Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.     

TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.     

The influence of peroxisome proliferator-activated receptor γ(1) during differentiation of mouse embryonic stem cells to neural cells

Peroxisome proliferator activated receptor γ, belongs to PPARs, which exerts various metabolic functions including differentiation process. To testify the importance of PPARγ in neural differentiation of mouse embryonic stem cells (mESCs), its expression level was assessed. Data revealed an elevation in expression level of PPARγ when neural precursors (NPs) are formed upon retinoic acid treatment. Thus, involvement of PPARγ in two stages of neural differentiation of mESCs, during and post-NPs formation was examined by application of its agonist and antagonist. Our results indicated that PPARγ inactivation via treatment with GW9662 during NPs formation, reduced expression of neural precursor and neural (neuronal and astrocytes) markers. However, PPARγ inactivation by antagonist treatment post-NPs formation stage only decreased the expression of mature astrocyte marker (Gfap) suggesting that inactivation of PPARγ by antagonist decreased astrocyte differentiation. Here, we have demonstrated the stage dependent role of PPARγ modulation on neural differentiation of mESCs by retinoic acid treatment for the first time.     

Development of an optimal formulation for oxidative stability of walnut-beverage emulsions based on gum arabic and xanthan gum using response surface methodology

The susceptibility of lipids to oxidation is one of the most fundamental problems in oil-in-water emulsions. A response surface methodology 5-level-3-factor central-composite rotatable design was applied to study the effects of key formula ingredients including walnut oil (WO, 3-6%, w/w), gum arabic (GA, 5-10%, w/w) and xanthan gum (XG, 0.05-0.15%, w/w) on lipid oxidation in walnut-beverage emulsions. During 30 days’ storage, the oxidation process was monitored by evaluating the peroxide value, anisidine value and total oxidation (Totox) value in different emulsion formulations. Use of XG as a stabilizer at high concentrations considerably inhibited the oxidation of WO in the prepared emulsions. The experimental data were satisfactorily fitted to quadratic models using multiple regression analysis. The optimum conditions to obtain the minimum peroxide (0.923 mequiv. O₂/kg oil), anisidine (0.500) and Totox (2.347) values are met when a walnut-beverage emulsion is formulated with 3% WO, 10% GA and 0.12% XG.     

Phylogenetic and population genetic analyses of Phaeosphaeria nodorum and its close relatives indicate cryptic species and an origin in the Fertile Crescent

The origin of the fungal wheat pathogen Phaeosphaeria nodorum remains unclear despite earlier intensive global population genetic and phylogeographical studies. We sequenced 1683 bp distributed across three loci in 355 globally distributed Phaeosphaeria isolates, including 74 collected in Iran near the center of origin of wheat. We identified nine phylogenetically distinct clades, including two previously unknown species tentatively named P1 and P2 collected in Iran. Coalescent analysis indicates that P1 and P2 are sister species of P. nodorum and the other Phaeosphaeria species identified in our analysis. Two species, P. nodorum and P. avenaria f. sp. tritici 1 (Pat1), comprised ～85% of the sampled isolates, making them the dominant wheat-infecting pathogens within the species complex. We designed a PCR-RFLP assay to distinguish P. nodorum from Pat1. Approximately 4% of P. nodorum and Pat1 isolates showed evidence of hybridization. Measures of private allelic richness at SSR and sequence loci suggest that the center of origin of P. nodorum coincides with its host in the Fertile Crescent. We hypothesize that the origin of this species complex is also in the Fertile Crescent, with four species out of nine found exclusively in the Iranian collections.     

Chlorophyll fluorescence as a tool for evaluation of drought stress in strawberry

The effect of water deficit on chlorophyll fluorescence, sugar content, and growth parameters of strawberry (Fragaria×ananassa Duch. cv. Elsanta) was studied. Drought stress caused significant reductions in leaf water potential, fresh and dry masses, leaf area, and leaf number. A gradual reduction of photochemical quenching (q_P) and quantum efficiency (Φ_PS2) was observed under drought stress while non-photochemical quenching (q_N) increased. Maximum efficiency of photosystem 2 (F_v/F_m) was not affected by drought stress.     

Comparison of Carnoy's solution and 96% ethyl alcohol fixation in bloody Pap smears

OBJECTIVE: To compare 2 methods of fixation in bloody Pap smears with Carnoy's solution and 96% ethyl alcohol. STUDY DESIGN: After observation of contact bleeding, 2 samples were prepared from cervical cells with conventional Pap smear. One sample was fixed in 96% ethyl alcohol and another sample was fixed in Carnoy's solution. RESULTS: Of 450 slides, 410 were selected for study. In study of cell adequacy, diagnosis of squamous cells and glandular cells was better in Carnoy's-fixed slides. Blood contamination of slides was reduced in Carnoy's-fixed slides (13.85% vs. 49.51%), and clearance of slides was increased in Carnoy's-fixed slides. Diagnosis of inflammatory cells and pathogenic microorganisms in was increased in Carnoy's-fixed slides, but no difference was seen in diagnosis of epithelial cell and glandular cell abnormalities. CONCLUSION: Carnoy's solution can be used as an effective fixative in bloody smears in conventional Pap tests.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> Alleviates Salinity by Boosting Redox Poise and Antioxidative Potential of Tomato

More than 20% of irrigated land has been influenced by salt stress, decreasing crop production. In this research, we investigated the effect of different levels of salinity (0, 50, 100 and 150 mM NaCl) and the efficiency of Piriformospora indica on growth, biochemical traits, antioxidative defense system in tomato (Solanum lycopersicum L.). NaCl stress reduced chlorophyll content, height and biomass of plants. Higher level of salinity (150 mM) declined the plant height by 22.65%, total dry weight by 56.44% and total chlorophyll by 44.34%, however, P. indica inoculation raised plant height by 43.47%, dry weight by 69.23% and total chlorophyll content by 48.09%. Salinity stress increased H₂O₂, malondialdehyde (MDA), superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) level in leaves and roots tomato seedlings. However, P. indica inoculation reduced H₂O₂, MDA and superoxide anion and enhanced DPPH compared to non-inoculated plants at all NaCl levels. The total phenol and flavonoids increased with NaCl treatment. On the other hand, the total phenolic and flavonoid increased more in P. indica inoculated plants compared to non-inoculated ones. Moreover, inoculation of P. indica implicated noteworthy improvement of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) activity in tomato upon salinity. Notably, colonization with P. indica significantly improved the content of reduced ascorbic acid (AsA), glutathione (GSH) and redox ratio in the tomato plants under salinity resulting in reduced redox state. Our findings confirmed that salinity had negative effect on tomato seedling; however, P. indica inoculation increased tolerance to salinity by improving the content of phenolic compounds, non-enzymatic antioxidants, and increasing the activity of antioxidant enzymes.     

Recombinant Production and Antimicrobial Assessment of <Emphasis Type="Italic">Beta</Emphasis> Casein- IbAMP<Subscript>4</Subscript> as a Novel Antimicrobial Polymeric Protein and its Synergistic Effects with Thymol

There is a growing research interest on products with antimicrobial activity. Antimicrobial polymers are one of the most surefire procedures to combat microbes. In the present study, the ability of Βeta-casein- one of the milk major self assembly proteins with high polymeric film production capability—as a fusion partner of Ib-AMP₄ antimicrobial peptide was investigated. Also, the antimicrobial activities of Βeta-casein- IbAMP₄ fusion protein antimicrobial against common food pathogens were assessed. The pET21a-BCN-Ib-AMP ₄ construct was transformed to Escherichia coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg mL^?1 fusion protein by ultrafiltration. 5 μg mL^?1 H₂O₂ was applied for accelerating the formation of two necessary disulfide bonds. Antimicrobial assays were performed against E. coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus and Candida albicans. Results of antimicrobial tests confirmed the efficiency of BCN-IbAMP₄ against all tested microorganisms. Overall, the combination of thymol plus BCN-IbAMP₄ increased their antimicrobial activities. MIC, MBC, MFC, FICI and FBCI values showed strong synergistic activity between the two examined compounds. Time kill and growth kinetic studies indicated significant reduction of cell viability during first period of exposure to BCN-IbAMP₄ and thymol combination.     

Evaluation of the toxicity effects of silk fibroin on human lymphocytes and monocytes

Silk fibroin nanoparticles (SFNPs) as a natural polymer have been utilized in biomedical applications such as suture, tissue engineering‐based scaffolds, and drug delivery carriers. Since there is little data regarding the toxicity effects on different cells and tissues, we aimed to determine the toxicity mechanisms of SFNPs on human lymphocytes and monocytes based on reliable methods. Our results showed that SFNPs (0.5, 1, and 2 mg/mL) induced oxidative stress via increasing reactive oxygen species production, mitochondrial membrane potential (?Ψ) collapse, which was correlated to cytochrome c release and Adenosine diphosphate (ADP)/Adenosine tri phosphate (ATP) ratio increase as well as lysosomal as another toxicity mechanism, which led to cytosolic release of lysosomal digestive proteases, phosphor lipases, and apoptosis signaling. Taken together, these data suggested that SFNPs toxicity was associated with mutual mitochondrial/lysosomal cross‐talk and oxidative stress on human lymphocytes and monocytes with activated apoptosis signaling.