Whole-Genome Shotgun Sequencing for Nasopharyngeal Microbiome in Pre-school Children With Recurrent Wheezing.

Microbiome mediates early life immune deviation in asthma development. Recurrent wheeze (RW) in pre-school years is a risk factor for asthma diagnosis in school-age children. Dysbiosis exists in asthmatic airways, while its origin in pre-school years and relationship to RW is not clearly defined. This study investigated metagenomics of nasopharyngeal microbiome in pre-school children with RW. We applied whole-genome shotgun sequencing and human rhinovirus (HRV) detection on nasopharyngeal samples collected from three groups of pre-school children: (i) RW group: 16 children at-risk for asthma who were hospitalized for RW, (ii) inpatient control (IC): 18 subjects admitted for upper respiratory infection, and (iii) community control (CC): 36 children without respiratory syndromes. Sequence reads were analyzed by MetaPhlAn2 and HUMAnN2 algorithm for taxonomic and functional identification. Linear discriminant analysis effect size (LEfSe) analysis was used to identify discriminative features. We identified that Moraxella catarrhalis and Dolosigranulum pigrum were predominant species in nasopharynx. RW had lower alpha diversity (Shannon diversity index) than CC (0.48 vs. 1.07; P adj = 0.039), characterized by predominant Proteobacteria. LEfSe analysis revealed D. pigrum was the only discriminative species across groups (LDA = 5.57, P = 0.002), with its relative abundance in RW, IC, and CC being 9.6, 14.2, and 37.3%, respectively (P < 0.05). LEfSe identified five (ribo)nucleotides biosynthesis pathways to be group discriminating. Adjusting for HRV status, pre-school children with RW have lower nasopharyngeal biodiversity, which is associated with Proteobacteria predominance and lower abundance of D. pigrum. Along with discriminative pathways found in RW and CC, these microbial biomarkers help to understand RW pathogenesis.

Whole genome sequencing data of 1110 Mycobacterium tuberculosis isolates identifies insertions and deletions associated with drug resistance.

BACKGROUND: Drug resistance in Mycobacterium tuberculosis (MTB) is one of the major challenges in tuberculosis (TB) treatment. However, known mutations cannot explain all of the cases of resistance and little research has focused on the relationship between insertions / deletions (indels) and drug resistance. RESULTS: Here, we retrieved whole genome sequencing data of 743 drug-resistant MTB strains and 367 pan-susceptible strains from TB patients from the public domain to identify novel genomic markers of drug resistance. A total of 20 region markers containing genes and intergenic regions (IGRs) with significant statistical correlation with antibiotic resistance were revealed, four of which have been previously reported to be associated with drug resistance. In addition, 83 point markers containing frameshift (FS) mutations and IGR indels were also identified independently based on differences in their incidence rates between drug-sensitive and -resistant strains. Among the 83 point markers, eight indels were detected in known drug-associated genes or IGRs. Furthermore, the overlap between 20 region markers and 83 point markers further indicated their associations with drug resistance. The markers identified were involved in essential bacterial metabolic functions, including cell wall and transmembrane transporter functions. A strong correlation between FS mutations and mutations in DNA repair genes including I21V in alkA, R48G in mutT4 and P2R in nth was also found. CONCLUSIONS: This study identified a set of novel genetic markers with FS mutations and IGR indels associated with MTB drug resistance, which greatly broadens the pool of mutations related to MTB drug resistance. This insight may be important in identifying novel mechanisms of drug resistance in MTB.

Phylogenetic and Structural Significance of Dihydrofolate Synthase (folC) Mutations in Drug-Resistant Mycobacterium tuberculosis.

The aim of this study is to investigate the mutation pattern of the folC gene in drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates of global and Hong Kong cohorts. The public sequence read archives of 1,124 MTB genomes from three independent studies were retrieved and folC mutations existing solely in drug-resistant MTB strains were identified. A phylogenetic tree was constructed to analyze the segregation of mutation-related amino acid residues in the FolC structure. These mutation sites were further supported by direct Sanger sequencing of the folC gene among 254 clinical MTB isolates in a Hong Kong cohort. Homology modeling of wild-type and mutated FolC was performed, and the predicted structures were docked with hydroxydihydropteroate, the metabolic derivative of para-aminosalicylic acid (PAS), to evaluate the resultant binding affinity changes. Combining the results of three previous cohorts and our cohort, E40, I43, S150, and E153 are the most frequently affected amino acid residues in resistant isolates. Based on the distribution of mutations in the genome-based phylogenetic tree, lineage-specific mutation patterns were observed. Regarding the segregation of affected amino acid residues, the four most frequently affected residues are all in close proximity of the binding pocket for the PAS derivative. Molecular modeling results showed that mutations at E40, I43, and S150 can alter the structure of FolC putative binding pocket, causing the PAS derivative to bind outside of the now deformed pocket. This might ablate the interaction between the protein and the PAS derivative. To conclude, this study is the first comprehensive mutation pattern and bioinformatics analysis of the folC gene in MTB drug-resistant isolates. The distribution of mutations in phylogenetic lineages and protein structure is reported, analyzed, and discussed.

Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Molecular changes in endometriosis-associated ovarian clear cell carcinoma.

BACKGROUND: Endometriosis is frequently associated with and thought of having propensity to develop into ovarian clear cell carcinoma (OCCC), although the molecular transformation mechanism is not completely understood. METHODS: We employed immunohistochemical (IHC) staining for marker expression along the potential progression continuum. Expression profiling of microdissected endometriotic and OCCC cells from patient-matched formalin-fixed, paraffin-embedded samples was performed to explore the carcinogenic pathways. Function of novel biomarkers was confirmed by knockdown experiments. RESULTS: PTEN was significantly lost in both endometriosis and invasive tumour tissues, while oestrogen receptor (ER) expression was lost in OCCC relative to endometriosis. XRCC5, PTCH2, eEF1A2 and PPP1R14B were significantly overexpressed in OCCC and associated endometriosis, but not in benign endometriosis (p ⩽ 0.004). Knockdown experiments with XRCC5 and PTCH2 in a clear cell cancer cell line resulted in significant growth inhibition. There was also significant silencing of a panel of target genes with histone H3 lysine 27 trimethylation, a signature of polycomb chromatin-remodelling complex in OCCC. IHC confirmed the loss of expression of one such polycomb target gene, the serous ovarian cancer lineage marker Wilms' tumour protein 1 (WT1) in OCCC, while endometriotic tissues showed significant co-expression of WT1 and ER. CONCLUSIONS: Loss of PTEN expression is proposed as an early and permissive event in endometriosis development, while the loss of ER and polycomb-mediated transcriptional reprogramming for pluripotency may play an important role in the ultimate transformation process. Our study provides new evidence to redefine the pathogenic programme for lineage-specific transformation of endometriosis to OCCC.

Draft Genome Sequence of Extensively Drug-Resistant Acinetobacter baumannii Strain CUAB1 from a Patient in Hong Kong, China.

We report the draft genome sequence of an extensively drug-resistant strain of Acinetobacter baumannii, CUAB1, isolated from a patient in a local Hong Kong hospital. MIC testing was performed, and genes previously associated with drug resistance were located.

Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis.

BACKGROUND: Panax notoginseng (Burk.) F.H. Chen is one of the most highly valued medicinal plants in the world. The major bioactive molecules are triterpene saponins, which are also known as ginsenosides. However, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. Hence, genomic and transcriptomic details about P. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now. RESULTS: In this study, RNA sequencing of three different P. notoginseng tissues was performed using next generation DNA sequencing. After assembling the high quality sequencing reads into 107,340 unigenes, biochemical pathways were predicted and 9,908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis. In addition, 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. In particular, one unigene predominantly expressed in the root was annotated as CYP716A53v2, which probably participates in the formation of protopanaxatriol from protopanaxadiol in P. notoginseng. The differential expression of this gene was further confirmed by real-time PCR. CONCLUSIONS: We have established a global transcriptome dataset for P. notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng.