Distinct Escherichia coli transcriptional profiles in the guts of recurrent UTI sufferers revealed by pangenome hybrid selection.

Low-abundance members of microbial communities are difficult to study in their native habitats, including Escherichia coli, a minor but common inhabitant of the gastrointestinal tract, and key opportunistic pathogen of the urinary tract. While multi-omic analyses have detailed interactions between uropathogenic Escherichia coli (UPEC) and the bladder mediating urinary tract infection (UTI), little is known about UPEC in its pre-infection reservoir, the gastrointestinal tract, partly due to its low relative abundance (<1%). To sensitively explore the genomes and transcriptomes of diverse gut E.

Distinct transcriptional profiles in the guts of recurrent UTI sufferers revealed by pangenome hybrid selection.

Low-abundance members of microbial communities are difficult to study in their native habitats. This includes , a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (<1% relative abundance).

Combining genomics and epidemiology to track mumps virus transmission in the United States.

Unusually large outbreaks of mumps across the United States in 2016 and 2017 raised questions about the extent of mumps circulation and the relationship between these and prior outbreaks. We paired epidemiological data from public health investigations with analysis of mumps virus whole genome sequences from 201 infected individuals, focusing on Massachusetts university communities. Our analysis suggests continuous, undetected circulation of mumps locally and nationally, including multiple independent introductions into Massachusetts and into individual communities.

Capturing sequence diversity in metagenomes with comprehensive and scalable probe design.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity.

Chikungunya Arthritis Mechanisms in the Americas: A Cross-Sectional Analysis of Chikungunya Arthritis Patients Twenty-Two Months After Infection Demonstrating No Detectable Viral Persistence in Synovial Fluid.

Objective: To determine if chikungunya virus persists in synovial fluid after infection, potentially acting as a causative mechanism of persistent arthritis.

Methods: We conducted a cross-sectional study of 38 Colombian participants with clinical chikungunya virus infection during the 2014-2015 epidemic who reported chronic arthritis and 10 location-matched controls without chikungunya virus or arthritis. Prior chikungunya virus infection status was serologically confirmed, and the presence of synovial fluid chikungunya virus, viral RNA, and viral proteins was determined by viral culture, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and mass spectrometry, respectively.

Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing.

We describe a patient with severe and progressive encephalitis of unknown etiology. We performed rapid metagenomic sequencing from cerebrospinal fluid and identified Powassan virus, an emerging tick-borne flavivirus that has been increasingly detected in the United States.

Evidence of Ebola Virus Replication and High Concentration in Semen of a Patient During Recovery.

In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen.

Zika virus evolution and spread in the Americas.

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations.

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA - including poly(rA) carrier and ribosomal RNA - from the viral RNA sample.

Ebola Virus Epidemiology and Evolution in Nigeria.

Containment limited the 2014 Nigerian Ebola virus (EBOV) disease outbreak to 20 reported cases and 8 fatalities. We present here clinical data and contact information for at least 19 case patients, and full-length EBOV genome sequences for 12 of the 20. The detailed contact data permits nearly complete reconstruction of the transmission tree for the outbreak.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples.

Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone.

The 2013-2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic.

Discovery of novel rhabdoviruses in the blood of healthy individuals from West Africa.

Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen's nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries.

Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak.

In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2000× coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic.

Double-stranded RNA-dependent ATPase DRH-3: insight into its role in RNAsilencing in Caenorhabditis elegans.

RNA helicases are proteins essential to almost every facet of RNA metabolism, including the gene-silencing pathways that employ small RNAs. A phylogenetically related group of helicases is required for the RNA-silencing mechanism in Caenorhabditis elegans. Dicer-related helicase 3 (DRH-3) is a Dicer-RIG-I family protein that is essential for RNA silencing and germline development in nematodes.

The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC.

Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways [1, 2]. In the RNA interference (RNAi) pathway [3], small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression [4].

Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.

In the Drosophila and mammalian RNA interference pathways, siRNAs direct the protein Argonaute2 (Ago2) to cleave corresponding mRNA targets, silencing their expression. Ago2 is the catalytic component of the RNAi enzyme complex, RISC. For each siRNA duplex, only one strand, the guide, is assembled into the active RISC; the other strand, the passenger, is destroyed.

A protein sensor for siRNA asymmetry.

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2.

Flavopiridol-induced apoptosis during S phase requires E2F-1 and inhibition of cyclin A-dependent kinase activity.

Transformed cells are selectively sensitized to apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol after their recruitment to S phase. During S phase, cyclin A-dependent kinase activity neutralizes E2F-1 allowing orderly S phase progression. Inhibition of cyclin A-dependent kinase by flavopiridol could cause inappropriately persistent E2F-1 activity during S phase traversal and exit.

Selective sensitization of transformed cells to flavopiridol-induced apoptosis following recruitment to S-phase.

Flavopiridol is a potent inhibitor of cyclin-dependent kinases (cdks). In a large proportion of solid tumor cell lines, the initial response to flavopiridol is cell cycle arrest. NCI-H661 non-small cell lung cancer cells are representative of a subset of more sensitive cell lines in which apoptosis is observed during the first 24 h of drug exposure.