Performances of rapid and connected salivary RT-LAMP diagnostic test for SARS-CoV-2 infection in ambulatory screening.

In the context of social events reopening and economic relaunch, sanitary surveillance of SARS-CoV-2 infection is still required. Here, we evaluated the diagnostic performances of a rapid, extraction-free and connected reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay on saliva. Nasopharyngeal (NP) swabs and saliva from 443 outpatients were collected simultaneously and tested by reverse-transcription quantitative PCR (RT-qPCR) as reference standard test. Seventy-one individuals (16.0%) were positive by NP and/or salivary RT-qPCR. Sensitivity and specificity of salivary RT-LAMP were 85.9% (95%CI 77.8-94.0%) and 99.5% (98.7-100%), respectively. Performances were similar for symptomatic and asymptomatic participants. Moreover, SARS-CoV-2 genetic variants were analyzed and no dominant mutation in RT-LAMP primer region was observed during the period of the study. We demonstrated that this RT-LAMP test on self-collected saliva is reliable for SARS-CoV-2 detection. This simple connected test with optional automatic results transfer to health authorities is unique and opens the way to secure professional and social events in actual context of economics restart.

Genetic Characterization of Salmonella Infantis with Multiple Drug Resistance Profiles Isolated from a Poultry-Farm in Chile.

Salmonella comprises over 2500 serotypes and foodborne contamination associated with this pathogen remains an important health concern worldwide. During the last decade, a shift in serotype prevalence has occurred as traditionally less prevalent serotypes are increasing in frequency of infections, especially those related to poultry meat contamination. S. Infantis is one of the major emerging serotypes, and these strains commonly display antimicrobial resistance and can persist despite cleaning protocols. Thus, this work aimed to isolate S. Infantis strains from a poultry meat farm in Santiago, Chile and to characterize genetic variations present in them. We determined their genomic and phenotypic profiles at different points along the production line. The results indicate that the strains encompass 853 polymorphic sites (core-SNPs) with isolates differing from one another by 0-347 core SNPs, suggesting variation among them; however, we found discrete correlations with the source of the sample in the production line. Furthermore, the pan-genome was composed of 4854 total gene clusters of which 2618 (53.9%) corresponds to the core-genome and only 181 (3.7%) are unique genes (those present in one particular strain). This preliminary analysis will enrich the surveillance of Salmonella, yet further studies are required to assess their evolution and phylogeny.

Arsenic Response of Three Altiplanic Exiguobacterium Strains With Different Tolerance Levels Against the Metalloid Species: A Proteomics Study.

Exiguobacterium is a polyextremophile bacterial genus with a physiology that allows it to develop in different adverse environments. The Salar de Huasco is one of these environments due to its altitude, atmospheric pressure, solar radiation, temperature variations, pH, salinity, and the presence of toxic compounds such as arsenic. However, the physiological and/or molecular mechanisms that enable them to prosper in these environments have not yet been described. Our research group has isolated several strains of Exiguobacterium genus from different sites of Salar de Huasco, which show different resistance levels to As(III) and As(V). In this work, we compare the protein expression patterns of the three strains in response to arsenic by a proteomic approach; strains were grown in absence of the metalloid and in presence of As(III) and As(V) sublethal concentrations and the protein separation was carried out in 2D electrophoresis gels (2D-GE). In total, 999 spots were detected, between 77 and 173 of which showed significant changes for As(III) among the three strains, and between 90 and 143 for As(V), respectively, compared to the corresponding control condition. Twenty-seven of those were identified by mass spectrometry (MS). Among these identified proteins, the ArsA [ATPase from the As(III) efflux pump] was found to be up-regulated in response to both arsenic conditions in the three strains, as well as the Co-enzyme A disulfide reductase (Cdr) in the two more resistant strains. Interestingly, in this genus the gene that codifies for Cdr is found within the genic context of the ars operon. We suggest that this protein could be restoring antioxidants molecules, necessary for the As(V) reduction. Additionally, among the proteins that change their expression against As, we found several with functions relevant to stress response, e.g., Hpf, LuxS, GLpX, GlnE, and Fur. This study allowed us to shed light into the physiology necessary for these bacteria to be able to tolerate the toxicity and stress generated by the presence of arsenic in their niche.

Local carotid atherosclerotic plaque proteins for the identification of circulating biomarkers in coronary patients.

OBJECTIVE: To identify circulating biomarkers that originate from atherosclerotic vulnerable plaques and that could predict future cardiovascular events. METHODS: After a protein enrichment step (combinatorial peptide ligand library approach), we performed a two-dimensional electrophoresis comparative analysis on human carotid plaque protein extracts (fibrotic and hemorrhagic atherosclerotic plaques). In silico analysis of the biological processes was applied on proteomic data. Luminex xMAP assays were used to quantify inflammatory components in carotid plaques. The systemic quantification of proteins originating from vulnerable plaques in blood samples from patients with stable and unstable coronary disease was evaluated. RESULTS: A total of 118 proteins are differentially expressed in fibrotic and hemorrhagic plaques, and allowed the identification of three biological processes related to atherosclerosis (platelet degranulation, vascular autophagy and negative regulation of fibrinolysis). The multiplex assays revealed an increasing expression of VEGF, IL-6, IL-8, IP-10 and RANTES in hemorrhagic as compared to fibrotic plaques (p<0.05). Measurement of protein expressions in plasmas from patients with stable and unstable coronary disease identified a combination of biomarkers, including proteins of the smooth muscle cell integrity (Calponin-1), oxidative stress (DJ-1) and inflammation (IL-8), that allows the accurate classification of patients at risk (p=0.0006). CONCLUSION: Using tissue protein enrichment technology, we validated proteins that are differentially expressed in hemorrhagic plaques as potential circulating biomarkers of coronary patients. Combinations of such circulating biomarkers could be used to stratify coronary patients.

Tmprss3 loss of function impairs cochlear inner hair cell Kcnma1 channel membrane expression.

Before acquiring their mature state, cochlear hair cells undergo a series of changes in expression of ion channels. How this complex mechanism is achieved is not fully understood. Tmprss3, a type II serine protease expressed in hair cells, is required for their proper functioning at the onset of hearing. To unravel the role of Tmprss3 in the acquisition of mature K(+) currents, we compared their function by patch-clamp technique in wild-type Tmprss3(WT) and Tmprss3(Y260X)-mutant mice. Interestingly, only outward K(+) currents were altered in Tmprss3(Y260X)-mutant mice. To determine by which mechanism this occurred, we compared the protein network of Tmprss3(WT) and Tmprss3(Y260X)-mutant mice using proteomic analysis. This led to the identification of a pathway related to potassium Kcnma1 channels. This pathway was validated by immunohistochemistry, focusing on the most downregulated protein that was identified as a cochlear Kcnma1-associated protein, APOA1. Finally, we show that, in contrast to Tmprss3(WT), Kcnma1 channels were absent at the neck of inner hair cells (IHCs) in Tmprss3(Y260X)-mutant mice. In conclusion, our data suggest that lack of Tmprss3 leads to a decrease in Kcnma1 potassium channels expression in (IHCs).

Carotid atherosclerotic plaques: proteomics study after a low-abundance protein enrichment step.

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin ß-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual.

Combined measurement of PEDF, haptoglobin and tau in cerebrospinal fluid improves the diagnostic discrimination between alzheimer's disease and other dementias.

Using proteomic approach in cerebrospinal fluid (CSF) we identified pigment epithelium-derived factor (PEDF) and Haptoglobin (Hp) as putative markers that could discriminate between AD and other dementias. ELISA assays were developed to measure the levels of PEDF and Hp in CSF from patients with AD (AD, n=27), non-AD (NAD, n=30) and in non-demented patients (ND, n=27). The combined assessment of PEDF, Hp and Tau levels, using Iterative Marginal Optimization, improved the differential diagnosis of AD, especially in patients with moderate to severe dementia (p<0.002). This pilot study highlights the probable different contribution of oxidative mechanisms in dementia.

A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics.

BACKGROUND: In current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user. RESULTS: We present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP) for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.). We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects. CONCLUSION: Sili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments.

Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope.

HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.

Pharmacoproteomic approach to the study of drug mode of action, toxicity, and resistance: applications in diabetes and cancer.

Proteomics is a powerful technique for investigating protein expression profiles in biological systems and their modifications in response to stimuli or to particular physiological or pathophysiological conditions. It is therefore a technique of choice for the study of drug mode of action, side-effects, toxicity and resistance. It is also a valuable approach for the discovery of new drug targets. All these proteomic applications to pharmacological issues may be called pharmacoproteomics. The pharmacoproteomic approach could be particularly useful for the identification of molecular alterations implicated in type 2 diabetes and for further characterization of existing or new drugs. In oncology, proteomics is widely used for the identification of tumour-specific protein markers, and pharmacoproteomics is used for the evaluation of chemotherapy, particularly for the characterization of drug-resistance mechanisms. The large amount of data generated by pharmacoproteomic screening requires the use of bioinformatic tools to insure a pertinent interpretation. Herein, we review the applications of pharmacoproteomics to the study of type 2 diabetes and to chemoresistance in different types of cancer and the current state of this technology in these pathologies. We also suggest a number of bioinformatic solutions for proteomic data management.

Hyaluronan, a major non-protein glycosaminoglycan component of the extracellular matrix in human bone marrow, mediates dexamethasone resistance in multiple myeloma.

Originating from a post-switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant multiple myeloma (MM) cells accumulate in the bone marrow of patients with MM. In this favourable microenvironment, their growth and survival are dependent upon both soluble factors and physical cell-to-cell and cell-to-extracellular-matrix contacts. In this study, hyaluronan (HA), a major non-protein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, acted as a survival factor against dexamethasone (Dex)-induced apoptosis in MM cell lines. These effects were mediated through an interleukin 6 (IL-6) autocrine pathway, involving signal transducers and activators of transcription-3 phosphorylation on IL-6-dependent XG-1 and XG-6 cell lines. HA promoted accumulation of IL-6 in the culture medium without affecting IL-6 gene expression, suggesting that HA protects, stabilizes and concentrates IL-6 close to its site of secretion, thus favouring its autocrine activity. In contrast, in the IL-6-independent RPMI8226 cell line, HA survival effect was mediated through a gp80-IL-6 receptor-independent pathway, resulting in the upregulation of Bcl-2 anti-apoptotic protein expression and nuclear factor-kappaB activation. Taken together, these data suggest that HA antagonizes Dex-induced apoptosis of MM cells by favouring the autocrine activity of different cytokines or growth factors. As HA is a major component of the bone marrow extracellular matrix, these findings support the idea that HA could play a major role in the survival of MM cells in vivo, and could explain why MM cells accumulate in the bone marrow of patients with MM and escape conventional chemotherapy.

Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies.

To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.