Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes.

Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor (Gata4, Nkx2-5 and Mef2c) and mature cardiac markers (Actc1, myh6) significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available.YK and AB thank the European Research Council (ERC Starting Grant 2013 to AB) for funding.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/cddiscovery.2016.

BACTEC MGIT 960 TM system for screening of Mycobacterium tuberculosis complex among cattle

Bovine tuberculosis remains a disease of economic and public health importance in developing countries. The largest number of new cases of tuberculosis usually occurs in South-East Asia region and Africa. This study was aimed to evaluate the recent technique (BACTEC MGIT 960 TM system) for screening of Mycobacterium tuberculosis complex among cattle in Egypt. From the 1180 cattle examined in three different Governorates (El-Sharkia, El-Gharbia and El-Monefeia) by single intradermal tuberculin test, 29 animals (2.46%) were positive reactors. The post mortem examination of the positive reactors showed that 22 animals (75.9%) had visible lesions [respiratory form (31.0%), digestive form (13.8%), mixed form (20.7%) and generalized form (10.3%)], while seven (24.1%) did not show visible lesions. The results of isolation and identification using conventional culture method (Lowenstein- Jensen medium) were 22 mycobacterial isolates (75.9%), 20 (68.97%) Mycobacterium bovis and 2 (6.9%) unidentified slow growth. The BACTEC MGIT 960 TM system was used for recovery of Mycobacteria and compared with conventional culture method (Lowenstein-Jensen medium). The recovery rate of BACTEC MGIT 960 TM system was 82.8%, while that of Lowenstein-Jensen medium was 75.9%. The mean time for detection of Mycobacteria was 17.8 ± 0.9 days and 46.5 ± 0.4 days for BACTEC MGIT 960 TM system and Lowenstein-Jensen medium, respectively while the contamination rate with BACTEC MGIT 960 TM system was 6.9% and 10.3% in Lowenstein-Jensen medium.Key words: Bovine tuberculosis, tuberculin test, Lowenstein-Jensen medium, BACTEC system

ZIF-8 Modified Polypropylene Membrane: A Biomimetic Cell Culture Platform with a View to the Improvement of Guided Bone Regeneration.

PurposeDespite the significant advances in modeling of biomechanical aspects of cell microenvironment, it remains a major challenge to precisely mimic the physiological condition of the particular cell niche. Here, the metal-organic frameworks (MOFs) have been introduced as a feasible platform for multifactorial control of cell-substrate interaction, given the wide range of physical and mechanical properties of MOF materials and their structural flexibility.ResultsIn situ crystallization of zeolitic imidazolate framework-8 (ZIF-8) on the polydopamine (PDA)-modified membrane significantly raised surface energy, wettability, roughness, and stiffness of the substrate. This modulation led to an almost twofold increment in the primary attachment of dental pulp stem cells (DPSCs) compare to conventional plastic culture dishes. The findings indicate that polypropylene (PP) membrane modified by PDA/ZIF-8 coating effectively supports the growth and proliferation of DPSCs at a substantial rate. Further analysis also displayed the exaggerated multilineage differentiation of DPSCs with amplified level of autocrine cell fate determination signals, like BSP1, BMP2, PPARG, FABP4, ACAN, and COL2A. Notably, osteogenic markers were dramatically overexpressed (more than 100-folds rather than tissue culture plate) in response to biomechanical characteristics of the ZIF-8 layer.ConclusionHence, surface modification of cell culture platforms with MOF nanostructures proposed as a powerful nanomedical approach for selectively guiding stem cells for tissue regeneration. In particular, PP/PDA/ZIF-8 membrane presented ideal characteristics for using as a barrier membrane for guided bone regeneration (GBR) in periodontal tissue engineering

The effect of industrial and waste fibres on concrete strength and structural behaviour of rc short columns

Concrete is a brittle substance; thus, it is reinforced with rebars and fibers to enhance its ductility. On the other hand, the presence of waste from various industries negatively impacts the environment. The ongoing reconstruction in Iraq has resulted in an abundance of locally produced rebar-connecting wire (RCW) and copper electric wire (CEW) waste. To minimize the environmental impact of these wastes, they can be reused in other industries, such as the concrete industry. Few studies have dealt with concrete's structural and mechanical properties containing these local residues. Therefore, this study included an experimental investigation of concrete columns with and without various types of industrial and waste fibers. Two types of industrial fibers (macro hooked-end; CH, and micro straight; CS) steel fibers and two types of waste fibers (RCW and CEW) were utilized. Six reinforced concrete (RC) columns (150 × 150 × 450 mm3) were cast: one control column without fibers and five columns with fibers. The fiber content within the columns was fixed at 0.75% of the concrete volume. The cracks pattern, load-deflection behavior and concrete strain for RC columns were investigated. Moreover, the mechanical properties in terms of compressive, splitting tensile, and flexural strengths tests were also conducted. The results revealed that all types of fibers used improved the mechanical and structural properties of the concrete. Moreover, although the hybrid synthetic fibers gave the best improvement compared to the reference sample, the waste fibers (especially RCW) showed a significant improvement that reached 30.91% in relation to the ultimate load and (10.1, 10.8 and 14.4%) in relation to the compressive, tensile, and flexural strengths respectively. Konkrit adalah material rapuh; oleh itu ianya dikuatkan dengan besi dan fiber bagi menguatkan kekuatannya. Dalam masa sama, kehadiran bahan buangan dalam pelbagai industri memberi kesan negatif kepada persekitaran. Penstrukturan semula Iraq yang sedang berlangsung memberi kesan kepada kebanjiran bahan buangan seperti besi penghubung litar (RCW) dan litar elektrik tembaga (CEW) buatan tempatan. Bagi mengurangkan kesan pencemaran terhadap alam sekitar, bahan-bahan ini boleh diguna balik dalam industri berbeza, seperti industri konkrit. Terdapat banyak kajian terhadap buangan tempatan yang melibatkan struktur bahan konkrit dan sifat mekanikal.  Oleh itu, kajian ini merupakan kajian eksperimen pasak konkrit dengan atau tanpa pelbagai jenis industri dan fiber buangan. Dua jenis fiber industri iaitu fiber besi (mikro hujung-penyangkut; CH dan mikro lurus; CS) dan dua jenis fiber buangan (RCW dan CEW) dipakai. Enam RC pasak konkrit (150 × 150 × 450 mm3) dihasilkan: satu pasak kawalan tanpa fiber dan lima pasak dengan fiber. Kandungan fiber dalam pasak di tetapkan pada 0.75% isipadu konkrit. Corak rekahan, ciri-ciri kesan beban dan tekanan konkrit pada pasak RC dikaji. Tambahan, kajian terhadap ciri-ciri mekanikal berdasarkan tekanan, rekahan tensil dan kekuatan anjalan telah dijalankan. Dapatan kajian menunjukkan kesemua fiber yang digunakan menambah baik ciri-ciri mekanikal dan struktur konkrit. Tambahan lagi, walaupun fiber sintetik hibrid menunjukkan paling baik berbanding sampel contoh, fiber buangan (terutama RCW) menunjukkan pembaharuan ketara mencapai 30.91% berbanding beban maksimum dan masing-masing menunjukkan 10.1, 10.8 dan 14.4% pada tekanan, rekahan tensil dan kekuatan anjalan

Isolation and differentiation of adipose-derived stem cells into odontoblast-like cells: a preliminary in vitro study

Objective The aim of present study was to isolate and differentiate human adipose-derived stem cells (ASCs) into odontoblast-like cells. Materials and Methods In this experimental study, human adipose tissues were taken from the buccal fat pad of three individuals (mean age: 24.6 ± 2.1 years). The tissues were transferred to a laboratory in a sterile culture medium, divided into small pieces and digested by collagenase I (2 mg/mL, 60-90 minutes). ASCs were isolated by passing the cell suspension through cell strainers (70 and 40 µm), followed by incubation at 37ºC and 5% CO2in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS 5%) and penicillin/streptomycin (P/S). After three passages, the ASCs were harvested. Subsequently, flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect expression levels of NANOG and OCT4 to evaluate stemness. Then, a differentiation medium that included high-glucose DMEM supplemented with 10% FBS, dexamethasone (10 nM), sodium β-glycerophosphate (5 mM) and ascorbic acid (100 µM) was added. The cells were cultivated for four weeks, and the odontogenic medium was changed every two days. Cell differentiation was evaluated with Alizarin red staining and expressions of collagen I (COL1A1), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1). Results The ASCs were effectively and easily isolated. They were negative for CD45 and positive for the CD105 and CD73 markers. The ASCs expressed OCT4 and NANOG. Differentiated cells highly expressed DSPP, COL1A1 and DMP1. Alizarin red staining revealed a positive reaction for calcium deposition. Conclusion ASCs were isolated successfully in high numbers from the buccal fat pad of human volunteers and were differentiated into odontoblast-like cells. These ASCs could be considered a new source of cells for use in regenerative endodontic treatments

The outcome of trachomatous trichiasis surgery in Ethiopia: risk factors for recurrence.

BACKGROUND: Over 1.2 million people are blind from trachomatous trichiasis (TT). Lid rotation surgery is the mainstay of treatment, but recurrence rates can be high. We investigated the outcomes (recurrence rates and other complications) of posterior lamellar tarsal rotation (PLTR) surgery, one of the two most widely practised TT procedures in endemic settings. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a two-year follow-up study of 1300 participants who had PLTR surgery, conducted by one of five TT nurse surgeons. None had previously undergone TT surgery. All participants received a detailed trachoma eye examination at baseline and 6, 12, 18 and 24 months post-operatively. The study investigated the recurrence rates, other complications and factors associated with recurrence. Recurrence occurred in 207/635 (32.6%) and 108/641 (16.9%) of participants with pre-operative major (>5 trichiatic lashes) and minor (5 lashes (major recurrence). Recurrence was greatest in the first six months after surgery: 172 cases (55%) occurring in this period. Recurrence was associated with major TT pre-operatively (OR 2.39, 95% CI 1.83-3.11), pre-operative entropic lashes compared to misdirected/metaplastic lashes (OR 1.99, 95% CI 1.23-3.20), age over 40 years (OR 1.59, 95% CI 1.14-2.20) and specific surgeons (surgeon recurrence risk range: 18%-53%). Granuloma occurred in 69 (5.7%) and notching in 156 (13.0%). CONCLUSIONS/SIGNIFICANCE: Risk of recurrence is high despite high volume, highly trained surgeons. However, the vast majority are minor recurrences, which may not have significant corneal or visual consequences. Inter-surgeon variation in recurrence is concerning; surgical technique, training and immediate post-operative lid position require further investigation

Dengue Virus Inhibits Immune Responses in Aedes aegypti Cells

The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV) with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

<p>Abstract</p> <p>Background</p> <p>The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).</p> <p>Methods</p> <p>A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 μm and width: 3.28+/-0.20 μm, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.</p> <p>Results</p> <p>No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).</p> <p>Conclusions</p> <p>The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.</p