Genome sequences of five bacteriophages that belong to the genus .

We describe the genomes of five lytic myophages, therapeutic candidates, that belong to the family and genus . The genomes ranged from 165,574 to 169,768 bp, with ca. 40% GC content, contained 289-300 coding sequences, had 15-16 tRNA genes, and no terminal repeats.

Complete genome sequences of 12 lytic phages against multidrug-resistant .

We report the genome sequences of 12 phages isolated in Kenya, belonging to the genus , , , and . They have double-stranded DNA with lengths varying from 17,979 to 147,374 bp and G+C content from 33.14% to 40.

Complete genome sequences of four lytic bacteriophages against multidrug-resistant .

We report the genome sequences of four phages isolated from environmental wastewater in Kenya. They are double-stranded DNA phages with genomes varying in length from 42,231 to 43,348 bp, with G+C contents ranging from 34.96% to 35.

Characterization and Anti-Biofilm Activity of Lytic Enterococcus Phage vB_Efs8_KEN04 against Clinical Isolates of Multidrug-Resistant in Kenya.

() is a growing cause of nosocomial and antibiotic-resistant infections. Treating drug-resistant requires novel approaches. The use of bacteriophages (phages) against multidrug-resistant (MDR) bacteria has recently garnered global attention.

Managing Microbiota Activity of with Probiotic (Bactocell) and Antimicrobial (Fumidil B) Treatments: Effects on Spring Colony Strength.

Against a backdrop of declining bee colony health, this study aims to gain a better understanding of the impact of an antimicrobial (Fumidil B, Can-Vet Animal Health Supplies Ltd., Guelph, ON, Canada) and a probiotic (Bactocell, Lallemand Inc., Montreal, QC, Canada) on bees' microbiota and the health of their colonies after wintering.

Environmental contamination across multiple hospital departments with multidrug-resistant bacteria pose an elevated risk of healthcare-associated infections in Kenyan hospitals.

Background: Healthcare-associated infections (HAIs) are often caused by multidrug-resistant (MDR) bacteria contaminating hospital environments which can cause outbreaks as well as sporadic transmission.

Methods: This study systematically sampled and utilized standard bacteriological culture methods to determine the numbers and types of MDR Enterococcus faecalis/faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) from high-touch environments of five Kenyan hospitals; level 6 and 5 hospitals (A, B, and C), and level 4 hospitals (D and E), in 2018. Six hundred and seventeen high-touch surfaces across six hospital departments; surgical, general, maternity, newborn, outpatient and pediatric were sampled.

Determination of and Antimicrobial Resistance and Virulence Factors and Their Association with Clinical and Demographic Factors in Kenya.

Background: Enterococci are clinically significant because of their increasing antibiotic resistance and their ability to cause severe infections due to an arsenal of virulence genes. Few studies in the developing world have examined virulence factors that may significantly impact patient outcomes. This study describes the antimicrobial resistance profiles and prevalence of five key genes , , , and in forty-four clinical and isolates in Kenya and their association with patients' demographic and clinical characteristics.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism.

Ten Thousand-Fold Higher than Acceptable Bacterial Loads Detected in Kenyan Hospital Environments: Targeted Approaches to Reduce Contamination Levels.

Microbial monitoring of hospital surfaces can help identify target areas for improved infection prevention and control (IPCs). This study aimed to determine the levels and variations in the bacterial contamination of high-touch surfaces in five Kenyan hospitals and identify the contributing modifiable risk factors. A total of 559 high-touch surfaces in four departments identified as high risk of hospital-acquired infections were sampled and examined for bacterial levels of contamination using standard bacteriological culture methods.

Discovery of physiological and cancer-related regulators of 3' UTR processing with KAPAC.

3' Untranslated regions (3' UTRs) length is regulated in relation to cellular state. To uncover key regulators of poly(A) site use in specific conditions, we have developed PAQR, a method for quantifying poly(A) site use from RNA sequencing data and KAPAC, an approach that infers activities of oligomeric sequence motifs on poly(A) site choice. Application of PAQR and KAPAC to RNA sequencing data from normal and tumor tissue samples uncovers motifs that can explain changes in cleavage and polyadenylation in specific cancers.

3' End Sequencing Library Preparation with A-seq2.

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3' untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3'UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3' end processing.

High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq.

High-throughput sequencing has greatly facilitated the discovery of long and short non-coding RNAs (ncRNAs), which frequently guide ribonucleoprotein complexes to RNA targets, to modulate their metabolism and expression. However, for many ncRNAs, the targets remain to be discovered. In this study, we developed computational methods to map C/D box snoRNA target sites using data from core small nucleolar ribonucleoprotein crosslinking and immunoprecipitation and from transcriptome-wide mapping of 2΄-O-ribose methylation sites.

A comprehensive analysis of 3' end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation.

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region (3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized.

Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data.

Background: Understanding the regulation of gene expression, including transcription start site usage, alternative splicing, and polyadenylation, requires accurate quantification of expression levels down to the level of individual transcript isoforms. To comparatively evaluate the accuracy of the many methods that have been proposed for estimating transcript isoform abundance from RNA sequencing data, we have used both synthetic data as well as an independent experimental method for quantifying the abundance of transcript ends at the genome-wide level.

Results: We found that many tools have good accuracy and yield better estimates of gene-level expression compared to commonly used count-based approaches, but they vary widely in memory and runtime requirements.

Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells.

Alternative polyadenylation is a cellular mechanism that generates mRNA isoforms differing in their 3' untranslated regions (3' UTRs). Changes in polyadenylation site usage have been described upon induction of proliferation in resting cells, but the underlying mechanism and functional significance of this phenomenon remain largely unknown. To understand the functional consequences of shortened 3' UTR isoforms in a physiological setting, we used 3' end sequencing and quantitative mass spectrometry to determine polyadenylation site usage, mRNA and protein levels in murine and human naive and activated T cells.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30.

Knowing what we do and doing what we should: quality assurance in hemodialysis.

An international group of around 50 nephrologists and scientists, including representatives from large dialysis provider organisations, formulated recommendations on how to develop and implement quality assurance measures to improve individual hemodialysis patient care, population health and cost effectiveness. Discussed were methods thought to be of highest priority, those clinical indicators which might be most related to meaningful patient outcomes, tools to control treatment delivery and the role of facilitating computerized expert systems. Emphasis was given to the use of new technologies such as measurement of online dialysance and ways of assessing fluid status.

Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors.

Expression of mature messenger RNAs (mRNAs) requires appropriate transcription initiation and termination, as well as pre-mRNA processing by capping, splicing, cleavage, and polyadenylation. A core 3'-end processing complex carries out the cleavage and polyadenylation reactions, but many proteins have been implicated in the selection of polyadenylation sites among the multiple alternatives that eukaryotic genes typically have. In recent years, high-throughput approaches to map both the 3'-end processing sites as well as the binding sites of proteins that are involved in the selection of cleavage sites and in the processing reactions have been developed.

Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases.

In eukaryotes, the poly(A) tail added at the 3' end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications.

Cleavage factor Im is a key regulator of 3' UTR length.

In eukaryotes, the 3' ends of RNA polymerase II-transcribed RNAs are generated in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. Through alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control and drastic changes in the length of mRNA 3' UTRs upon induction of proliferation in resting cells.

Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length.

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF I(m)25, CF I(m)59, and CF I(m)68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFI(m) proteins have much higher positional specificity compared to CPSF components.

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I.

Mammalian cleavage factor I (CF I(m)) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I(m)25, CF I(m)59, CF I(m)68). It is part of the cleavage and polyadenylation complex responsible for processing the 3' ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3' processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups.

Crystal structure of the 25 kDa subunit of human cleavage factor Im.

Cleavage factor I(m) is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor I(m) is an oligomer composed of a small 25 kDa subunit (CF I(m)25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein.

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases.

Poly(A) polymerases were identified almost 50 years ago as enzymes that add multiple AMP residues to the 3' ends of primer RNAs without use of a template from ATP as cosubstrate and with release of pyrophosphate. Based on sequence homology of a signature motif in the catalytic domain, poly(A) polymerases were later found to belong to a superfamily of nucleotidyl transferases acting on a very diverse array of substrates. Enzymes belonging to the superfamily can add from single nucleotides of AMP, CMP or UMP to RNA, antibiotics and proteins but also homopolymers of many hundred residues to the 3' ends of RNA molecules.