Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.     

Effects of combining releases of non-viable host eggs with insecticide application on Riptortus pedestris population and its egg parasitoids

Our previous study demonstrated that the release of refrigerated non-viable eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) enhanced parasitism rates in soybean fields but did not result in the reduction of R. pedestris populations. This study was further conducted using an open-cage exclusion design in a soybean field in order to evaluate the compatibility of combining releases of non-viable host eggs with a single pre-harvest application of insecticide for the control of R. pedestris. Refrigerated eggs of R. pedestris were released twice in treatment plots, and fresh (< 1 day old) eggs of R. pedestris were deployed in all experimental arenas, every 6 days, for host resource and measurement of field parasitism. The releases of host eggs did not reduce the number of R. pedestris in any life stage except the adult stage on two sampling dates. However, parasitism by Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae) was higher in treated plots (9–25%) than in the control plots (1–9%). Statistical significant reduction was not found in the pest population, but parasitism rates significantly increased. Pesticide application did not reduce the bug population but did affect the parasitoids population. Pest management tactics, using both artificially deployed host eggs and insecticide, are discussed.     

Complex Structure of Engineered Modular Domains Defining Molecular Interaction between ICAM-1 and Integrin LFA-1

Intermolecular contacts between integrin LFA-1 (α(L)β(2)) and ICAM-1 derive solely from the integrin α(L) I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the α(L) I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine α(L) I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the α(L) I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to α(L) I domain but reduced interaction with HRV.     

Characterization of PINK1 processing, stability, and subcellular localization

Mutations found in PTEN-induced putative kinase 1 (PINK1), a putative mitochondrial serine/threonine kinase of unknown function, have been linked to autosomal recessive Parkinson's disease. It is suggested that mutations can cause a loss of PINK1 kinase activity and eventually lead to mitochondrial dysfunction. In this report, we examined the subcellular localization of PINK1 and the dynamic kinetics of PINK1 processing and degradation. We also identified cytosolic chaperone heat-shock protein 90 (Hsp90) as an interacting protein of PINK1 by PINK1 co-immunoprecipitation. Immunofluorescence of PINK1 protein and mitochondrial isolation show that the precursor form of PINK1 translocates to the mitochondria and is processed into two cleaved forms of PINK1, which in turn localize more to the cytosolic than mitochondrial fraction. The cleavage does not occur and the uncleaved precursor stays associated with the mitochondria when the mitochondrial membrane potential is disrupted. Metabolic labeling analyses show that the PINK1 processing is rapid and the levels of cleaved forms are tightly regulated. Furthermore, cleaved forms of PINK1 are stabilized by Hsp90 interaction as the loss of Hsp90 activity decreases PINK1 level after mitochondrial processing. Lastly, we also find that cleaved forms of PINK1 are degraded by the proteasome, which is uncommon for mitochondrial proteins. Our findings support a dual subcellular localization, implying that PINK1 can reside in the mitochondria and the cytosol. This raises intriguing functional roles that bridge these two cellular compartments.     

Pulmonary Toxocariasis Mimicking Invasive Aspergillosis in a Patient with Ulcerative Colitis

A 45-year-old-male who had underlying ulcerative colitis and presented with fever and dry cough. Initially, the patient was considered to have invasive aspergillosis due to a positive galactomannan assay. He was treated with amphotericin B followed by voriconazole. Nevertheless, the patient deteriorated clinically and radiographically. The lung biopsy revealed eosinophilic pneumonia, and ELISA for Toxocara antigen was positive, leading to a diagnosis of pulmonary toxocariasis. After a 10-day treatment course with albendazole and adjunctive steroids, the patient recovered completely without any sequelae. Pulmonary toxocariasis may be considered in patients with subacute or chronic pneumonia unresponsive to antibiotic agents, particularly in cases with eosinophilia.     

The emu oil emulsified in egg lecithin and butylated hydroxytoluene enhanced the proliferation,stemness gene expression,and in vitro wound healing of adipose-derived stem cells

In recent decades, mesenchymal stem cells originated from adipose tissue (adipose-derived stem cells, ASCs) have gained increased attention for production of cell-based therapeutics. Emu oil as a natural compound showed antioxidant effects in previous studies. The goal of this study was to investigate the effect of crude emu oil on the proliferation, cell cycle progression, stemness genes expression, and in vitro wound healing potential of ASCs. An emulsion of emu oil was prepared using egg lecithin and butylated hydroxytoluene to improve bioavailability and solubility of emu oil in the expansion medium. The ASCs were treated using a series of emu oil concentrations in emulsion form, diluted in expansion medium (0.03–3 mg/ml). The emu oil-free emulsion was used as control treatment. The results revealed that emu oil (1.25 mg/ml) in emulsion form significantly (p?<?0.001) increased ASCs proliferation and colony formation. Additionally, emu oil caused upregulation of stemness marker genes (Sox2, Oct4, Nanog, and Nestin) (p?<?0.05). The cell cycle analysis after emu oil treatments showed an increase in the population of ASCs in S-phase of the cell cycle. Besides, an accelerated in vitro scratch wound healing was observed in emu oil-treated ASCs. Emu oil enhanced proliferation, colony formation, stemness genes expression, and in vitro wound healing of ASCs. These findings suggest that emu oil treatment could maintain the stemness of ex vivo cultivated ASCs and enhance their regenerative potential.     

The Effects of Resveratrol,Metformin, Cold and Strength Training on the Level of Perilipin 5 in the Heart,Skeletal Muscle and Brown Adipose Tissues in Mouse

The high accumulation of lipid droplets in the cell is related to metabolic disorders, such as obesity. Perilipin 5 (Plin5), plays an important role in triglyceride hydrolysis in the lipid droplets. In this study, this protein has been evaluated in different tissues and conditions in mice. Fifty male mice were divided into 5 groups and treated for 45 days with Resveratrol, Metformin, strength training, and 4?°C cold. Brown adipose tissue (BAT), gastrocnemius skeletal muscle and heart were isolated for RNA extraction. The Plin5 gene expression was evaluated, using Real-Time PCR, and the plin5 was analyzed at the protein level, using western blot. In BAT, Resveratrol significantly reduced the plin5 protein level and gene expression (p?<?0.05). In heart tissue, Resveratrol and strength training, decreased (p?<?0.05) the plin5 expression, but Metformin increased the gene expression (p?<?0.05). In skeletal muscle, resveratrol, strength training, cold and Metformin significantly increased the plin5 expression at the gene and protein level (p?<?0.05). In BAT, Resveratrol has a greater effect in decreasing lipid deposits, compared with the strength training and cold; thus, it can play a better role in preventing lipid accumulation. In heart tissue, Resveratrol probably decreases insulin resistance, due to the increased expression of plin5 in skeletal muscle.     

Anti-hypercholesterolemic impacts of barley and date palm fruits on the ovary of Wistar albino rats and their offspring

A high cholesterol diet is related to ovarian dysfunction and infertility which has been increased among young ages consuming processed food products. The present study was conducted to evaluate the role of a high cholesterol diet on the ovaries of young female rats via assessments of histopathology, immunohistochemistry, oxidative stress and apoptic markers. Also, mating of hypercholesterolemic female rats was carried out to measure the fertility and numbers of their offspring. At the same time, phytotherapy was carried out through supplementing the diet with barley and/ or date palm fruits (10%) during the experiment to assess the phyto-therapeutic impacts in attenuation of drastic hypercholesterolemic effects.Hypercholesterolemic diet-fed rats exhibited damage of the ovarian follicles and increased follicular atresia. Furthermore, expression of cleaved caspase-3 was upregulated, while PCNA was downregulated in granulosa, theca and stroma cells. Hypercholesterolemic female rats showed marked depletion of antioxidative enzymes, increased lipid peroxidation and apoptotic markers. Alterations to the female serum hormones were detected. Offspring maternally fed on hypercholesterolemic diet showed a significant decrease of body weight and altered sex ratio. However, concomitant supplementation of barley and or date fruits to hypercholesterolemic groups revealed marked improvement of ovarian structure and function.On the basis of these evidences, it is believed that the enhanced synergistic effects of barley and/or date palm fruits in the amelioration of ovarian structure and functions were elicited by the potential antioxidant activity of their phytomicronutrients, polyphenols, β-glucan and trace elements. These materials scavenge free radicals from inflamed cells that can be used to establish an effective and novel therapeutic strategy for activating ovarian cell regeneration.