Arginine methyltransferases as regulators of RNA-binding protein activities in pathogenic Kinetoplastids

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a "regulatory code". Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality

PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation

Life in plastic, it’s fantastic! How Leishmania exploit genome instability to shape gene expression

Leishmania are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, Leishmania regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, Leishmania are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations. Typically, such events indicate genome instability in other eukaryotes, coinciding with genetic abnormalities, but for Leishmania, exploiting these products of genome instability can provide selectable substrates to catalyse necessary gene expression changes by modifying gene copy number. Unorthodox DNA replication, DNA repair, replication stress factors and DNA repeats are recognised in Leishmania as contributors to this intrinsic instability, but how Leishmania regulate genome plasticity to enhance fitness whilst limiting toxic under- or over-expression of co-amplified and co-transcribed genes is unclear. Herein, we focus on fresh, and detailed insights that improve our understanding of genome plasticity in Leishmania. Furthermore, we discuss emerging models and factors that potentially circumvent regulatory issues arising from polycistronic transcription. Lastly, we highlight key gaps in our understanding of Leishmania genome plasticity and discuss future studies to define, in higher resolution, these complex regulatory interactions

Aspects of Benthic Decapod Diversity and Distribution from Rocky Nearshore Habitat at Geographically Widely Dispersed Sites

Relationships of diversity, distribution and abundance of benthic decapods in intertidal and shallow subtidal waters to 10 m depth are explored based on data obtained using a standardized protocol of globally-distributed samples. Results indicate that decapod species richness overall is low within the nearshore, typically ranging from one to six taxa per site (mean = 4.5). Regionally the Gulf of Alaska decapod crustacean community structure was distinguishable by depth, multivariate analysis indicating increasing change with depth, where assemblages of the high and mid tide, low tide and 1 m, and 5 and 10 m strata formed three distinct groups. Univariate analysis showed species richness increasing from the high intertidal zone to 1 m subtidally, with distinct depth preferences among the 23 species. A similar depth trend but with peak richness at 5 m was observed when all global data were combined. Analysis of latitudinal trends, confined by data limitations, was equivocal on a global scale. While significant latitudinal differences existed in community structure among ecoregions, a semi-linear trend in changing community structure from the Arctic to lower latitudes did not hold when including tropical results. Among boreal regions the Canadian Atlantic was relatively species poor compared to the Gulf of Alaska, whereas the Caribbean and Sea of Japan appeared to be species hot spots. While species poor, samples from the Canadian Atlantic were the most diverse at the higher infraordinal level. Linking 11 environmental variables available for all sites to the best fit family-based biotic pattern showed a significant relationship, with the single best explanatory variable being the level of organic pollution and the best combination overall being organic pollution and primary productivity. While data limitations restrict conclusions in a global context, results are seen as a first-cut contribution useful in generating discussion and more in-depth work in the still poorly understood field of biodiversity distribution

Effective genome editing in Leishmania (Viannia) braziliensis stably expressing Cas9 and T7 RNA polymerase

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite

Prevalence and correlates of frailty in an older rural African population:findings from the HAALSI cohort study

Background: Frailty is a key predictor of death and dependency, yet little is known about frailty in sub-Saharan Africa despite rapid population ageing. We describe the prevalence and correlates of phenotypic frailty using data from the Health and Aging in Africa: Longitudinal Studies of an INDEPTH Community cohort. Methods: We analysed data from rural South Africans aged 40 and over. We used low grip strength, slow gait speed, low body mass index, and combinations of self-reported exhaustion, decline in health, low physical activity and high self-reported sedentariness to derive nine variants of a phenotypic frailty score. Each frailty category was compared with self-reported health, subjective wellbeing, impairment in activities of daily living and the presence of multimorbidity. Cox regression analyses were used to compare subsequent all-cause mortality for non-frail (score 0), pre-frail (score 1–2) and frail participants (score 3+). Results: Five thousand fifty nine individuals (mean age 61.7 years, 2714 female) were included in the analyses. The nine frailty score variants yielded a range of frailty prevalences (5.4% to 13.2%). For all variants, rates were higher in women than in men, and rose steeply with age. Frailty was associated with worse subjective wellbeing, and worse self-reported health. Both prefrailty and frailty were associated with a higher risk of death during a mean 17 month follow up for all score variants (hazard ratios 1.29 to 2.41 for pre-frail vs non-frail; hazard ratios 2.65 to 8.91 for frail vs non-frail). Conclusions: Phenotypic frailty could be measured in this older South African population, and was associated with worse health, wellbeing and earlier death

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract\ud \ud \ud \ud Background\ud \ud Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.\ud \ud \ud \ud Results\ud \ud ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups.\ud \ud \ud \ud Conclusion\ud \ud These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.We thank Lucile FloeterWinter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii).We thank Lucile Floeter-Winter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii)

Comparison of Pathway Analysis Approaches Using Lung Cancer GWAS Data Sets

Pathway analysis has been proposed as a complement to single SNP analyses in GWAS. This study compared pathway analysis methods using two lung cancer GWAS data sets based on four studies: one a combined data set from Central Europe and Toronto (CETO); the other a combined data set from Germany and MD Anderson (GRMD). We searched the literature for pathway analysis methods that were widely used, representative of other methods, and had available software for performing analysis. We selected the programs EASE, which uses a modified Fishers Exact calculation to test for pathway associations, GenGen (a version of Gene Set Enrichment Analysis (GSEA)), which uses a Kolmogorov-Smirnov-like running sum statistic as the test statistic, and SLAT, which uses a p-value combination approach. We also included a modified version of the SUMSTAT method (mSUMSTAT), which tests for association by averaging χ2 statistics from genotype association tests. There were nearly 18000 genes available for analysis, following mapping of more than 300,000 SNPs from each data set. These were mapped to 421 GO level 4 gene sets for pathway analysis. Among the methods designed to be robust to biases related to gene size and pathway SNP correlation (GenGen, mSUMSTAT and SLAT), the mSUMSTAT approach identified the most significant pathways (8 in CETO and 1 in GRMD). This included a highly plausible association for the acetylcholine receptor activity pathway in both CETO (FDR≤0.001) and GRMD (FDR = 0.009), although two strong association signals at a single gene cluster (CHRNA3-CHRNA5-CHRNB4) drive this result, complicating its interpretation. Few other replicated associations were found using any of these methods. Difficulty in replicating associations hindered our comparison, but results suggest mSUMSTAT has advantages over the other approaches, and may be a useful pathway analysis tool to use alongside other methods such as the commonly used GSEA (GenGen) approach

Introductory programming: a systematic literature review

As computing becomes a mainstream discipline embedded in the school curriculum and acts as an enabler for an increasing range of academic disciplines in higher education, the literature on introductory programming is growing. Although there have been several reviews that focus on specific aspects of introductory programming, there has been no broad overview of the literature exploring recent trends across the breadth of introductory programming. This paper is the report of an ITiCSE working group that conducted a systematic review in order to gain an overview of the introductory programming literature. Partitioning the literature into papers addressing the student, teaching, the curriculum, and assessment, we explore trends, highlight advances in knowledge over the past 15 years, and indicate possible directions for future research

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London