Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells

yesThe gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer

Reduction of hyperglycemia in STZ-induced diabetic mice by prophylactic treatment with heat-killed Mycobacterium aurum: possible effects on glucose utilization, mitochondrial uncoupling, and oxidative stress in liver and skeletal muscle

BackgroundIn addition to conventional treatment and modifications in physical activity and diet, alternative strategies have been investigated to manage, prevent, or delay diabetes in humans. In this regard, one strategy has relied on the immunomodulatory properties of mycobacteria, whereby Bacillus Calmette–Guerin, an attenuated live strain of Mycobacterium bovis, has been shown to improve glycemic control in patients with diabetes and to alleviate hyperglycemia in selected murine models of diabetes. A novel heat-killed (HK) whole-cell preparation of Mycobacterium aurum (M. aurum) is currently under development as a potential food supplement; nevertheless, its potential bioactivity remains largely unknown. Thus, the present study investigated the potential prophylactic anti-diabetic effects of HK M. aurum in streptozotocin (STZ)–induced diabetic mice.MethodsMice were divided into three groups: the STZ-induced diabetic group was injected with a single intraperitoneal high dose of STZ, the HK M. aurum–treated diabetic group was prophylactically treated with three doses of HK M. aurum 6 weeks before STZ injection, and the control non-diabetic group was given three intradermal injections of borate-buffered saline and an intraperitoneal injection of citrate buffer. Liver lactate dehydrogenase (LDH), uncoupling protein 2 (UCP2), and glucose transporter 2 (GLUT2) and skeletal muscle LDH, UCP3, and GLUT4 protein expression levels in different mouse groups were determined by Western blot.ResultsOur results indicated that HK M. aurum did not cause any significant changes in glycemic levels of normal non-diabetic mice. Prophylactic administration of three doses of HK M. aurum to diabetic mice resulted in a significant reduction in their blood glucose levels when compared to those in control diabetic mice. Prophylactic treatment of diabetic mice with HK M. aurum significantly restored their disturbed protein expression levels of liver UCP2 and LDH as well as of skeletal muscle UCP3. On the other hand, prophylactic treatment of diabetic mice with HK M. aurum had no significant effect on their liver GLUT2 and skeletal muscle GLUT4 and LDH protein expression levels.ConclusionsOur findings provide the first evidence that HK M. aurum possesses a hyperglycemia-lowering capacity and might support its future use as a food supplement for the amelioration of diabetes

Leveraging Pleiotropy to Discover and interpret Gwas Results For Sleep-Associated Traits

Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required phenotyping. This reduces statistical power and limits discovery of multiple genetic associations. We present a strategy to leverage pleiotropy between traits to both discover new loci and to provide mechanistic hypotheses of the underlying pathophysiology. Specifically, we combine a colocalization test with a locus-level test of pleiotropy. In simulations, we show that this approach is highly selective for identifying true pleiotropy driven by the same causative variant, thereby improves the chance to replicate the associations in underpowered validation cohorts and leads to higher interpretability. Here, as an exemplar, we use Obstructive Sleep Apnea (OSA), a common disorder diagnosed using overnight multi-channel physiological testing. We leverage pleiotropy with relevant cellular and cardio-metabolic phenotypes and gene expression traits to map new risk loci in an underpowered OSA GWAS. We identify several pleiotropic loci harboring suggestive associations to OSA and genome-wide significant associations to other traits, and show that their OSA association replicates in independent cohorts of diverse ancestries. By investigating pleiotropic loci, our strategy allows proposing new hypotheses about OSA pathobiology across many physiological layers. For example, we identify and replicate the pleiotropy across the plateletcrit, OSA and an eQTL of DNA primase subunit 1 (PRIM1) in immune cells. We find suggestive links between OSA, a measure of lung function (FEV1/FVC), and an eQTL of matrix metallopeptidase 15 (MMP15) in lung tissue. We also link a previously known genome-wide significant peak for OSA in the hexokinase 1 (HK1) locus to hematocrit and other red blood cell related traits. Thus, the analysis of pleiotropic associations has the potential to assemble diverse phenotypes into a chain of mechanistic hypotheses that provide insight into the pathogenesis of complex human diseases

F-Spondin/spon1b Expression Patterns in Developing and Adult Zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate

Possible Role of PTEN Expression in Discriminating Benign Endometrial Hyperplasia from Atypical Hyperplasia/Endometrial Intraepithelial Neoplasia in a Series of Egyptian Patients

Background: Endometrial hyperplasia represents a heterogeneous group of lesions in response to the unopposed growth-promoting action of estrogen. WHO classified endometrial hyperplastic lesions into Benign Hyperplasia (BH) and atypical hyperplasia/ endometrial intraepithelial neoplasia AH/EIN. Phosphatase and tensin homolog (PTEN) is one of the earliest and most common genetic abnormalities detected in endometrioid adenocarcinoma (type I) and even in its precursors. This study aimed at histological evaluation of hyperplastic endometrial lesions according to WHO 2014 and investigating the role of PTEN expression in highlighting the precancerous group (AH/EIN). Patient and Method: This study included a series of 70 Egyptian patients suffered from hyperplastic endometrial lesions. They were previously diagnosed according to WHO1994 schema simple endometrial hyperplasia without atypia (n=18), simple endometrial hyperplasia with atypia (n=2), complex hyperplasia without atypia (n=25), complex hyperplasia with atypia (n=5) and hyperplastic endometrial polyps (n=20). Results: Cases were histologically re-evaluated according to WHO 2014 classification; BH (62 cases) and eight cases of AH/EIN. A significant difference in PTEN expression (regarding percentage and intensity of staining) in relation to histopathological diagnosis was detected (P-value 0.02 and &lt;0.05, respectively). The sensitivity and specificity of the absence of diffuse PTEN protein expression (&gt;50%) to detect AH/EIN were 100% and 77.4%, respectively. Conclusion: Diffuse, dim or loss of immunohistochemical expression of PTEN protein is significantly correlated with the new WHO classification segregation of AH/EIN as precancerous lesions. However, further studies are recommended to confirm this association. </jats:sec