A Complex Proteomic Response of the Parasitic Nematode Anisakis simplex s.s. to Escherichia coliLipopolysaccharide.

Helminths are masters at manipulating host's immune response. Especially, parasitic nematodes have evolved strategies that allow them to evade, suppress, or modulate host's immune response to persist and spread in the host's organism. While the immunomodulatory effects of nematodes on their hosts are studied with a great commitment, very little is known about nematodes' own immune system, immune response to their pathogens, and interactions between parasites and bacteria in the host's organism. To illustrate the response of the parasitic nematode Anisakis simplex s.s. during simulated interaction with Escherichia coli, different concentrations of lipopolysaccharide (LPS) were used, and the proteomic analysis with isobaric mass tags for relative and absolute quantification (tandem mass tag-based LC-MS/MS) was performed. In addition, gene expression and biochemical analyses of selected markers of oxidative stress were determined. The results revealed 1148 proteins in a group of which 115 were identified as differentially regulated proteins, for example, peroxiredoxin, thioredoxin, and macrophage migration inhibitory factor. Gene Ontology annotation and Reactome pathway analysis indicated that metabolic pathways related to catalytic activity, oxidation-reduction processes, antioxidant activity, response to stress, and innate immune system were the most common, in which differentially regulated proteins were involved. Further biochemical analyses let us confirm that the LPS induced the oxidative stress response, which plays a key role in the innate immunity of parasitic nematodes. Our findings, to our knowledge, indicate for the first time, the complexity of the interaction of parasitic nematode, A. simplex s.s. with bacterial LPS, which mimics the coexistence of helminth and gut bacteria in the host. The simulation of this crosstalk led us to conclude that the obtained results could be hugely valuable in the integrated systems biology approach to describe a relationship between parasite, host, and its commensal bacteria.

Analysis of changes in the proteomic profile of porcine corpus luteum during different stages of the oestrous cycle: effects of PPAR gamma ligands.

CONTEXT: The corpus luteum (CL) is an endocrine gland in the ovary of mature females during the oestrous cycle and pregnancy. There is evidence of a relationship between the secretory function of the CL and PPARs. AIMS: In this study, we investigated the changes in the proteome of the CL in relation to the phase of the oestrous cycle and the impact of PPARγ ligands on the proteomic profile of the CL during the mid- and late-luteal phase of the oestrous cycle. METHODS: The porcine CL explants were incubated in vitro for 6h in the presence of PPARγ ligands (agonist pioglitazone, antagonist T0070907) or without ligands. Global proteomic analysis was performed using the TMT-based LC-MS/MS method. KEY RESULTS: The obtained results showed the disparity in proteomic profile of the untreated CL - different abundance of 23 and 28 proteins for the mid- and late-luteal phase, respectively. Moreover, seven proteins were differentially regulated in the CL tissue treated with PPARγ ligands. In the mid-luteal phase, one protein, CAND1, was downregulated after treatment with T0070907. In the late-luteal phase, the proteins SPTAN1, GOLGB1, TP53BP1, MATR3, RRBP1 and SRRT were upregulated by pioglitazone. CONCLUSIONS: Comparative proteomic analysis revealed that certain proteins constitute a specific proteomic signature for each examined phase. Moreover, the study showed that the effect of PPARγ ligands on the CL proteome was rather limited. IMPLICATIONS: The results provide a broader insight into the processes that may be responsible for the structural luteolysis of the porcine CL, in addition to apoptosis and autophagy.

Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis.

Anisakis simplex s. s. is a parasitic nematode of marine mammals and causative agent of anisakiasis in humans. The cuticle and intestine of the larvae are the tissues most responsible for direct and indirect contact, respectively, of the parasite with the host. At the L4 larval stage, tissues, such as the cuticle and intestine, are fully developed and functional, in contrast to the L3 stage. As such, this work provides for the first time the tissue-specific proteome of A. simplex s. s. larvae in the L4 stage. Statistical analysis (FC ≥ 2; p-value ≤ 0.01) showed that 107 proteins were differentially regulated (DRPs) between the cuticle and the rest of the larval body. In the comparison between the intestine and the rest of the larval body at the L4 stage, 123 proteins were identified as DRPs. Comparison of the individual tissues examined revealed a total of 272 DRPs, with 133 proteins more abundant in the cuticle and 139 proteins more abundant in the intestine. Detailed functional analysis of the identified proteins was performed using bioinformatics tools. Glycolysis and the tricarboxylic acid cycle were the most enriched metabolic pathways by cuticular and intestinal proteins, respectively, in the L4 stage of A. simplex s. s. The presence of two proteins, folliculin (FLCN) and oxoglutarate dehydrogenase (OGDH), was confirmed by Western blot, and their tertiary structure was predicted and compared with other species. In addition, host-pathogen interactions were identified, and potential new allergens were predicted. The result of this manuscript shows the largest number of protein identifications to our knowledge using proteomics tools for different tissues of L4 larvae of A. simplex s. s. The identified tissue-specific proteins could serve as targets for new drugs against anisakiasis.

Recent Advances in Proteomics-Based Approaches to Studying Age-Related Macular Degeneration: A Systematic Review.

Age-related macular degeneration (AMD) is a common ocular disease characterized by degeneration of the central area of the retina in the elderly population. Progression and response to treatment are influenced by genetic and non-genetic factors. Proteomics is a powerful tool to study, at the molecular level, the mechanisms underlying the progression of the disease, to identify new therapeutic targets and to establish biomarkers to monitor progression and treatment effectiveness. In this work, we systematically review the use of proteomics-based approaches for the study of the molecular mechanisms underlying the development of AMD, as well as the progression of the disease and on-treatment patient monitoring. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) reporting guidelines were followed. Proteomic approaches have identified key players in the onset of the disease, such as complement components and proteins involved in lipid metabolism and oxidative stress, but also in the progression to advanced stages, including factors related to extracellular matrix integrity and angiogenesis. Although anti-vascular endothelial growth factor (anti-VEGF)-based therapy has been crucial in the treatment of neovascular AMD, it is necessary to deepen our understanding of the underlying disease mechanisms to move forward to next-generation therapies for later-stage forms of this multifactorial disease.

Is the "Habsburg jaw" related to inbreeding?

Background: The "Habsburg jaw" has long been associated with inbreeding due to the high prevalence of consanguineous marriages in the Habsburg dynasty. However, it is thought that mandibular prognathism (MP) is under the influence of a dominant major gene.Aim: To investigate the relationship between the "Habsburg jaw" and the pedigree-based inbreeding coefficient (F) as a relative measure of genome homozygosity.Subjects and methods: The degree of MP and maxillary deficiency (MD) of 15 members of the Habsburg dynasty was quantified through the clinical analysis of 18 dysmorphic features diagnosed from 66 portraits.Results: A statistically significant correlation (r = 0.711, p = 0.003) between MP and MD was observed among individuals. Only MP showed a statistically significant positive regression on F as evidenced from univariate analysis (b = 6.36 ± 3.34, p = 0.040) and multivariate analysis (PCA) performed from single dysmorphic features (b = 14.10 ± 6.62, p = 0.027, for the first PC).Conclusion: Both MP and MD are generally involved in the "Habsburg jaw." The results showed a greater sensitivity to inbreeding for the lower third of the face and suggest a positive association between the "Habsburg jaw" and homozygosity and therefore a basically recessive inheritance pattern.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis.

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.

3, 3', 5-triiodo-L-thyronine Increases In Vitro Chondrogenesis of Mesenchymal Stem Cells From Human Umbilical Cord Stroma Through SRC2.

Our group focuses on the study of mesenchymal stem cells (MSCs) from human umbilical cord stroma or Warthons jelly and their directed differentiation toward chondrocyte-like cells capable of regenerating damaged cartilage when transplanted into an injured joint. This study aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 3', 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3ß gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. J. Cell. Biochem. 117: 2097-2108, 2016. © 2016 Wiley Periodicals, Inc.

Quantitative proteomic analysis of host--pathogen interactions: a study of Acinetobacter baumannii responses to host airways.

BACKGROUND: Acinetobacter baumannii is a major health problem. The most common infection caused by A. baumannii is hospital acquired pneumonia, and the associated mortality rate is approximately 50%. Neither in vivo nor ex vivo expression profiling has been performed at the proteomic or transcriptomic level for pneumonia caused by A. baumannii. In this study, we characterized the proteome of A. baumannii under conditions that simulate those found in the airways, to gain some insight into how A. baumannii adapts to the host and to improve knowledge about the pathogenesis and virulence of this bacterium. A clinical strain of A. baumannii was grown under different conditions: in the presence of bronchoalveolar lavage fluid from infected rats, of RAW 264.7 cells to simulate conditions in the respiratory tract and in control conditions. We used iTRAQ labelling and LC-MALDI-TOF/TOF to investigate how A. baumannii responds on exposure to macrophages/BALF. RESULTS: 179 proteins showed differential expression. In both models, proteins involved in the following processes were over-expressed: (i) pathogenesis and virulence (OmpA, YjjK); (ii) cell wall/membrane/envelope biogenesis (MurC); (iii) energy production and conversion (acetyl-CoA hydrolase); and (iv) translation (50S ribosomal protein L9). Proteins involved in the following were under-expressed: (i) lipid metabolism (short-chain dehydrogenase); (ii) amino acid metabolism and transport (aspartate aminotransferase); (iii) unknown function (DNA-binding protein); and (iv) inorganic ion transport and metabolism (hydroperoxidase). CONCLUSIONS: We observed alterations in cell wall synthesis and identified 2 upregulated virulence-associated proteins with >15 peptides/protein in both ex vivo models (OmpA and YjjK), suggesting that these proteins are fundamental for pathogenesis and virulence in the airways. This study is the first comprehensive overview of the ex vivo proteome of A. baumannii and is an important step towards identification of diagnostic biomarkers, novel drug targets and potential vaccine candidates in the fight against pneumonia caused by A. baumannii.

Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA.

Cryoconservation of peptide extracts from trypsin digestion of proteins for proteomic analysis in a hospital biobank facility.

We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Low-complexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC-MALDI-TOF/TOF analysis, purified ß-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated -20 °C freezer in the Biobank facility. Samples were kept dried at -20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC-MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins - serum albumin and serotransferrin - were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC-MS/MS, ß-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage - up to 3 months - of dried peptide extracts.

Molecular mechanisms involved in the response to desiccation stress and persistence in Acinetobacter baumannii.

Desiccation tolerance contributes to the maintenance of bacterial populations in hospital settings and may partly explain its propensity to cause outbreaks. Identification and relative quantitation of proteins involved in bacterial desiccation tolerance was made using label-free quantitation and iTRAQ labeling. Under desiccating conditions, the population of the Acinetobacter baumannii clinical strain AbH12O-A2 decreased in the first week, and thereafter, a stable population of 0.5% of the original population was maintained. Using label-free quantitation and iTRAQ labeling, 727 and 765 proteins, respectively, were detected; 584 of them by both methods. Proteins overexpressed under desiccation included membrane and periplasmic proteins. Proteins associated with antimicrobial resistance, efflux pumps, and quorum quenching were overexpressed in the samples subjected to desiccation stress. Electron microscopy revealed clear morphological differences between desiccated and control bacteria. We conclude that A. baumannii is able to survive long periods of desiccation through the presence of cells in a dormant state, via mechanisms affecting control of cell cycling, DNA coiling, transcriptional and translational regulation, protein stabilization, antimicrobial resistance, and toxin synthesis, and that a few surviving cells embedded in a biofilm matrix are able to resume growth and restore the original population in appropriate environmental conditions following a "bust-and-boom" strategy.

Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells.

Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.

Pharmacoproteomic study of three different chondroitin sulfate compounds on intracellular and extracellular human chondrocyte proteomes.

Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 µg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies.

Liver stiffness predicts clinical outcome in human immunodeficiency virus/hepatitis C virus-coinfected patients with compensated liver cirrhosis.

UNLABELLED: Our aim was to assess the predictive value of liver stiffness (LS), measured by transient elastography (TE), for clinical outcome in human immunodeficiency virus / hepatitis C virus (HIV/HCV)-coinfected patients with compensated liver cirrhosis. This was a prospective cohort study of 239 consecutive HIV/HCV-coinfected patients with a new diagnosis of cirrhosis, done by TE, and no previous decompensation of liver disease. The time from diagnosis to the first liver decompensation and death from liver disease, as well as the predictors of these outcomes, were evaluated. After a median (Q1-Q3) follow-up of 20 (9-34) months, 31 (13%, 95% confidence interval [CI]: 9%-17%) patients developed a decompensation. The incidence of decompensation was 6.7 cases per 100 person-years (95% CI, 4.7-9-6). Fourteen (8%) out of 181 patients with a baseline LS < 40 kPa developed a decompensation versus 17 (29%) out of 58 with LS ≥ 40 kPa (P = 0.001). Factors independently associated with decompensation were Child-Turcotte-Pugh (CTP) class B versus A (hazard ratio [HR] 7.7; 95% CI 3.3-18.5; P < 0.0001), log-plasma HCV RNA load (HR 2.1; 95% CI 1.2-3.6; P = 0.01), hepatitis B virus coinfection (HR, 10.3; 95% CI, 2.1-50.4; P = 0.004) and baseline LS (HR 1.03; 95% CI 1.01-1.05; P = 0.02). Fifteen (6%, 95% CI: 3.5%-9.9%) patients died, 10 of them due to liver disease, and one underwent liver transplantation. CTP class B (HR 16.5; 95% CI 3.4-68.2; P < 0.0001) and previous exposure to HCV therapy (HR 7.4; 95% CI 1.7-32.4, P = 0.007) were independently associated with liver-related death; baseline LS (HR 1.03; 95% CI 0.98-1.07; P = 0.08) was of borderline significance. CONCLUSION: LS predicts the development of hepatic decompensations and liver-related mortality in HIV/HCV-coinfection with compensated cirrhosis and provides additional prognostic information to that provided by the CTP score.

Dimethylarginine dimethylaminohydrolase 2, a newly identified mitochondrial protein modulating nitric oxide synthesis in normal human chondrocytes.

OBJECTIVE: The mitochondrion is known to be important to chondrocyte survival. This study was undertaken to analyze protein expression profiles in chondrocyte mitochondria that are affected by interleukin-1ß (IL-1ß). METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from subjects with no history of joint disease. Cells were incubated for 48 hours with or without IL-1ß (5 ng/ml). Proteins were separated by 2-dimensional electrophoresis and stained with Sypro Ruby, Coomassie brilliant blue, or silver. Qualitative and quantitative analyses were carried out using PDQuest software. Proteins were identified by mass spectrometry using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology. The proteomic results were validated by real-time polymerase chain reaction, Western blotting, and microscopy. Nitric oxide (NO) was quantified using Griess reagent. RESULTS: Comparative analysis revealed differential expression of signal transduction proteins that regulate cytoskeleton, transcription, metabolic, and stress-related pathways. In total extracts, dimethylarginine dimethylaminohydrolase 2 (DDAH-2) did not show any change in expression after stimulation with IL-1ß. However, in mitochondrial extracts, DDAH-2 expression was significantly increased after exposure to IL-1ß. Conventional immunofluorescence and confocal microscopy revealed the presence of DDAH-2 in the mitochondria of IL-1ß-stimulated chondrocytes. These results were reproducible in cartilage explants treated with IL-1ß. In addition, we demonstrated that inhibition of the expression of DDAH-2, as well as interruption of its translocation to the mitochondria, reduced the NO production induced by IL-1ß. DDAH-2 protein expression was higher in osteoarthritic (OA) cartilage than in normal cartilage. CONCLUSION: In the present study, the presence of DDAH-2 in normal human chondrocytes and cartilage was identified for the first time. DDAH-2 could play an important role in IL-1ß-induced NO production and in OA pathogenesis.

Sustained virological response to pegylated interferon plus ribavirin leads to normalization of liver stiffness in hepatitis C virus-infected patients.

INTRODUCTION: Pegylated interferon plus ribavirin (Peg-IFN/RBV) therapy leads to improvements in liver stiffness measurements (LSM) in hepatitis C virus (HCV)-infected patients. However, the rate of LSM return to normal values in response to Peg-IFN/RBV is unclear. Thus, our aim was to assess the probability and factors associated with LSM normalization in HCV-infected patients receiving Peg-IFN/RBV. METHODS: This prospective observational longitudinal study included 160 HCV-infected patients, 111 (69%) with human immunodeficiency virus and receiving Peg-IFN/RBV, with baseline LSM ≥ 7kPa. The outcome variable was LSM normalization, i.e. a stable decrease in LSM below 7kPa after starting Peg-IFN/RBV. RESULTS: After starting Peg-IFN/RBV, 56 [35%, 95% confidence interval (95% CI): 28-42%] patients showed LSM normalization. The probability of LSM normalization was 21% (95% CI: 13.2-32.4%) at 12 months, and 51.3% (95% CI: 39.9-63.9%) at 24 months after Peg-INF/RBV initiation for individuals with sustained virological response (SVR), and 8.3% (95% CI: 4-16.6%) at 12 months and 11.3% (95% CI: 6-20.7%) at 24 months for those without SVR (p<0.001). For individuals with LSM ≥ 7kPa 24 weeks after the pre-planned end of treatment, LSM normalizations were only observed among those with SVR. Achievement of SVR [Hazard ratio (HR, 95% CI): 6.84 (3.39-13.81)] and lack of baseline cirrhosis [HR (95% CI): 4.17 (1.69-10)] were independently associated with LSM normalization after starting Peg-IFN/RBV. CONCLUSIONS: LSM normalizations during Peg-IFN/RBV treatment are more likely, and occur earlier among patients with SVR. In addition, LSM normalizations continue 24 weeks after the scheduled end of therapy, but only among individuals who reach SVR.

Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior.

BACKGROUND: A challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation. METHODS: Mesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP. RESULTS: Syndecan-1 (SDC1) was the most decreased protein in MSC-miR21--derived EV, and it was involved in inflammation and EV production. MSC-miR21--derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV. CONCLUSION: This work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.

Ampicillin Stability in a Portable Elastomeric Infusion Pump: A Step Forward in Outpatient Parenteral Antimicrobial Therapy.

Outpatient parenteral antimicrobial therapy (OPAT) with continuous infusion pumps is postulated as a very promising solution to treat complicated infections, such as endocarditis or osteomyelitis, that require patients to stay in hospital during extended periods of time, thus reducing their quality of life and increasing the risk of complications. However, stability studies of drugs in elastomeric devices are scarce, which limits their use in OPAT. Therefore, we evaluated the stability of ampicillin in sodium chloride 0.9% at two different concentrations, 50 and 15 mg/mL, in an elastomeric infusion pump when stored in the refrigerator and subsequently in real-life conditions at two different temperatures, 25 and 32 °C, with and without the use of a cooling device. The 15 mg/mL ampicillin is stable for up to 72 h under refrigeration, allowing subsequent dosing at 25 °C for 24 h with and without a cooling device, but at 32 °C its concentration drops below 90% after 8 h. In contrast, 50 mg/mL ampicillin only remains stable for the first 24 h under refrigeration, and subsequent administration at room temperature is not possible, even with the use of a cooling system. Our data support that 15 mg/mL AMP is suitable for use in OPAT if the volume and rate of infusion are tailored to the dosage needs of antimicrobial treatments.

Metabolic labeling of human bone marrow mesenchymal stem cells for the quantitative analysis of their chondrogenic differentiation.

Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.

Extracellular proteome of a highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii.

The study of the extracellular proteomes of pathogenic bacteria is essential for gaining insights into the mechanisms of pathogenesis and for the identification of virulence factors. Through the use of different proteomic approaches, namely Nano-LC and 2DE combined with MALDI-TOF/TOF, we have characterized the extracellular proteome of a highly invasive, multidrug-resistant strain of A. baumannii (clone AbH12O-A2). This study focused on two main protein fractions of the extracellular proteome: proteins that are exported by outer membrane vesicles (OMVs) and freely soluble extracellular proteins (FSEPs) present in the culture medium of A. baumannii. Herein, a total of 179 nonredundant proteins were identified in the OMV protein fraction and a total of 148 nonredundant proteins were identified in FSEP fraction. Of the OMV proteins, 39 were associated with pathogenesis and virulence, including proteins associated with attachment to host cells (e.g., CsuE, CsuB, CsuA/B) and specialized secretion systems for delivery of virulence factors (e.g., P. pilus assembly and FilF), whereas the FSEP fraction possesses extracellular enzymes with degradative activity, such as alkaline metalloprotease. Furthermore, among the FSEP we have detected at least 18 proteins with a known role in oxidative stress response (e.g., catalase, thioredoxin, oxidoreductase, superoxide dismutase). Further assays demonstrated that in the presence of FSEPs, bacterial cells withstand much higher concentrations of H2O2 showing higher survival rate (approximately 2.5 fold) against macrophages. In this study we have identified an unprecedented number of novel extracellular proteins of A. baumannii and we provide insight into their potential role in relevant processes such as oxidative stress response and defense against macrophage attack.