Dual RNA-seq of pathogen and host

Abstract | A comprehensive understanding of host-pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing 'dual RNA-seq', in which gene expression changes in both the pathogen and the host are analysed simultaneously. R E V I E W S 618 | SEPTEMBER 2012 | VOLUME 10 www.nature.com/reviews/micro R E V I E W S © 2012 Macmillan Publishers Limited. All rights reserved Nature Reviews | Microbiology Compare with target genome (or genomes), and enumerate sequence

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.Spanish Ministry of Economy and Competitiveness BIO2010-15417 BIO2013-44220-R AGL2013-45339-RRecerCaixa program 2012/ACUP/00048Catalonian government 2017SGR49

Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs

Transgene regulation in plants by alternative splicing of a suicide exon

Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence.

Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available 'omics' datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria

Management of Bleeding and Hemolysis During Percutaneous Microaxial Flow Pump Support A Practical Approach

© 2023 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Percutaneous ventricular assist devices (pVADs) are increasingly being used because of improved experience and availability. The Impella (Abiomed), a percutaneous microaxial, continuous-flow, short-term ventricular assist device, requires meticulous postimplantation management to avoid the 2 most frequent complications, namely, bleeding and hemolysis. A standardized approach to the prevention, detection, and treatment of these complications is mandatory to improve outcomes. The risk for hemolysis is mostly influenced by pump instability, resulting from patient- or device-related factors. Upfront echocardiographic assessment, frequent monitoring, and prompt intervention are essential. The precarious hemostatic balance during pVAD support results from the combination of a procoagulant state, due to critical illness and contact pathway activation, together with a variety of factors aggravating bleeding risk. Preventive strategies and appropriate management, adapted to the impact of the bleeding, are crucial. This review offers a guide to physicians to tackle these device-related complications in this critically ill pVAD-supported patient population.Peer reviewe

TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction

Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on β-adrenergic versus Angiotensin II (Ang II)-dependent (Gs vs. Gαq mediated) modulation of Ca2+i-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca2+ currents and Ca2+i transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca2+ currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca2+/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, β-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca2+-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca2+i-dependent hypertrophic growth response to Ang II, but not to β-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT1 signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for β-adrenergic Ca2+i-stimulation in adult myocytes

Deceleration of Fusion–Fission Cycles Improves Mitochondrial Quality Control during Aging

Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the ‘mitochondrial infectious damage adaptation’ (MIDA) model according to which a deceleration of fusion–fission cycles reflects a systemic adaptation increasing life span