Test Groups, Not Individuals: A Review of the Pooling Approaches for SARS-CoV-2 Diagnosis.

Massive molecular testing for SARS-CoV-2 diagnosis is mandatory to manage the spread of COVID-19. Diagnostic screening should be performed at a mass scale, extended to the asymptomatic population, and repeated over time. An accurate diagnostic pipeline for SARS-CoV-2 that could massively increase the laboratory efficiency, while being sustainable in terms of time and costs, should be based on a pooling strategy. In the past few months, researchers from different disciplines had this same idea: test groups, not individuals. This critical review intends to highlight both the general consents-even if the results from different publications have been obtained with different protocols-and the points of disagreement that are creating some interpretative/comprehension difficulties. Different pooling schemes and technical aspects associated to the type of pooling adopted are described and discussed. We hope that this review can consolidate information to support researchers in designing optimized COVID-19 testing protocols based on pooling.

Informed sequential pooling approach to detect SARS-CoV-2 infection.

The alarming spread of the pandemic coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus requires several measures to reduce the risk of contagion. Every successful strategy in controlling the SARS-CoV-2 infection depends on timely diagnosis, which should include testing of asymptomatic carriers. Consequently, increasing the throughput for clinical laboratories for the purposes of conducting large-scale diagnostic testing is urgently needed. Here we support the hypothesis that standard diagnostic protocol for SARS-CoV-2 virus could be conveniently applied to pooled samples obtained from different subjects. We suggest that a two-step sequential pooling procedure could identify positive subjects, ensuring at the same time significant benefits of cost and time. The simulation data presented herein were used to assess the efficiency, in terms of number of required tests, both for random assignment of the subjects to the pools and for situations in which epidemiological and clinical data are used to create "informed" pools. Different scenarios were simulated to measure the effect of different pool sizes and different values for virus frequency. Our results allow for a customization of the pooling strategy according to the specific characteristics of the cohort being tested.

l-Arginine prevents inflammatory and pro-calcific differentiation of interstitial aortic valve cells.

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.

NETosis Delays Diabetic Wound Healing in Mice and Humans.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.

Lumican is overexpressed in lung adenocarcinoma pleural effusions.

Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value.

Identification of potential protein markers of noble rot infected grapes.

The evaluation of Botrytis cinerea as noble rot on withered grapes is of great importance to predict the wine sensory/organoleptic properties and to manage the winemaking process of Amarone, a passito dry red wine. This report describes the first proteomic analysis of grapes infected by noble rot under withering conditions to identify possible markers of fungal infection. 2-D gel electrophoresis revealed that protein profiles of infected and not infected grape samples are significantly different in terms of number of spots and relative abundance. Protein identification by MS analysis allowed to identify only in infected berries proteins of B. cinerea that represent potential markers of the presence of the fungus in the withered grapes.

Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.

CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.

OFFGEL fractionation of peptides: where really is your sample?

Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker.

AIMS/HYPOTHESIS: Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. METHODS: Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. RESULTS: Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. CONCLUSIONS/INTERPRETATION: We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

No peptide left behind: the "out of range" recovery in IPG-IEF fractionation.

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.

Improvements to polar 2-D electrophoresis for proteomic applications.

Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7 cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18-24 cm) on 2-PE and report some representative images for a qualitative assessment.

Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.

Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

Pros and cons of peptide isolectric focusing in shotgun proteomics.

In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations.

Roles of insulin, age, and asymmetric dimethylarginine on nitric oxide synthesis in vivo.

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after (15)N-arginine intravenous infusion and on asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations in conditions reportedly associated with altered NO availability, i.e., aging, hypertension, hypercholesterolemia, and type 2 diabetes mellitus (T2DM). A total of 26 male subjects (age 23-71 years, BMI 23-33 kg/m(2)), some of whom were affected by mixed pathologic features, were enrolled. NOx fractional synthesis rate (FSR) was lower in elderly (P < 0.015) and T2DM subjects (P < 0.03) than in matched control subjects. Hyperinsulinemia generally increased both NOx FSR and absolute synthesis rate (ASR) and reduced NOx, ADMA, and SDMA concentrations. Insulin sensitivity was impaired only in T2DM. With use of simple linear regression analysis across all subjects, age was inversely correlated with both NOx FSR (R(2) = 0.23, P < 0.015) and ASR (R(2) = 0.21, P < 0.02). NOx FSR inversely correlated with both ADMA and SDMA. With use of multiple regression analysis and various models, NOx FSR remained inversely associated with age and ADMA, whereas ASR was inversely associated with age and diabetes. No association with insulin sensitivity was found. We conclude that whole-body NOx production is decreased in aging and T2DM. Age, ADMA concentration, and T2DM, but not insulin resistance, appear as negative regulators of whole-body NOx production.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing.

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes.

Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach.

Antimicrobial photodynamic therapy (PDT) is a promising tool to combat antibiotic-resistant bacterial infections. During PDT, bacteria are killed by reactive oxygen species generated by a visible light absorbing photosensitizer (PS). We used a classical proteomic approach that included two-dimensional gel electrophoresis and mass spectrometry analysis, to identify some proteins of Staphylococcus aureus that are damaged during PDT with the cationic PS meso-tetra-4-N-methyl pyridyl porphine (T4). Suspensions of S. aureus cells were incubated with selected T4 concentrations and irradiated with doses of blue light that reduced the survival to about 60% or 1%. Proteomics analyses of a membrane proteins enriched fraction revealed that these sub-lethal PDT treatments affected the expression of several functional classes of proteins, and that this damage is selective. Most of these proteins were found to be involved in metabolic activities, in oxidative stress response, in cell division and in the uptake of sugar. Subsequent analyses revealed that PDT treatments delayed the growth and considerably reduced the glucose consumption capacity of S. aureus cells. This investigation provides new insights towards the characterization of PDT induced damage and mechanism of bacterial killing using, for the first time, a proteomic approach.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

Polar electrophoresis: shape of two-dimensional maps is as important as size.

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.