Differentiation of definitive endoderm from human induced pluripotent stem cells on hMSCs feeder in a defined medium

Background: The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium. Methods: Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3- ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry. Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences. Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine. © 2016, Avicenna Journal of Medical Biotechnology. All rights reserved

Ruminal and post-ruminal barley grain digestion and starch granule morphology under three heat methods

La editorial tiene los derechos de autor y le solicito a hacer visibles la fecha técnica del artículo.This study was designed to investigate the effect of three heating methods on the ruminal and post-ruminal nutrient degradability and starch granule morphology of barley grain (BG - Hordeum vulgare). Treatments were Control (CG): without processing; Roasted (RG): roasted BG for 300 s at 130°C; Microwaved (MG): irradiated BG for 120 s at 1200 W; and Steam flaked (SG): BG misted for 30 min under steam flow of boiling water and flaked. Gas production and in situ techniques were used to evaluate the ruminal degradability of treatments, and a modified three-step method was utilised to estimate the total-tract digestibility. Morphological changes of starch granules were determined by field emission scanning electron microscopy (FESEM). Ruminal gas production and dry matter disappearance were increased (p < .05) in SG vs. CG. Heat processing had different effects on starch and crude protein degradability; however, starch degradability increased (p < .05) from CG to SG. Post-ruminal disappearance of dry matter in CG was greater (p < .05) than other treatments. These results validated by FESEM images that explained high barley grain degradability in relation to the number of holes on the surface of starch granules. Heat processing can enhance ruminal and post-ruminal utilisation efficiency of barley grain, resulting in improvement of total-tract digestibility

Histidine substitution in the most flexible fragments of firefly luciferase modifies its thermal stability.

Molecular dynamics (MD) at two temperatures of 300 and 340 K identified two histidine residues, His461 and His489, in the most flexible regions of firefly luciferase, a light emitting enzyme. We therefore designed four protein mutants H461D, H489K, H489D and H489M to investigate their enzyme kinetic and thermodynamic stability changes. Substitution of His461 by aspartate (H461D) decreased ATP binding affinity, reduced the melting temperature of protein by around 25 degrees C and shifted its optimum temperature of activity to 10 degrees C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Similar changes were observed for H489K and, to a lesser degree, H489M mutations. Based on our results we conclude that the MD simulation-based rational substitution of histidines by salt-bridge forming residues can modulate conformational dynamics in luciferase and shift its optimal temperature activity

The general behavior of NLONLO unintegrated parton distributions based on the single-scale evolution and the angular ordering constraint

To overcome the complexity of generalized two hard scale ($k_t$,$\mu$) evolution equation, well known as the $Ciafaloni$, $Catani$, $Fiorani$ and $Marchsini$ ($CCFM$) evolution equations, and calculate the unintegrated parton distribution functions ($UPDF$), $Kimber$, $Martin$ and $Ryskin$ ($KMR$) proposed a procedure based on ($i$) the inclusion of single-scale ($\mu$) only at the last step of evolution and ($ii$) the angular ordering constraint ($AOC$) on the $DGLAP$ terms (the $DGLAP$ collinear approximation), to bring the second scale, $k_t$ into the $UPDF$ evolution equations. In this work we intend to use the $MSTW 2008$ (Martin et al) parton distribution functions (PDF) and try to calculate $UPDF$ for various values of $x$ (the longitudinal fraction of parton momentum), $\mu$ (the probe scale) and $k_t$ (the parton transverse momentum) to see the general behavior of three dimensional $UPDF$ at the $NLO$ level up to the $LHC$ working energy scales ($\mu^2)$. It is shown that there exits some pronounced peaks for the three dimensional $UPDF$ $(f_a(x,k_t))$ with respect to the two variables $x$ and $k_t$ at various energies ($\mu$). These peaks get larger and move to larger values of $k_t$, as the energy ($\mu$) is increased. We hope these peaks could be detected in the $LHC$ experiments at $CERN$ and other laboratories in the less exclusive processes

Sources of academic stress among Iranian adolescents: a multilevel study from Qazvin City, Iran

Background: Academic stress can cause mental and physical problems and affect adolescents’ healthy development. This study aimed to estimate academic stress and explore its sources at the individual- and school levels among school-going adolescents in the city of Qazvin, Iran. Results: This cross-sectional study used a stratified cluster sampling to recruit 1724 students aged 12–19 years from 53 schools in Qazvin City. Data were collected using a validated self-administered questionnaire. The mean academic stress score was 45.7 (95% CI 45.2, 46.3). The stress level was statistically higher among older 47.5 (95% CI 46.7, 48.3) than younger 44.1 (95% CI 43.4, 44.9) adolescents. The main academic stressors included: future uncertainty 69.7 (95% CI 68.8, 70.7), academic competition 58.5 (95% CI 57.3, 59.6), and interaction with teachers 56.1 (95% CI 55.3, 56.9). Gender, educational period, school type, family socioeconomic status, and father’s education were associated with academic stress. Conclusions: We conducted a multilevel study using a random sample of male and female students in the city of Qazvin, Iran. Results indicated moderate levels of stress among Iranian adolescents. The academic stress was associated with several individual and school-level variables. Students and their families and teachers need education on stress prevention methods and coping mechanisms. Future research should focus on developing and testing multilevel policies and interventions to improve students’ mental health and academic performance. Keywords: Academic stress, Adolescence, Students, Multilevel analysis, Ira

Sources of academic stress among Iranian adolescents: a multilevel study from Qazvin City, Iran

Background: Academic stress can cause mental and physical problems and affect adolescents’ healthy development. This study aimed to estimate academic stress and explore its sources at the individual- and school levels among school-going adolescents in the city of Qazvin, Iran. Results: This cross-sectional study used a stratified cluster sampling to recruit 1724 students aged 12–19 years from 53 schools in Qazvin City. Data were collected using a validated self-administered questionnaire. The mean academic stress score was 45.7 (95% CI 45.2, 46.3). The stress level was statistically higher among older 47.5 (95% CI 46.7, 48.3) than younger 44.1 (95% CI 43.4, 44.9) adolescents. The main academic stressors included: future uncertainty 69.7 (95% CI 68.8, 70.7), academic competition 58.5 (95% CI 57.3, 59.6), and interaction with teachers 56.1 (95% CI 55.3, 56.9). Gender, educational period, school type, family socioeconomic status, and father’s education were associated with academic stress. Conclusions: We conducted a multilevel study using a random sample of male and female students in the city of Qazvin, Iran. Results indicated moderate levels of stress among Iranian adolescents. The academic stress was associated with several individual and school-level variables. Students and their families and teachers need education on stress prevention methods and coping mechanisms. Future research should focus on developing and testing multilevel policies and interventions to improve students’ mental health and academic performance. Keywords: Academic stress, Adolescence, Students, Multilevel analysis, Ira

The expression pattern of VISTA in the PBMCs of relapsing-remitting multiple sclerosis patients: A single-cell RNA sequencing-based study

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Dysregulated immune responses have been implicated in MS development. Growing evidence has indicated that inhibitory immune checkpoint molecules can substantially regulate immune responses and maintain immune tolerance. V-domain Ig suppressor of T cell activation (VISTA) is a novel inhibitory immune checkpoint molecule that can suppress immune responses; however, its expression pattern in the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) has not thoroughly been studied. Herein, we evaluated Vsir expression in PBMCs of RRMS patients and characterized the expression pattern of the Vsir in the PBMCs of MS patients. Besides, we investigated the effect of fingolimod, IFNβ-1α, glatiramer acetate (GA), and dimethyl fumarate (DMF) on Vsir expression in PBMCs of RRMS patients. Our results have shown that Vsir expression is significantly downregulated in the PBMCs of patients with RRMS. Besides, the single-cell RNA sequencing results have demonstrated that Vsir expression is downregulated in classical monocyte, intermediate monocytes, non-classical monocytes, myeloid DCs (mDC), Plasmacytoid dendritic cells (pDCs), and naive B-cells of PBMCs of MS patients compared to the control. In addition, DMF, IFNβ-1α, and GA have significantly upregulated Vsir expression in the PBMCs of RRMS patients. Collectively, the current study has shed light on Vsir expression in the PBMCs of MS patients; however, further studies are needed to elucidate the significance of VISTA in the mentioned immune cells

Electrospun PLLA Nanofiber Scaffolds and Their Use in Combination with BMP-2 for Reconstruction of Bone Defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus