Transposon insertion mapping with PIMMS, Pragmatic Insertional Mutation Mapping System

The PIMMS (Pragmatic Insertional Mutation Mapping System) pipeline has beendeveloped for simple conditionally essential genome discovery experiments in bacteria.Capable of using raw sequence data files alongside a FASTA sequence of thereference genome and GFF file, PIMMS will generate a tabulated output of each codingsequence with corresponding mapped insertions accompanied with normalized resultsenabling streamlined analysis. This allows for a quick assay of the genome to identifyconditionally essential genes on a standar d desktop computer prioritizing results forfurther investigation

Transposon insertion mapping with PIMMS, Pragmatic Insertional Mutation Mapping System

The PIMMS (Pragmatic Insertional Mutation Mapping System) pipeline has beendeveloped for simple conditionally essential genome discovery experiments in bacteria.Capable of using raw sequence data files alongside a FASTA sequence of thereference genome and GFF file, PIMMS will generate a tabulated output of each codingsequence with corresponding mapped insertions accompanied with normalized resultsenabling streamlined analysis. This allows for a quick assay of the genome to identifyconditionally essential genes on a standar d desktop computer prioritizing results forfurther investigation

Complete genome sequence of Streptococcus agalactiae strain 01173, isolated from Kuwaiti wild fish

© 2020 Santi et al. Here, we report the complete genome of piscine Streptococcus agalactiae 01173 serotype Ia, which was generated using long-read sequencing technology. The bacteria were isolated from wild fish displaying signs of streptococcosis, from a fish kill incident in Kuwait

Vru (Sub0144) controls expression of proven and putative virulence determinants and alters the ability of Streptococcus uberis to cause disease in dairy cattle

The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696)

Nectar chemistry modulates the impact of an invasive plant on native pollinators

1. Invasive species are considered a main driver of pollinator declines, yet the direct effects of invasive alien plants on pollinators are poorly understood. 2. Abundant, invasive plant species can provide a copious nectar resource for native pollinators. However, the nectar of some plants contains secondary compounds, usually associated with defence against herbivores. The impacts of these compounds on pollinators are often unknown. 3. We compared how consumption of grayanotoxin I and III, natural secondary compounds in the nectar of invasive Rhododendron ponticum L., affected three native bee species: a honeybee, (Apis mellifera L.), a solitary mining bee (Andrena carantonica, Pérez) and a bumblebee, (Bombus terrestris, L.). 4. Survival of the solitary bee and the bumblebee species was not affected by either grayanotoxin, but honeybees were ∼20× more likely to die when fed solutions containing grayanotoxin I. Furthermore, solitary bees were deterred from feeding and exhibited malaise behaviours indicative of sublethal toxicity in response to consumption of grayanotoxin I. In contrast, grayanotoxins did not affect bumblebee survival or behaviour, even when bees were subjected to multiple stressors (parasite infection or food stress). 5. Our experiments suggest that while R. ponticum provides abundant floral nectar, it is only available as a food resource to pollinators that tolerate grayanotoxins. Pollinators whose health is negatively affected by grayanotoxins may experience negative impacts from R. ponticum invasion directly (if they consume R. ponticum nectar) or indirectly (if native floral resources are replaced by R. ponticum). 6. Our study makes a novel comparison of the effects of a natural nectar secondary compound on three pollinator species and clearly demonstrates drastic variation in the responses of different key pollinator taxa to a nectar toxin. Our findings are thus in congruence with literature demonstrating the varying effects of invasive plant chemistry on native foliar herbivores, and our work demonstrates that nectar chemistry should be taken into account when determining the impacts of plant invasion for native pollinators

PIMMS-Dash: Accessible analysis, interrogation, and visualisation of high-throughput transposon insertion sequencing (TIS) data

MotivationCurrent methods for visualising and interrogating high-throughput transposon insertion mutagenesis sequencing (TIS) data requires a significant time investment in learning bioinformatics, often producing static figures that do not facilitate real time analysis.SummaryWe have created an accessible web-based browser tool for visualisation and downstream analysis of high-throughput TIS data results generated by the PIMMS analysis pipeline. This includes multiple interactive and sortable tables to aid the user to identify genes of interest, enabling the user to gain a greater understanding of the genes contributing to fitness in their experimental work. PIMMS-Dash permits researchers, with any level of bioinformatics knowledge, to interrogate data sets and generate publication quality figures.AvailabilityPIMMS-Dash is freely available and is accessible online at https://pimms-dashboard-uon.azurewebsites.net and a Docker containerised version is available at https://github.com/Streptococcal-Research-Group/PIMMS-Dash to run locally

Bovine Neonatal Monocytes Display Phenotypic Differences Compared With Adults After Challenge With the Infectious Abortifacient Agent Neospora caninum

The neonatal period represents a window of susceptibility for ruminants given the abundance of infectious challenges in their environment. Maternal transfer of immunity does not occur in utero but post-parturition, however this does not compensate for potential deficits in the cellular compartment. Here we present a cellular and transcriptomic study to investigate if there is an age-related difference in the monocyte response in cattle during intra-cellular protozoan infection. We utilized Neospora caninum, an obligate intracellular protozoan parasite that causes abortion and negative economic impacts in cattle worldwide, to study these responses. We found neonatal animals had a significant greater percentage of CD14+ monocytes with higher CD80 cell surface expression. Adult monocytes harbored more parasites compared to neonatal monocytes; additionally greater secretion of IL-1β was observed in neonates. Microarray analysis revealed neonates have 535 genes significantly upregulated compared to adult with 23 upregulated genes. Biological pathways involved in immune response were evaluated and both age groups showed changes in the upregulation of tyrosine phosphorylation of STAT protein and JAK-STAT cascade pathways. However, the extent to which these pathways were upregulated in neonates was much greater. Our findings suggest that neonates are more resistant to cellular invasion with protozoan parasites and that the magnitude of the responses is related to significant changes in the JAK-STAT network

Arginine dependence of acute myeloid leukaemia blast proliferation: a novel therapeutic target

Acute myeloid leukemia (AML) is one of the most common acute leukemias in adults and children, yet significant numbers of patients relapse and die of disease. In this study, we identify the dependence of AML blasts on arginine for proliferation. We show that AML blasts constitutively express the arginine transporters CAT-1 and CAT-2B, and that the majority of newly diagnosed patients' blasts have deficiencies in the arginine-recycling pathway enzymes argininosuccinate synthase and ornithine transcarbamylase, making them arginine auxotrophic. BCT-100, a pegylated human recombinant arginase, leads to a rapid depletion in extracellular and intracellular arginine concentrations, resulting in arrest of AML blast proliferation and a reduction in AML engraftment in vivo. BCT-100 as a single agent causes significant death of AML blasts from adults and children, and acts synergistically in combination with cytarabine. Using RNA sequencing, 20 further candidate genes which correlated with resistance have been identified. Thus, AML blasts are dependent on arginine for survival and proliferation, as well as depletion of arginine with BCT-100 of clinical value in the treatment of AML

A paradox in bacterial pathogenesis: Activation of the local macrophage inflammasome is required for virulence of streptococcus uberis

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection

Variants in Hormone Biosynthesis Genes and Risk of Endometrial Cancer.

We investigated the risk associated with variants in three genes involved in estrogen biosynthesis, CYP11A1, CYP17A1, and CYP19A1, in the population-based case-control study of Estrogen, Diet, Genetics, and Endometrial Cancer. This study was conducted in New Jersey in 2001-2006 with 417 cases and 402 controls. For CYP11A1, there was no association between the number of [TTTTA]( n ) repeats (D15S520) and risk. For CYP17A1, risk was somewhat lower among women with the C/C genotype at T-34C (rs743572) (adjusted OR = 0.65, 95% CI 0.41-1.02). For CYP19A1, risk was lower among women homozygous for the 3-bp deletion (rs11575899) in exon 4 (adjusted OR = 0.44, 95% CI 0.26-0.76), while the number of [TTTA]( n ) repeats was not significantly related to risk: the adjusted OR for n = 7/7 repeats versus n \u3e 7/\u3e7 repeats was 0.81 (95% CI 0.54-1.23). In stratified analyses, results for CYP19A1 were stronger among women with higher (\u3e or =27.4) body mass index: for the homozygous deletion, OR = 0.30 (95% CI 0.15-0.62); for the n = 7/7 genotype, OR = 0.49 (95% CI 0.26-0.93). The interaction between the n = 7/7 genotype and BMI was statistically significant (p = 0.01). The insertion/deletion variant in CYP19A1 appears to be related to risk of endometrial cancer; risk associated with variants in this gene may vary according to BMI