Molecular epidemiology of dengue viruses from complete genome sequences

The availability of the complete genetic blueprint of the dengue virus is essential in molecular epidemiological studies to uncover the role of the virus in dengue pathogenesis. During the course of this project, over two hundred complete genomes of the dengue virus were generated from clinical samples collected in three dengue-endemic Southeast Asian countries. In addition, a bioinformatics platform integrating a sequence database, sequence retrieval tools, sequence annotation data and a variety of analysis tools was developed for easy management, manipulation and analysis of dengue virus sequence data. Whereas previous studies have mostly focused on epidemiological events in the Americas and Thailand, sequence data recovered from dengue epidemics in Indonesia, Malaysia and Singapore in this study have uncovered some of the dengue virus diversity circulating in the region. The three countries appear to share similar pool of dengue viruses, with some viral lineages in sustained circulation since at least the 1970s. Sequencing of historical virus isolates prior to the 2004/2005 epidemics in Indonesia and Singapore revealed that adaptive viral evolution played little or no role in triggering those epidemics. Lastly, a method that utilised all available sequence data from Malaysia was devised to reconstruct the history of dengue virus in that country since the 1960s

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to capture more than 90% of sequences in the Greengenes database and with nearly twice the resolution of existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the diversity of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.Comment: 17 pages, 2 tables, 2 figures, supplementary materia

Outcompeting p53-Mutant Cells in the Normal Esophagus by Redox Manipulation.

As humans age, normal tissues, such as the esophageal epithelium, become a patchwork of mutant clones. Some mutations are under positive selection, conferring a competitive advantage over wild-type cells. We speculated that altering the selective pressure on mutant cell populations may cause them to expand or contract. We tested this hypothesis by examining the effect of oxidative stress from low-dose ionizing radiation (LDIR) on wild-type and p53 mutant cells in the transgenic mouse esophagus. We found that LDIR drives wild-type cells to stop proliferating and differentiate. p53 mutant cells are insensitive to LDIR and outcompete wild-type cells following exposure. Remarkably, combining antioxidant treatment and LDIR reverses this effect, promoting wild-type cell proliferation and p53 mutant differentiation, reducing the p53 mutant population. Thus, p53-mutant cells can be depleted from the normal esophagus by redox manipulation, showing that external interventions may be used to alter the mutational landscape of an aging tissue

AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kβ/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors

Cancer therapeutic resistance; Target identificationResistencia terapéutica contra el cáncer; Identificación de objetivosResistència terapèutica contra el càncer; Identificació d'objectiusThe PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kβi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.This work was funded by the Wellcome Trust (WT206194) to KY and MJG and by AstraZeneca to KY, BRD, STB and JTL

Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

UNLABELLED: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure. IMPORTANCE: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production of Escherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h−1. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h−1) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C

KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype

KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype

Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth

NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer

Spread, circulation, and evolution of the Middle East respiratory syndrome coronavirus

The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections. Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10−3 substitutions per site per year (95% credible interval [95% CI], 8.76 × 10−4; 1.37 × 10−3) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012). Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection. Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number (R0) for the virus that is less than 1