIVdb algorithm to infer the status on drug resistance. Results UDS method was performed on 90 ART-na?ve subjects that seeking service of care,in Hunan. In total,42.2%(38/90)of the subjects showed major NRTI or nonnucleoside reverse transcriptase inhibitor NNRTI TDRs by UDS technique,at a HIV variant frequency level of≥1%,15.6%(14/90)showed NRTI TDR,16.7%(15/90) showed a major NNRTI TDR and 10%(9/90)were both resistant to NRTI and NNRTI when variants were analyzed by Stanford HIVdb. Conclusion ART-na?ve subjects from Hunan province, which had been predominately infected by subtype AE,would frequently possess HIV variants with NRTI/NNRTI TDRs that would affect the use of first line ART in the region,identified by the UDS technique. Further studies were needed to describe the prevalence of TDRs and to gather related information.

Identification of Culex Flavivirus by deep sequencing approach in Xinjiang, China / 中华微生物学和免疫学杂志

Objective To investigate the diversity of mosquito-borne viruses in Xinjiang , China, and to identify mosquitos-borne viruses of medical importance rapidly .Methods The virus-derived RNAs in mosquitos captured in wild were screened and confirmed by using Illumina deep sequencing approach and reverse transcription PCR , respectively .The alignment analysis was performed by using gene sequences from GenBank.Results One hundred and forty-four Culex Flavivirus ( CxFV, Flavivirus genus, Flaviviridae) specific sequences were identified .The overlapping reads were assembled into 7 uncontinuous viral genomic contigs.The gaps between the contigs were further filled by RT-PCR products, which resulted in reconstruc-tion of viral genomic 5′and 3′terminus (687 nt and 411 nt).Phylogenetic analysis showed that the newly identified CxFV belonged to America/Asian genotype , which specifically clustered into a clade with other CxFV strains from China mainland ,sharing 98.2%-99.5%homologies in nucleotide sequences and 99.5%in amino acids sequences among them .Conclusion Illumina deep sequencing approach was successfully applied to arthropod-borne virus surveillance .The recently emerged Culex Flavivirus was detected for the first time in Xinjiang, China.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.