Optimization of expression, purification and handling anti bacteria feature protein of bovine neutrophil B-defensing BNBD2

زمینه و هدف: دیفنسین‎ها یکی از بزرگ‎ترین خانواده‎ی پپتید‎های ضد میکروب می‎باشند که به واسطه‎ی فعالیت بر علیه باکتری‎ها، قارچ‎ها و بسیاری از ویروس‎ها به عنوان آنتی‎بیوتیک‎های نسل جدید منفعت بسیاری دارند. هدف از این مطالعه بهینه سازی بیان، تخلیص و بررسی خاصیت ضد میکروبی پروتئین بتا دیفنسین 2 نوتروفیل‎های گاو (BNBD2) بوده است. روش بررسی: در این مطالعه‎ی تجربی-آزمایشگاهی باکتری اشرشیاکلی B‏L21(DE3) حامل وکتور pET-32a(+) که ژن BNBD2 در آن همسانه سازی شده بود جهت مطالعات مورد استفاده قرار گرفت. بیان پروتئین BNBD2 با تغییر در پارامترهای دانسیته‎ی سلولی، دمای رشد، مدت زمان القاء با استفاده از سیستم الکتروفورز عمودی (SDS-PAGE) و تست برادفورد به صورت کمی و کیفی بررسی گردید. مراحل تخلیص پروتئین نوترکیب با کمک روش شیمیایی شکافت در جایگاه فرمیک اسید و عبور از سانتریکون و اثر ضد باکتری پروتئین تخلیص شده بر چند نمونه‎ی باکتریایی گرم مثبت و گرم منفی مورد بررسی قرار گرفت. یافته ها: با استفاده از محیط کشت Luria–Bertani، شروع القاء در جذب نوری 8/0 در طول موج 600 نانومتر، غلظت یک میلی مولار ماده‎ی القاء کننده‎ی IPTG، دمای رشد 30 درجه و مدت زمان 4 ساعت پس از القاء بیشترین میزان بیان پروتئین به دست آمد. پروتئین نوترکیب با استفاده از شکافت در جایگاه فرمیک اسید و عبور از سانتریکون تخلیص گردید. نتایج آزمایش وسترن بلاتینگ نیز نشان داد که پروتئین نوترکیب به طور اختصاصی به آنتی‎بادی mouse anti-(His)6 peroxidase متصل می‎گردد. تشکیل هاله‎ی عدم رشد در محیط‎های کشت مولر هینتون آگار حاوی کشت سطحی باکتری های مورد آزمایش خاصیت ضد باکتری این پروتئین را نشان داد. نتیجه گیری: با توجه به خاصیت ضد میکروبی پروتئین BNBD2و امکان بیان پروتئین در باکتری E. coli می توان به تولید انبوه این پروتئین نوترکیب اقدام نمود

How do general practitioners experience providing care to refugees with mental health problems? A qualitative study from Denmark

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A comparative assessment of the physical and microstructural properties of waste garnet generated from automated and manual blasting process

Cold mix asphalt (CMA) is an eco-friendly sustainable asphalt mixture, mostly for asphalt surface treatments (ASTs). However, material compatibility and poor adhesion leading to high voids, moisture damage susceptibility, and weak early strength remain challenging. Efforts to solve this limitation is beamed towards binder improvement and modification with modifiers, adhesion promoters, or polymers. Other forms of AST mixture improvement entail supplementary cementitious reinforcing or pozzolanic agents in the form of by-products. In this study, the physio-mechanical and microstructural desirability of spent garnet for use as fine aggregate in CMA was explored. Spent garnet is a by-product of abrasive blasting, often produced in large quantities and disposed of in landfills. Often, spent garnet waste gets contaminated with toxic elements either during usage or in landfills. This study aimed to investigate the properties of Automatically (AG) and Manually generated (MG) spent garnet grades. The physio-mechanical, morphologic, and chemical parameters of spent garnet were assessed to achieve this aim. The result compared with relevant specifications on cold mixtures plus Jabatan Kerja Raya (JKR) requirement. Moreover, crystallinity and composition were studied using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and X-ray Fluorescence (XRF). The presence of toxic heavy metals that often contaminate spent garnet deposits in landfills was evaluated too. Results suggested that both AG and MG's high sand equivalent and least water absorption of 98 %, 89 %, and 0.14 %, 0.23 % accordingly, and can replace sand in CMA. However, MG spent garnet is not desirable for chemically sensitive materials. The AG garnet was found to be Pyrope while the MG spent garnet is largely Almandine garnet – the strongest form of garnet, including traces of other garnet forms

Interobserver agreement of whole-body magnetic resonance imaging is superior to whole-body computed tomography for assessing disease burden in patients with multiple myeloma

Objectives Whole-body MRI (WB-MRI) is recommended by the International Myeloma Working Group for all patients with asymptomatic myeloma and solitary plasmacytoma and by the UK NICE guidance for all patients with suspected myeloma. Some centres unable to offer WB-MRI offer low-dose whole-body CT (WB-CT). There are no studies comparing interobserver agreement and disease detection of contemporary WB-MRI (anatomical imaging and DWI) versus WB-CT. Our primary aim is to compare the interobserver agreement between WB-CT and WB-MRI in the diagnosis of myeloma. Methods Consecutive patients with newly diagnosed myeloma imaged with WB-MRI and WB-CT were prospectively reviewed. For each body region and modality, two experienced and two junior radiologists scored disease burden with final scores by consensus. Intraclass correlation coefficients (ICC), median scores, Wilcoxon signed-rank test and Spearman’s correlation coefficients were calculated. Results There was no significant difference in overall observer scores between WB-MRI and WB-CT (p = 0.87). For experienced observers, interobserver agreement for WB-MRI was superior to WB-CT overall and for each region, without overlap in whole-skeleton confidence intervals (ICC 0.98 versus 0.77, 95%CI 0.96–0.99 versus 0.45–0.91). For inexperienced observers, although there is a trend for a better interobserver score for the whole skeleton on WB-MRI (ICC 0.95, 95%CI 0.72–0.98) than on WB-CT (ICC 0.72, 95%CI 0.34–0.88), the confidence intervals overlap. Conclusions WB-MRI offers excellent interobserver agreement which is superior to WB-CT for experienced observers. Although the overall burden was similar across both modalities, patients with lower disease burdens where MRI could be advantageous are not included in this series

The Endogenous Th17 Response in NO<inf>2</inf>-Promoted Allergic Airway Disease Is Dispensable for Airway Hyperresponsiveness and Distinct from Th17 Adoptive Transfer

Severe, glucocorticoid-resistant asthma comprises 5-7% of patients with asthma. IL-17 is a biomarker of severe asthma, and the adoptive transfer of Th17 cells in mice is sufficient to induce glucocorticoid-resistant allergic airway disease. Nitrogen dioxide (NO2) is an environmental toxin that correlates with asthma severity, exacerbation, and risk of adverse outcomes. Mice that are allergically sensitized to the antigen ovalbumin by exposure to NO2 exhibit a mixed Th2/Th17 adaptive immune response and eosinophil and neutrophil recruitment to the airway following antigen challenge, a phenotype reminiscent of severe clinical asthma. Because IL-1 receptor (IL-1R) signaling is critical in the generation of the Th17 response in vivo, we hypothesized that the IL-1R/Th17 axis contributes to pulmonary inflammation and airway hyperresponsiveness (AHR) in NO2-promoted allergic airway disease and manifests in glucocorticoid-resistant cytokine production. IL-17A neutralization at the time of antigen challenge or genetic deficiency in IL-1R resulted in decreased neutrophil recruitment to the airway following antigen challenge but did not protect against the development of AHR. Instead, IL-1R-/- mice developed exacerbated AHR compared to WT mice. Lung cells from NO2-allergically inflamed mice that were treated in vitro with dexamethasone (Dex) during antigen restimulation exhibited reduced Th17 cytokine production, whereas Th17 cytokine production by lung cells from recipient mice of in vitro Th17-polarized OTII T-cells was resistant to Dex. These results demonstrate that the IL-1R/Th17 axis does not contribute to AHR development in NO2-promoted allergic airway disease, that Th17 adoptive transfer does not necessarily reflect an endogenously-generated Th17 response, and that functions of Th17 responses are contingent on the experimental conditions in which they are generated. © 2013 Martin et al

Regulation of the High Affinity IgE Receptor (FcεRI) in Human Neutrophils: Role of Seasonal Allergen Exposure and Th-2 Cytokines

The high affinity IgE receptor, FcεRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional FcεRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of FcεRI-α chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, FcεRI-α chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/FcγRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced FcεRI-α chain surface expression. In conclusion, these results suggest that enhanced FcεRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma

Effect of polymer-modified bitumen and reclaimed asphalt pavement on the physical properties of bitumen

The high cost of road construction due to the energy required for new material production and the environmental impact necessitates the use of reclaimed asphalt pavement (RAP) and other waste materials. This paper examined the effect of using RAP with a PET additive as a rejuvenator. PET additive (2%) was added to the virgin binder (VB) of 60/70 penetration grade to form a Polymer modified bitumen (PMB). RAP percentages used were 30, 40, and 50%, respectively. The physical properties were examined using the penetration, softening point, and ductility. The results show that the physical properties of RAP were improved by utilizing Rap binder (RB) and PMB mix ratio. The penetration and ductility values increased by 46.4% and 66.7%, respectively, with the PMB (30): RB (70) ratio blend, compared to RB: VB blend. This suggests a reduction in brittle behavior and less stiffness with the addition of PMB in the blend, enhancing the flexibility of the RAP. Although both mixtures blended with RB exhibited softening points within the acceptable range, improved performance was observed with RB/PMB mix blend over RB/VB blend when used in hot asphalt mixtur

Effect of polymer-modified bitumen and reclaimed asphalt pavement on the physical properties of bitumen

The high cost of road construction due to the energy required for new material production and the environmental impact necessitates the use of reclaimed asphalt pavement (RAP) and other waste materials. This paper examined the effect of using RAP with a PET additive as a rejuvenator. PET additive (2%) was added to the virgin binder (VB) of 60/70 penetration grade to form a Polymer modified bitumen (PMB). RAP percentages used were 30, 40, and 50%, respectively. The physical properties were examined using the penetration, softening point, and ductility. The results show that the physical properties of RAP were improved by utilizing Rap binder (RB) and PMB mix ratio. The penetration and ductility values increased by 46.4% and 66.7%, respectively, with the PMB (30): RB (70) ratio blend, compared to RB: VB blend. This suggests a reduction in brittle behavior and less stiffness with the addition of PMB in the blend, enhancing the flexibility of the RAP. Although both mixtures blended with RB exhibited softening points within the acceptable range, improved performance was observed with RB/PMB mix blend over RB/VB blend when used in hot asphalt mixtur

Performance of asphaltic concrete incorporating fly ash under low temperature

One of the most common asphalt concrete pavement distresses is low temperature cracking, also known as thermal cracking. Characterizations of low temperature cracking and formulation for pavement design have taken a lot of effort. Asphalt binder has viscoelastic behaviour, so asphalt mixture behaviour changes as the temperature changes. At high and low temperatures, the asphalt binder shows viscoelastic plastic behaviour and elastic behaviour. Low temperature cracks that grow day by day due to the movement of vehicles are the most significant pavement cracks caused by cold climates. It needs early and premature repairs to build and expand low temperature cracks. The aim of this research is to perform Low Temperature Cracking analysis of asphalt materials (laboratory and analytical assessment), in light of the latest update of binder cracking temperature. The role of basic material properties in low-temperature cracking was studied in this work. As a result, statistical analysis in the cohesive failure condition revealed that the asphalt mixture aggregate's free energy was ineffective in this cohesion failure. Fly ash had been used in the other type of asphalt mixture. It was proven that the addition of fly ash as an additive can improves the low temperature resistance of the asphalt mix. The binder with 60/70 penetration grade was used. The different amount of fly ash (0%, 1%, 3% and 5%) was added to the asphalt mixture. Marshall Stability and flow, resilient modulus and dynamic creep were carried out to investigate the mechanisms of cracking at low temperature. From the results obtained, there are significant effect comes from the addition of the fly ash. The result show that the addition of 5% fly ash produce the best outcomes for the density, stability, stiffness, resilient modulus and dynamic creep. Thus, it can conclude that the existence of fly ash in the mixture is able to enhance the mechanical performance of the AC14 dense-graded asphalt