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Summary Recent advances of long-term time-lapse microscopy have made it easy for researchers to quantify cell behavior and molecular dynamics at single-cell resolution. However, the lack of easy-to-use software tools optimized for customized research is still a major challenge for quantitatively understanding biological processes through microscopy images. Here, we present CellTracker, a highly integrated graphical user interface software, for automated cell segmentation and tracking of time-lapse microscopy images. It covers essential steps in image analysis including project management, image pre-processing, cell segmentation, cell tracking, manually correction and statistical analysis such as the quantification of cell size and fluorescence intensity, etc. Furthermore, CellTracker provides an annotation tool and supports model training from scratch, thus proposing a flexible and scalable solution for customized dataset analysis. Availability and implementation CellTracker is an open-source software under the GPL-3.0 license. It is implemented in Python and provides an easy-to-use graphical user interface. The source code, instruction manual and demos can be found at https://github.com/WangLabTHU/CellTracker. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR] more...

WD repeat domain 5 (WDR5) serves an important role in various biological functions through the epigenetic regulation of gene transcription. Aberrant expression of WDR5 has been observed in various types of human cancer, including prostate cancer, breast cancer and leukemia. However, the role of WDR5 expression and its clinical implications in hepatocellular carcinoma (HCC) remain largely unknown. The present study investigated the WDR5 expression pattern in HCC. It was demonstrated that the mRNA and protein levels of WDR5 were upregulated in HCC cancer tissues compared with normal adjacent tissues using reverse transcription-quantitative polymerase chain reaction and western blotting. Furthermore, the elevated WDR5 protein level was significantly associated with the histological grade (P=0.038), tumor size (P=0.023), tumor-node-metastasis stage (P=0.035) and reduced long-term survival time. Additionally, it was demonstrated through the shRNA-mediated knockdown of WDR5 in HCC cells in vitro that WDR5 expression promotes cell proliferation using an MTT assay. Taken together, the results suggested that WDR5 overexpression may have an oncogenic effect in HCC, and may be a promising biomarker for the diagnosis and prognosis of HCC. [ABSTRACT FROM AUTHOR] more...

Objectives Lung adenocarcinoma (LUAD) is a common subtype of non-small cell lung cancer prevalent in Asia. There is a dearth of understanding regarding the transcriptome landscape of LUAD without primary known driver mutations. In this study, LUAD samples without well-known driver mutations occurring in EGFR , KRAS , ALK , ROS1 or RET (quintuple-negative) were used for transcriptome study with a focus on long noncoding RNAs (lncRNAs), alternative splicing and gene fusions. Materials and methods 24 pairs of LUAD and adjacent normal samples and 13 tumor-only samples derived from 37 quintuple-negative patients were used. Differentially expressed lncRNA transcripts were detected by paired t -test and were validated by qPCR. Functions of lncRNAs were predicted by co-expressed mRNAs. Aberrant splicing events in LUAD were identified using MISO. In addition, gene fusions were screened by SOAPfuse. Results and conclusion In total, 90 and 153 up- or down-regulated lncRNA transcripts were detected in LUAD samples in comparison with the adjacent normal samples. The most significantly differentially expressed lncRNA transcript was ENST00000598996.1 ( FENDRR ) down-regulated in LUAD. By lncRNA-mRNA co-expression analysis, functions of 14 lncRNAs were predicted. The predicted functions included vasculature development, immune response, cell cycle and respiratory gaseous exchange. Furthermore, six co-expressed pairs of lncRNAs and their nearby protein coding genes were identified as associated with lung development. This study also identified two highly recurrent (22 in 24) differential exon skipping events occurring in MYH14 and ESYT2 with exon including isoforms of both genes up-regulated in isoform percentage in LUAD samples. On the other hand, two out of 24 LUAD samples possessed the driver mutation exon 14 skipping of MET . The transcriptional alterations of LUAD samples without well-known driver mutations identified in the study can be used as references for future research. The translational values of these transcriptional changes are also worthy of further investigation. [ABSTRACT FROM AUTHOR] more...

DNA methylation is an important epigenetic mechanism for phenotypic diversification in all forms of life. We previously described remarkable cell-to-cell heterogeneity in epigenetic pattern within a clonal population of Streptococcus pneumoniae, a leading human pathogen. We here report that the epigenetic diversity is caused by extensive DNA inversions among hsdSA , hsdSB , and hsdSC, three methyltransferase hsdS genes in the Spn556II type-I restriction modification (R-M) locus. Because hsdSA encodes the sequence recognition subunit of this type-I R-M DNA methyltransferase, these site-specific recombinations generate pneumococcal cells with variable HsdSA alleles and thereby diverse genome methylation patterns. Most importantly, the DNA methylation pattern specified by the HsdSA1 allele leads to the formation of opaque colonies, whereas the pneumococci lacking HsdSA1 produce transparent colonies. Furthermore, this HsdSA1-dependent phase variation requires intact DNA methylase activity encoded by hsdM in the Spn556II (renamed olony pacity eterminant or cod) locus. Thus, the DNA inversion-driven ON/OFF switch of the hsdSA1 allele in the cod locus and resulting epigenetic switch dictate the phase variation between the opaque and transparent phenotypes. Phase variation has been well documented for its importance in pneumococcal carriage and invasive infection, but its molecular basis remains unclear. Our work has discovered a novel epigenetic cause for this significant pathobiology phenomenon in S. pneumoniae. Lastly, our findings broadly represents a significant advancement in our understanding of bacterial R-M systems and their potential in shaping epigenetic and phenotypic diversity of the prokaryotic organisms because similar site-specific recombination systems widely exist in many archaeal and bacterial species. [ABSTRACT FROM AUTHOR] more...

• Novel hybrid self-supervised visual representation learning method tailored for H&E-stained histopathological images. • Generative and discriminative self-supervised learning can complement and enhance each other. • Good rationality by leveraging domain-specific knowledge in histopathology. • Good versatility for different kinds of computational histopathology tasks. Visual representation extraction is a fundamental problem in the field of computational histopathology. Considering the powerful representation capacity of deep learning and the scarcity of annotations, self-supervised learning has emerged as a promising approach to extract effective visual representations from unlabeled histopathological images. Although a few self-supervised learning methods have been specifically proposed for histopathological images, most of them suffer from certain defects that may hurt the versatility or representation capacity. In this work, we propose CS-CO, a hybrid self-supervised visual representation learning method tailored for H&E-stained histopathological images, which integrates advantages of both generative and discriminative approaches. The proposed method consists of two self-supervised learning stages: cross-stain prediction (CS) and contrastive learning (CO). In addition, a novel data augmentation approach named stain vector perturbation is specifically proposed to facilitate contrastive learning. Our CS-CO makes good use of domain-specific knowledge and requires no side information, which means good rationality and versatility. We evaluate and analyze the proposed CS-CO on three H&E-stained histopathological image datasets with downstream tasks of patch-level tissue classification and slide-level cancer prognosis and subtyping. Experimental results demonstrate the effectiveness and robustness of the proposed CS-CO on common computational histopathology tasks. Furthermore, we also conduct ablation studies and prove that cross-staining prediction and contrastive learning in our CS-CO can complement and enhance each other. Our code is made available at https://github.com/easonyang1996/CS-CO. [ABSTRACT FROM AUTHOR] more...

Here, we report an unconventional Chinese pedigree consisting of three branches all segregating prelingual hearing loss (HL) with unclear inheritance pattern. After identifying the cause of one branch as maternally inherited aminoglycoside-induced HL, targeted next generation sequencing (NGS) was applied to identify the genetic causes for the other two branches. One affected subject from each branch was subject to targeted NGS whose genomic DNA was enriched either by whole-exome capture (Agilent SureSelect All Exon 50 Mb) or by candidate genes capture (Agilent SureSelect custom kit). By NGS analysis, we identified that patients from Branch A were compound heterozygous for p.E1006K and p.D1663V in the CDH23 (DFNB12) gene; and patients from Branch B were homozygous for IVS7-2A>G in the SLC26A4 (DFNB4) gene. Both CDH23 mutations altered conserved calcium binding sites of the extracellular cadherin domains. The co-occurrence of three different genetic causes in this family was exceedingly rare but fully compatible with the mutation spectrum of HL. Our study has also raised several technical and analytical issues when applying the NGS technique to genetic testing. [ABSTRACT FROM AUTHOR] more...

Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive. High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole-genome linkage analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known ADNSHL genes and shared by the sequenced samples: c.328C>A in COCH (DFNA9) resulting in a p.Q110K substitution and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs*2. The pathogenicity of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co-segregation with phenotype in the pedigree and in light of established genotype-phenotype correlations. The frameshift deletion in MYO6 was confirmed as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the linkage scan. Our clinical data also supported the emerging genotype-phenotype correlation for DFNA22. [ABSTRACT FROM AUTHOR] more...

We describe a large four-generational Chinese pedigree segregating MYH9-related disease caused by a V1516L mutation. The clinical findings supported previously established genotype-phenotype correlations, and also demonstrated interindividual variability of disease manifestations even within the same family. The same mutation was previously reported in another Chinese pedigree but resulting from a different DNA substitution. Analyzing the patterns of previously reported mutations revealed a limited spectrum of pathogenic variants. The implications of this finding are discussed. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR] more...

Abstract: Recent study revealed that most human genes have alternative splicing and can produce multiple isoforms of transcripts. Differences in the relative abundance of the isoforms of a gene can have significant biological consequences. Identifying genes that are differentially spliced between two groups of RNA-sequencing samples is an important basic task in the study of transcriptomes with next-generation sequencing technology. We use the negative binomial (NB) distribution to model sequencing reads on exons, and propose a NB-statistic to detect differentially spliced genes between two groups of samples by comparing read counts on all exons. The method opens a new exon-based approach instead of isoform-based approach for the task. It does not require information about isoform composition, nor need the estimation of isoform expression. Experiments on simulated data and real RNA-seq data of human kidney and liver samples illustrated the method''s good performance and applicability. It can also detect previously unknown alternative splicing events, and highlight exons that are most likely differentially spliced between the compared samples. We developed an NB-statistic method that can detect differentially spliced genes between two groups of samples without using a prior knowledge on the annotation of alternative splicing. It does not need to infer isoform structure or to estimate isoform expression. It is a useful method designed for comparing two groups of RNA-seq samples. Besides identifying differentially spliced genes, the method can highlight on the exons that contribute the most to the differential splicing. We developed a software tool called DSGseq for the presented method available at http://bioinfo.au.tsinghua.edu.cn/software/DSGseq. [Copyright &y& Elsevier] more...

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis. [ABSTRACT FROM AUTHOR] more...

Abstract: High-throughput RNA sequencing (RNA-seq) technology provides a revolutionary approach to studying splicing events de novo. However, identifying splice junctions with high sensitivity and specificity remains a challenge. In the present study, we proposed a new tool named SeqSaw to detect splice junctions with or without the canonical GT–AG splicing signal. SeqSaw was applied to two ENCODE RNA-seq datasets and also compared with two existing methods. It was shown that the proposed method obtained better results on finding novel splice junctions. Experiments also revealed that the current sequencing depth has not yet reached saturation to detect novel transcripts. Moreover, by comparing the number of supporting reads, we demonstrated that many un-annotated splicing events can be tissue specific. [Copyright &y& Elsevier] more...

CpG island methylation plays important role in various biological processes. To investigate methylation landscape of all CpG islands on the human genome, we develop a model for predicting the CpG island methylation status. This model outperforms other existing methods. We apply the model on the whole human genome and predict the landscape of DNA methylation of all CpG islands. Based on the methylation profile, we find that about 31% of CpG islands are methylation-prone and CpG islands located in promoter regions are seldom methylated. There is no significant difference in the CpG island methylation level between R and G bands among the chromosomes. The occupancy of RNA polymerase II is significantly higher in methylation-resistant promoter CpG islands, indicating that genes with such promoter CpG islands tend to be more active. [ABSTRACT FROM AUTHOR] more...

Abstract: The liver performs a number of essential functions for life. The development of such a complex organ relies on finely regulated gene expression profiles which change over time in the development and determine the phenotype and function of the liver. We used high-density oligonucleotide microarrays to study the gene expression and transcription regulation at 14 time points across the C57/B6 mouse liver development, which include E11.5 (embryonic day 11.5), E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, Day0 (the day of birth), Day3, Day7, Day14, Day21, and normal adult liver. With these data, we made a comprehensive analysis on gene expression patterns, functional preferences and transcriptional regulations during the liver development. A group of uncharacterized genes which might be involved in the fetal hematopoiesis were detected. [Copyright &y& Elsevier] more...

MicroRNAs (miRNAs) are a class of approximately 22-nt small RNAs that regulate posttranscriptional gene expression. Thousands of expressed sequence tags (ESTs) have been identified by using upstream 2500-nt and downstream 4000-nt flanking sequences to BLAST in the dbEST database. The cotranscription of the miRNAs and their flanking sequences covered by the matched ESTs is verified by RT-PCR. It directly reveals that a large portion of mammalian intergenic miRNAs are first transcribed as long primary transcripts (pri-miRNAs). Also, the transcripts’ ranges of tens of pri-miRNAs are predicted by the EST-extension method. We then extracted the tissue-specific expression information from the annotations of the matched ESTs and established the expression profile of the studied miRNAs for tens of tissues. This provided a new way to establish the expression profiles of miRNAs. Results show that the human brain, lung, liver, and eye and the mouse brain, eye, and mammary gland are tissues in which enriched numbers of miRNAs are expressed. [ABSTRACT FROM AUTHOR] more...

Abstract: The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 5′ splice site (5′ss) selection in human genome. The results show that free energy exerts different effects on alternative 5′ss choice in different situations and −8.1kcal/mol is a threshold. When both free energies of two competing 5′ss are larger than −8.1kcal/mol, the 5′ss with lower free energy is more frequently used. However, in other pairs of 5′ss, lower-free-energy 5′ss does not seem to be favored and even the other 5′ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 5′ splicing with short alternative exons (<50nt), which implies a complex mechanism of 5′ss selection involving both U1 snRNA binding free energy and regulatory factors. [Copyright &y& Elsevier] more...

The systematic analysis of very short alternative splicing (VSAS) in the human genome showed that VSAS might contribute more to protein-function diversity than expected. More than 65% of VSAS fragments have different secondary structures from flanking regions. They tend to have a non-loop structure and have an important influence on protein functions, as shown by the predicted 3D structure of human IL-4δ2. The observed VSAS events can be classified into two groups depending on whether they insert new structure domains in the proteins, and they might be of different evolutionary status. [Copyright &y& Elsevier] more...

• Analyzed the taxonomic and functional dynamics of microbial community to algal blooms. • Constructed 161 high-quality metagenome-assembled genomes by binning analysis. • Cyanobacterial strains showed diverse energy pathways, transporters and prokaryotic defense systems. • Nitrogen and phosphorus influence the microbial structure by affecting co-pathways. Freshwater lakes are threatened by harmful cyanobacterial blooms, whose basic unit is Cyanobacterial Aggregate (CA). CA-attached bacteria play a significant role through different blooming stages with substantial variation of their taxonomic structure. However, little is known about their functional variations and functional links with cyanobacteria due to the lack of reference genomes. In this longitudinal study, we collected 16 CA samples from Lake Taihu, one of China's largest freshwater lakes, from April 2015 to February 2016, and sequenced their V4 region of 16S rRNA genes, full metagenomes (MG), and metatranscriptomes (MT). The analysis of these data revealed the dynamics of microbial taxonomic and functional structure in CAs, influenced by both external environmental factors and internal metabolism. 55 OTUs, 456 genes, and 37 transcripts showed significantly differential abundance across the early, middle, and late blooming stages (ANOVA test, P < 0.05). Total nitrogen and total phosphorus were proved to be the most important environmental drivers of microbial taxonomic and functional variations in CAs (Mantel's r > 0.25, P < 0.05). We constructed 161 high-quality metagenome-assembled genomes (MAGs), out of which 22 were cyanobacterial strains with diverse energy pathways, transporters and prokaryotic defense systems. Based on these MAGs, we constructed a cyanobacteria-bacteria co-nitrogen-pathway and a cyanobacteria-bacteria co-phosphorus-pathway, by which we demonstrated how nitrogen and phosphorus influence the dynamics of the microbial structure to a certain extent by affecting these co-pathways. Overall, these results characterized the taxonomic, functional, and transcriptional variations of microbes in CAs through different blooming stages. Genome assembly and metabolic analysis of cyanobacteria and their attached bacteria suggested that the material exchange and signal transduction do, indeed, exist among them. Our understanding of the underlying molecular pathways for cyanobacterial blooms could lead to the control of blooms by interventional strategies to disrupt critical microbes' expression. [Display omitted] [ABSTRACT FROM AUTHOR] more...