A key challenge in analyzing single cell RNA-sequencing data is the large number of false zeros, where genes actually expressed in a given cell are incorrectly measured as unexpressed. We present a ...method based on low-rank matrix approximation which imputes these values while preserving biologically non-expressed genes (true biological zeros) at zero expression levels. We provide theoretical justification for this denoising approach and demonstrate its advantages relative to other methods on simulated and biological datasets.
Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local ...microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
Abstract
T
H
17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is ...merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of T
H
17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against
S
.
aureus
. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in T
H
17 cells. Our data thus indicate a key function of T
H
17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of T
H
17 cells with regard to environmental changes.
The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts
. Intestinal stem cells are closely ...associated with a diverse but poorly characterized network of mesenchymal cell types
. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E
(PGE
). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE
drives the expansion οf a population of Sca-1
reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1
cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE
promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1
cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE
-Ptger4. Analyses of patient-derived organoids established that PGE
-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE
-Ptger4-Yap signalling axis.
The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator ...of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential
. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial
, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.
Influenza A virus (IAV) causes up to half a million deaths worldwide annually, 90% of which occur in older adults. We show that IAV-infected monocytes from older humans have impaired antiviral ...interferon production but retain intact inflammasome responses. To understand the in vivo consequence, we used mice expressing a functional Mx gene encoding a major interferon-induced effector against IAV in humans. In Mx1-intact mice with weakened resistance due to deficiencies in Mavs and TIr7, we found an elevated respiratory bacterial burden. Notably, mortality in the absence of Mavs and TIr7 was independent of viral load or MyD88-dependent signaling but dependent on bacterial burden, caspase-1/11, and neutrophil-dependent tissue damage. Therefore, in the context of weakened antiviral resistance, vulnerability to IAV disease is a function of caspase-dependent pathology.
Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic ...infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²⁺ extracellular concentration at 37°C and a peak in Fe³⁺ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P. ...aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm‐associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases.
Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial ...nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This paper aims to draw scientific attention to the significance and usefulness of socio- logical approach, namely higher education (HE) graduates’ perceptions survey as a one of the mechanisms to ...collect and use graduate feedback facilitating the study programmes’ evaluation, and in turn, enlarging our knowledge on the enhancement of HE quality, the improvement of graduate employability, and effective ways of smoothing the transition from education to work. The emphasis is being placed on critical description of the analytical framework set out by authors of the international research project - DEHEMS on HE graduates feedback. The DEHEMS project uses secondary - processed data based on primary - raw data as generated by two Europe-wide and large-scale research projects: REFLEX and HEGESCO.
The paper comprises four parts. Firstly, the basic conceptual question is analysed, that is, the role and the need for the use of feedback from graduates collected through national and international surveys perceived as a measure of HE programme evaluation (pro- gramme performance, teaching effectiveness). A particular attention is given to its two main elements: programme activities/components and programme outcomes/impact, as per programmes logic model. The second part is devoted to the discussion on the analyt- ical framework and methodological approach adopted in the DEHEMS study. The third part contains key elements of analytical framework presented in schematic form. Finally, some brief conclusions and further study directions follow.
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