Rescue of a familial dysautonomia mouse model by AAV9-Exon-specific U1 snRNA.

Familial dysautonomia (FD) is a currently untreatable, neurodegenerative disease caused by a splicing mutation (c.2204+6T>C) that causes skipping of exon 20 of the elongator complex protein 1 (ELP1) pre-mRNA. Here, we used adeno-associated virus serotype 9 (AAV9-U1-FD) to deliver an exon-specific U1 (ExSpeU1) small nuclear RNA, designed to cause inclusion of ELP1 exon 20 only in those cells expressing the target pre-mRNA, in a phenotypic mouse model of FD. Postnatal systemic and intracerebral ventricular treatment in these mice increased the inclusion of ELP1 exon 20. This also augmented the production of functional protein in several tissues including brain, dorsal root, and trigeminal ganglia. Crucially, the treatment rescued most of the FD mouse mortality before one month of age (89% vs 52%). There were notable improvements in ataxic gait as well as renal (serum creatinine) and cardiac (ejection fraction) functions. RNA-seq analyses of dorsal root ganglia from treated mice and human cells overexpressing FD-ExSpeU1 revealed only minimal global changes in gene expression and splicing. Overall then, our data prove that AAV9-U1-FD is highly specific and will likely be a safe and effective therapeutic strategy for this debilitating disease.

Serum from COVID-19 patients promotes endothelial cell dysfunction through protease-activated receptor 2.

BACKGROUND: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. OBJECTIVE: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. METHODS AND RESULTS: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. CONCLUSION: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.

Link between organic nanovescicles from vegetable kingdom and human cell physiology: intracellular calcium signalling.

BACKGROUND: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. RESULTS: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. CONCLUSIONS: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.

Genome-wide DNA methylation and transcriptomic analysis of liver tissues subjected to early ischemia/reperfusion injury upon human liver transplantation.

INTRODUCTION AND OBJECTIVES: Epigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. PATIENTS AND METHODS: 12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. RESULTS: Significant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. CONCLUSIONS: In the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI.

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma.

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

The Deep Proteomics Approach Identified Extracellular Vesicular Proteins Correlated to Extracellular Matrix in Type One and Two Endometrial Cancer.

Among gynecological cancers, endometrial cancer is the most common in developed countries. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that contain proteins involved in immune response and apoptosis. A deep proteomic approach can help to identify dysregulated extracellular matrix (ECM) proteins in EVs correlated to key pathways for tumor development. In this study, we used a proteomics approach correlating the two acquisitions-data-dependent acquisition (DDA) and data-independent acquisition (DIA)-on EVs from the conditioned medium of four cell lines identifying 428 ECM proteins. After protein quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 67 proteins. Our bioinformatic analysis identified 26 pathways associated with the ECM. Western blotting analysis on 13 patients with type 1 and type 2 EC and 13 endometrial samples confirmed an altered abundance of MMP2. Our proteomics analysis identified the dysregulated ECM proteins involved in cancer growth. Our data can open the path to other studies for understanding the interaction among cancer cells and the rearrangement of the ECM.

A urokinase-associated outbreak of Ralstonia mannitolilytica bloodstream infections in haemodialysis patients in north-eastern Italy, January to April 2023.

An outbreak of Ralstonia mannitolilytica bloodstream infections occurred in four hospitals in north-eastern Italy, involving 20 haemodialysis patients with tunnelled central vascular catheter access. We identified as the outbreak source a batch of urokinase vials imported from India contaminated with R. mannitolilytica. Whole genome sequences of the clinical and urokinase strains were highly related, and only urokinase-treated patients were reported with R. mannitolilytica infections (attack rate = 34%; 95% confidence interval: 22.1-47.4). Discontinuation of the contaminated urokinase terminated the outbreak.

Mitochondrial Metabolism and EV Cargo of Endothelial Cells Is Affected in Presence of EVs Derived from MSCs on Which HIF Is Activated.

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) have attracted growing interest as a possible novel therapeutic agent for the management of different cardiovascular diseases (CVDs). Hypoxia significantly enhances the secretion of angiogenic mediators from MSCs as well as sEVs. The iron-chelating deferoxamine mesylate (DFO) is a stabilizer of hypoxia-inducible factor 1 and consequently used as a substitute for environmental hypoxia. The improved regenerative potential of DFO-treated MSCs has been attributed to the increased release of angiogenic factors, but whether this effect is also mediated by the secreted sEVs has not yet been investigated. In this study, we treated adipose-derived stem cells (ASCs) with a nontoxic dose of DFO to harvest sEVs (DFO-sEVs). Human umbilical vein endothelial cells (HUVECs) treated with DFO-sEVs underwent mRNA sequencing and miRNA profiling of sEV cargo (HUVEC-sEVs). The transcriptomes revealed the upregulation of mitochondrial genes linked to oxidative phosphorylation. Functional enrichment analysis on miRNAs of HUVEC-sEVs showed a connection with the signaling pathways of cell proliferation and angiogenesis. In conclusion, mesenchymal cells treated with DFO release sEVs that induce in the recipient endothelial cells molecular pathways and biological processes strongly linked to proliferation and angiogenesis.

Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.

A Multi-Omics Approach Revealed Common Dysregulated Pathways in Type One and Type Two Endometrial Cancers.

Endometrial cancer (EC) is the most frequent gynecologic cancer in postmenopausal women. Pathogenetic mechanisms that are related to the onset and progression of the disease are largely still unknown. A multi-omics strategy can help identify altered pathways that could be targeted for improving therapeutical approaches. In this study we used a multi-omics approach on four EC cell lines for the identification of common dysregulated pathways in type 1 and 2 ECs. We analyzed proteomics and metabolomics of AN3CA, HEC1A, KLE and ISHIKAWA cell lines by mass spectrometry. The bioinformatic analysis identified 22 common pathways that are in common with both types of EC. In addition, we identified five proteins and 13 metabolites common to both types of EC. Western blotting analysis on 10 patients with type 1 and type 2 EC and 10 endometria samples confirmed the altered abundance of NPEPPS. Our multi-omics analysis identified dysregulated proteins and metabolites involved in EC tumor growth. Further studies are needed to understand the role of these molecules in EC. Our data can shed light on common pathways to better understand the mechanisms involved in the development and growth of EC, especially for the development of new therapies.

Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.

The rice foot rot pathogen Dickeya zeae alters the in-field plant microbiome.

Studies on bacterial plant diseases have thus far been focused on the single bacterial species causing the disease, with very little attention given to the many other microorganisms present in the microbiome. This study intends to use pathobiome analysis of the rice foot rot disease, caused by Dickeya zeae, as a case study to investigate the effects of this bacterial pathogen to the total resident microbiome and to highlight possible interactions between the pathogen and the members of the community involved in the disease process. The microbiome of asymptomatic and the pathobiome of foot-rot symptomatic field-grown rice plants over two growing periods and belonging to two rice cultivars were determined via 16S rRNA gene amplicon sequencing. Results showed that the presence of D. zeae is associated with an alteration of the resident bacterial community in terms of species composition, abundance and richness, leading to the formation of microbial consortia linked to the disease state. Several bacterial species were significantly co-presented with the pathogen in the two growing periods suggesting that they could be involved in the disease process. Besides, culture-dependent isolation and in planta inoculation studies of a bacterial member of the pathobiome, identified as positive correlated with the pathogen in our in silico analysis, indicated that it benefits from the presence of D. zeae. A similar microbiome/pathobiome experiment was also performed in a symptomatically different rice disease evidencing that not all plant diseases have the same consequence/relationship with the plant microbiome. This study moves away from a pathogen-focused stance and goes towards a more ecological perception considering the effect of the entire microbial community which could be involved in the pathogenesis, persistence, transmission and evolution of plant pathogens.

Rescue of spinal muscular atrophy mouse models with AAV9-Exon-specific U1 snRNA.

Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA.

In Planta Colonization and Role of T6SS in Two Rice Kosakonia Endophytes.

Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant-bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant-bacteria interaction.

Microprocessor-dependent processing of splice site overlapping microRNA exons does not result in changes in alternative splicing.

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These splice site overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in noncoding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were up-regulated during differentiation and the host mRNAs were down-regulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream from SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing.

TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are critical for the clearance of myelin debris in areas of demyelination, a key step to allow remyelination. TREM2 is expressed by microglia and promotes microglial survival, proliferation, and phagocytic activity. Herein we demonstrate that TREM2 was highly expressed on myelin-laden phagocytes in active demyelinating lesions in the CNS of subjects with MS. In gene expression studies, macrophages from subjects with TREM2 genetic deficiency displayed a defect in phagocytic pathways. Treatment with a new TREM2 agonistic antibody promoted the clearance of myelin debris in the cuprizone model of CNS demyelination. Effects included enhancement of myelin uptake and degradation, resulting in accelerated myelin debris removal by microglia. Most importantly, antibody-dependent TREM2 activation on microglia increased density of oligodendrocyte precursors in areas of demyelination, as well as the formation of mature oligodendrocytes thus enhancing remyelination and axonal integrity. These results are relevant as they propose TREM2 on microglia as a potential new target to promote remyelination.

Pathobiomes Revealed that Pseudomonas fuscovaginae and Sarocladium oryzae Are Independently Associated with Rice Sheath Rot.

Rice sheath rot has been mainly associated with the bacterial pathogen Pseudomonas fuscovaginae and in some cases to the fungal pathogen Sarocladium oryzae; it is yet unclear if they are part of a complex disease. The bacterial and fungal community associated with rice sheath rot symptomatic and asymptomatic rice plants was determined/studied with the main aim to shed light on the pathogen(s) causing rice sheath rot. Plant samples were collected from different rice varieties in two locations (highland and lowland) in two rice-growing seasons (wet and dry season) in Burundi. Our results showed that the bacterial Pseudomonas genus was prevalent in highland in both rice-growing seasons and was not affected by rice plant varieties. Pseudomonas sequence reads displayed a significant high similarity to Pseudomonas fuscovaginae indicating that it is the causal agent of rice sheath rot as previously reported. The fungal Sarocladium genus was on the other hand prevalent in lowland only in the wet season; the sequence reads were most significantly similar to Sarocladium oryzae. These studies showed that plant microbiome analysis is very useful in determining the microorganisms involved in a plant disease. P. fuscovaginae and S. oryzae were prevalent in symptomatic samples in highland and lowland respectively being present independently and hence are not part of a complex disease. The significant presence of other bacterial and fungal taxa in symptomatic samples is also discussed possibly making this disease more complex. Finally, we also report the microbial communities that are associated with the plant sheath in symptomatic and asymptomatic plants from the same rice fields.

DNA Methylome Distinguishes Head and Neck Cancer from Potentially Malignant Oral Lesions and Healthy Oral Mucosa.

There is a strong need to find new, good biomarkers of head and neck squamous cell carcinoma (HNSCC) because of the bad prognoses and high mortality rates. The aim of this study was to identify the potential biomarkers in HNSCC that have differences in their DNA methylome and potentially premalignant oral lesions, in comparison to healthy oral mucosa. In this study, 32 oral samples were tested: nine healthy oral mucosae, 13 HNSCC, and 10 oral lesions for DNA methylation by the Infinium MethylationEPIC BeadChip. Our findings showed that a panel of genes significantly hypermethylated in their promoters or specific sites in HNSCC samples in comparison to healthy oral samples, which are mainly oncogenes, receptor, and transcription factor genes, or genes included in cell cycle, transformation, apoptosis, and autophagy. A group of hypomethylated genes in HNSCC, in comparison to healthy oral mucosa, are mainly involved in the host immune response and transcriptional regulation. The results also showed significant differences in gene methylation between HNSCC and potentially premalignant oral lesions, as well as differently methylated genes that discriminate between oral lesions and healthy mucosa. The given methylation panels point to novel potential biomarkers for early diagnostics of HNSCC, as well as potentially premalignant oral lesions.

Hepatitis C Virus NS5A Targets Nucleosome Assembly Protein NAP1L1 To Control the Innate Cellular Response.

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A.IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.

Quorum Sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: Role in Virulence and Interspecies Interactions in the Olive Knot.

The olive knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi. P. savastanoi pv. savastanoi in the olive knot undergoes interspecies interactions with the harmless endophyte Erwinia toletana; P. savastanoi pv. savastanoi and E. toletana colocalize and form a stable community, resulting in a more aggressive disease. P. savastanoi pv. savastanoi and Etoletana produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal, and they share AHLs in planta In this work, we have further studied the AHL QS systems of P. savastanoi pv. savastanoi and Etoletana in order to determine possible molecular mechanism(s) involved in this bacterial interspecies interaction/cooperation. The AHL QS regulons of P. savastanoi pv. savastanoi and Etoletana were determined, allowing the identification of several QS-regulated genes. Surprisingly, the P. savastanoi pv. savastanoi QS regulon consisted of only a few loci whereas in Etoletana many putative metabolic genes were regulated by QS, among which are several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which was found to be essential for both colocalization of P. savastanoi pv. savastanoi and Etoletana cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome.IMPORTANCE This is a report on studies of the quorum sensing (QS) systems of the olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive knot cooperator Erwinia toletana These two bacterial species form a stable community in the olive knot, share QS signals, and cooperate, resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well as by studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease.