In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup

Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed.     

A novel polyclonal antibody library for expression profiling of poorly characterized, membrane and secreted human proteins

The YOMICS? antibody library (http://www.yomics.com/) presented in this article is a new collection of 1559 murine polyclonal antibodies specific for 1287 distinct human proteins. This antibody library is designed to target marginally characterized membrane-associated and secreted proteins. It was generated against human proteins annotated as transmembrane or secreted in GenBank, EnsEMBL, Vega and Uniprot databases, described in no or very few dedicated PubMed-linked publications. The selected proteins/protein regions were expressed in E. coli, purified and used to raise antibodies in the mouse. The capability of YOMICS? antibodies to specifically recognize their target proteins either as recombinant form or as expressed in cells and tissues was confirmed through several experimental approaches, including Western blot, confocal microscopy and immunohistochemistry (IHC). Moreover, to show the applicability of the library for biomarker investigation by IHC, five antibodies against proteins either known to be expressed in some cancers or homologous to tumor-associated proteins were tested on tissue microarrays carrying tumor and normal tissues from breast, colon, lung, ovary and prostate. A consistent differential expression in cancer was observed. Our results indicate that the YOMICS? antibody library is a tool for systematic protein expression profile analysis that nicely complements the already available commercial antibody collections.     

Diagnostic accuracy, effectiveness and cost for cognitive impairment and dementia screening of three short cognitive tests applicable to illiterates

Background

Illiteracy, a universal problem, limits the utilization of the most widely used short cognitive tests. Our objective was to assess and compare the effectiveness and cost for cognitive impairment (CI) and dementia (DEM) screening of three short cognitive tests applicable to illiterates.

Methods

Phase III diagnostic test evaluation study was performed during one year in four Primary Care centers, prospectively including individuals with suspicion of CI or DEM. All underwent the Eurotest, Memory Alteration Test (M@T), and Phototest, applied in a balanced manner. Clinical, functional, and cognitive studies were independently performed in a blinded fashion in a Cognitive Behavioral Neurology Unit, and the gold standard diagnosis was established by consensus of expert neurologists on the basis of these results. Effectiveness of tests was assessed as the proportion of correct diagnoses (diagnostic accuracy [DA]) and the kappa index of concordance (k) with respect to gold standard diagnoses. Costs were based on public prices at the time and hospital accounts.

Results

The study included 139 individuals: 47 with DEM, 36 with CI, and 56 without CI. No significant differences in effectiveness were found among the tests. For DEM screening: Eurotest (k = 0.71 [0.59–0.83], DA = 0.87 [0.80–0.92]), M@T (k = 0.72 [0.60–0.84], DA = 0.87 [0.80–0.92]), Phototest (k = 0.70 [0.57–0.82], DA = 0.86 [0.79–0.91]). For CI screening: Eurotest (k = 0.67 [0.55–0.79]; DA = 0.83 [0.76–0.89]), M@T (k = 0.52 [0.37–0.67]; DA = 0.80 [0.72–0.86]), Phototest (k = 0.59 [0.46–0.72]; DA = 0.79 [0.71–0.86]). There were no differences in the cost of DEM screening, but the cost of CI screening was significantly higher with M@T (330.7±177.1€, mean±sd) than with Eurotest (294.1±195.0€) or Phototest (296.0±196.5€). Application time was shorter with Phototest (2.8±0.8 min) than with Eurotest (7.1±1.8 min) or M@T (6.8±2.2 min).

Conclusions

Eurotest, M@T, and Phototest are equally effective. Eurotest and Phototest are both less expensive options but Phototest is the most efficient, requiring the shortest application time.     

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells

Background

Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P₂ in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane_. For controlled and reversible depletion of PtdIns(4,5)P₂, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.

Methods and Results

Expression and correct targeting of ECFP-PH-PLCδ1_, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P₂ in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K⁺ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P₂ was identified.

Conclusions

Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.     

PTPN22 1858C>T polymorphism distribution in Europe and association with rheumatoid arthritis: case-control study and meta-analysis

Objective

The PTPN22 rs2476601 polymorphism is associated with rheumatoid arthritis (RA); nonetheless, the association is weaker or absent in some southern European populations. The aim of the study was to evaluate the association between the PTPN22 rs2476601 polymorphism and RA in Italian subjects and to compare our results with those of other European countries, carrying out a meta-analysis of European data.

Methods

A total of 396 RA cases and 477 controls, all of Italic ancestry, were genotyped for PTPN22 rs2476601 polymorphism. Patients were tested for autoantibodies positivity. The meta-analysis was performed on 23 selected studies.

Results

The PTPN22 T1858 allele was significantly more frequent in RA patients compared to controls (5.7% vs. 3.7%, p = 0.045). No clear relationship arose with the autoantibodies tested. The 1858T allele frequency in Italian RA patients was lower than the one described in northern European populations and similar to the frequency found in Spain, Turkey, Greece, Tunisia. A clear-cut North-South gradient arose from the analysis.

Conclusions

The PTPN22 T1858 allele is associated with RA in the Italian population. A North-South gradient of the allele frequency seems to exist in Europe, with a lower prevalence of the mutation in the Mediterranean area.     

Recognition of morphometric vertebral fractures by artificial neural networks: analysis from GISMO Lombardia Database

Background

It is known that bone mineral density (BMD) predicts the fracture''s risk only partially and the severity and number of vertebral fractures are predictive of subsequent osteoporotic fractures (OF). Spinal deformity index (SDI) integrates the severity and number of morphometric vertebral fractures. Nowadays, there is interest in developing algorithms that use traditional statistics for predicting OF. Some studies suggest their poor sensitivity. Artificial Neural Networks (ANNs) could represent an alternative. So far, no study investigated ANNs ability in predicting OF and SDI. The aim of the present study is to compare ANNs and Logistic Regression (LR) in recognising, on the basis of osteoporotic risk-factors and other clinical information, patients with SDI≥1 and SDI≥5 from those with SDI = 0.

Methodology

We compared ANNs prognostic performance with that of LR in identifying SDI≥1/SDI≥5 in 372 women with postmenopausal-osteoporosis (SDI≥1, n = 176; SDI = 0, n = 196; SDI≥5, n = 51), using 45 variables (44 clinical parameters plus BMD). ANNs were allowed to choose relevant input data automatically (TWIST-system-Semeion). Among 45 variables, 17 and 25 were selected by TWIST-system-Semeion, in SDI≥1 vs SDI = 0 (first) and SDI≥5 vs SDI = 0 (second) analysis. In the first analysis sensitivity of LR and ANNs was 35.8% and 72.5%, specificity 76.5% and 78.5% and accuracy 56.2% and 75.5%, respectively. In the second analysis, sensitivity of LR and ANNs was 37.3% and 74.8%, specificity 90.3% and 87.8%, and accuracy 63.8% and 81.3%, respectively.

Conclusions

ANNs showed a better performance in identifying both SDI≥1 and SDI≥5, with a higher sensitivity, suggesting its promising role in the development of algorithm for predicting OF.     

Safety, immunogenicity and dose ranging of a new Vi-CRM₁₉₇ conjugate vaccine against typhoid fever: randomized clinical testing in healthy adults

Background

Typhoid fever causes more than 21 million cases of disease and 200,000 deaths yearly worldwide, with more than 90% of the disease burden being reported from Asia. Epidemiological data show high disease incidence in young children and suggest that immunization programs should target children below two years of age: this is not possible with available vaccines. The Novartis Vaccines Institute for Global Health developed a conjugate vaccine (Vi-CRM₁₉₇) for infant vaccination concomitantly with EPI vaccines, either starting at 6 weeks with DTP or at 9 months with measles vaccine. We report the results from a Phase 1 and a Phase 2 dose ranging trial with Vi-CRM₁₉₇ in European adults.

Methodology

Following randomized blinded comparison of single vaccination with either Vi-CRM₁₉₇ or licensed polysaccharide vaccines (both containing 25·0 µg of Vi antigen), a randomised observer blinded dose ranging trial was performed in the same center to compare three concentrations of Vi-CRM₁₉₇ (1·25 µg, 5·0 µg and 12·5 µg of Vi antigen) with the polysaccharide vaccine.

Principal Findings

All vaccines were well tolerated. Compared to the polysaccharide vaccine, Vi-CRM₁₉₇ induced a higher incidence of mild to moderate short lasting local pain. All Vi-CRM₁₉₇ formulations induced higher Vi antibody levels compared to licensed control, with clear dose response relationship.

Conclusions

Vi-CRM₁₉₇ did not elicit safety concerns, was highly immunogenic and is therefore suitable for further clinical testing in endemic populations of South Asia.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01123941","term_id":"NCT01123941"}}NCT01123941 {"type":"clinical-trial","attrs":{"text":"NCT01193907","term_id":"NCT01193907"}}NCT01193907     

Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement.