Hyperglycemia impedes definitive endoderm differentiation of human embryonic stem cells by modulating histone methylation patterns.

Exposure to maternal diabetes during fetal growth is a risk factor for the development of type II diabetes (T2D) in later life. Discovery of the mechanisms involved in this association should provide valuable background for therapeutic treatments. Early embryogenesis involves epigenetic changes including histone modifications. The bivalent histone methylation marks H3K4me3 and H3K27me3 are important for regulating key developmental genes during early fetal pancreas specification. We hypothesized that maternal hyperglycemia disrupted early pancreas development through changes in histone bivalency. A human embryonic stem cell line (VAL3) was used as the cell model for studying the effects of hyperglycemia upon differentiation into definitive endoderm (DE), an early stage of the pancreatic lineage. Hyperglycemic conditions significantly down-regulated the expression levels of DE markers SOX17, FOXA2, CXCR4 and EOMES during differentiation. This was associated with retention of the repressive histone methylation mark H3K27me3 on their promoters under hyperglycemic conditions. The disruption of histone methylation patterns was observed as early as the mesendoderm stage, with Wnt/ß-catenin signaling being suppressed during hyperglycemia. Treatment with Wnt/ß-catenin signaling activator CHIR-99021 restored the expression levels and chromatin methylation status of DE markers, even in a hyperglycemic environment. The disruption of DE development was also found in mouse embryos at day 7.5 post coitum from diabetic mothers. Furthermore, disruption of DE differentiation in VAL3 cells led to subsequent impairment in pancreatic progenitor formation. Thus, early exposure to hyperglycemic conditions hinders DE development with a possible relationship to the later impairment of pancreas specification.

Pre-stroke physical activity is associated with fewer post-stroke complications, lower mortality and a better long-term outcome.

BACKGROUND AND PURPOSE: Physical activity is associated with a reduced incidence of first-time stroke. However, few studies have examined the effect of pre-stroke physical activity on post-stroke complications and clinical outcomes. METHODS: A total of 39 835 cases of stroke registered in the nationwide stroke registry system of Taiwan between 2006 and 2009 were analyzed according to five levels of severity as determined by National Institutes of Health Stroke Scale score upon hospital admission. Pre-stroke physical activity was defined in the Taiwan Stroke Registry as dedicated leisure-time physical activity for at least 30 min/day for 3 days/week for more than 6 months. A Cox model was used to compare complications and outcomes between active and inactive groups. RESULTS: The active and inactive groups were similar in age distribution and stroke type distribution, but the active group had better National Institutes of Health Stroke Scale scores upon admission. The active group also had significantly fewer post-stroke complications. Active patients had lower hospital mortality and better functional outcomes upon discharge as per the modified Rankin Scale. Improved functional status in the active group was significant at 1, 3 and 6 months post-stroke. CONCLUSION: Dedicated leisure-time physical activity for at least 30 min/day, at least three times per week for more than 6 months was associated with decreased stroke severity, fewer post-stroke complications, lower mortality and better outcomes.

Population-based comparison of prognostic factors in invasive micropapillary and invasive ductal carcinoma of the breast.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a variant of breast carcinoma with a higher propensity for lymph node metastases compared with invasive ductal carcinoma (IDC). METHODS: Retrospective analysis of 636 IMPC and 297 735 IDC cases in the Surveillance, Epidemiology and End RESULTS database comparing disease-specific survival (DSS) and overall survival (OS) between IMPC and IDC. RESULTS: A higher percentage of IMPC cases (52.0%) had nodal metastases compared with IDC cases (34.6%). The 5-year DSS and OS for IMPC was 91.8% and 82.9%, respectively compared with 88.6% and 80.5% for IDC, respectively. For both IMPC and IDC, oestrogen-receptor positivity was associated with better survival, while having four or more positive lymph nodes or larger tumour size correlated with worse survival. Radiotherapy provided a survival benefit for both histological types. CONCLUSIONS: Despite IMPC's higher propensity for lymph node metastasis, IMPC has DSS and OS that compare favourably with IDC.

Microstructural remodeling of articular cartilage following defect repair by osteochondral autograft transfer.

OBJECTIVE: To assess collagen network alterations occurring with flow and other abnormalities of articular cartilage at medial femoral condyle (MFC) sites repaired with osteochondral autograft (OATS) after 6 and 12 months, using quantitative polarized light microscopy (qPLM) and other histopathological methods. DESIGN: The collagen network structure of articular cartilage of OATS-repaired defects and non-operated contralateral control sites were compared by qPLM analysis of parallelism index (PI), orientation angle (α) relative to the local tissue axes, and retardance (Γ) as a function of depth. qPLM parameter maps were also compared to ICRS and Modified O'Driscoll grades, and cell and matrix sub-scores, for sections stained with H&E and Safranin-O, and for Collagen-I and II. RESULTS: Relative to non-operated normal cartilage, OATS-repaired regions exhibited structural deterioration, with low PI and more horizontal α, and unique structural alteration in adjacent host cartilage: more aligned superficial zone, and reoriented deep zone lateral to the graft, and matrix disorganization in cartilage overhanging the graft. Shifts in α and PI from normal site-specific values were correlated with histochemical abnormalities and co-localized with changes in cell organization/orientation, cloning, or loss, indicative of cartilage flow, remodeling, and deterioration, respectively. CONCLUSIONS: qPLM reveals a number of unique localized alterations of the collagen network in both adjacent host and implanted cartilage in OATS-repaired defects, associated with abnormal chondrocyte organization. These alterations are consistent with mechanobiological processes and the direction and magnitude of cartilage strain.

Chinese American adolescents: perceived parenting styles and adolescents' psychosocial health.

BACKGROUND: Asian Americans are one of the fastest-growing minority groups in the USA, and Chinese constitute the largest group. Evidence suggests that Asian American adolescents experience higher levels of depressive symptoms than their same-gender white counterparts. Quantitative findings suggest associations between parenting factors and Chinese American adolescents' mental health. A qualitative understanding regarding Chinese American adolescents' perceived parenting styles and its relationship with adolescents' psychosocial health is warranted. AIM: To gain an in-depth understanding of Chinese American adolescents' perceived parenting styles and how parenting styles might influence adolescents' psychosocial health. METHODS: In this qualitative study, we recruited 15 Chinese American adolescents aged 12-17 years in a southwest metropolitan area. We conducted two focus group interviews. Participants also filled out a brief questionnaire that included their socio-demographic information, immigration history and level of acculturation. RESULTS: Participants reported perceiving that parents had high expectations about academic performance and moral values. They also perceived stricter family rules regarding choices of friends compared with their non-Asian peers. Parents tended to be more protective of girls than of boys. Both Chinese American boys and girls reported poor or ineffective communication with their parents, which contributed to increased conflict between parents and adolescents and emotional distress of the adolescents. CONCLUSIONS: The findings provide evidence for nurses to develop linguistically and culturally tailored resources (e.g. parent support groups, programs aimed to improving parent-child communication) or connect these families with existing resources to enhance parenting skills and consequently reduce emotional distress of their adolescent children.

Trans-stromal epithelial cyst after multiple lamellar keratoplasties - Case report and review of literature.

DESIGN: Single Observational Case Report. SETTING: A 67-year-old male of Persian descent had a complex systemic and ocular history prior to a right penetrating keratoplasty (PK) reported here. The clinical diagnoses leading to the PK included Cogan's syndrome, chronic uveitis, secondary glaucoma, and corneal stromal scarring, presumed secondary to a corneal ulcer diagnosed on the second visit to our clinic. The specimen described here had been in place for 11 months and 17 days after the third failed Descemet stripping endothelial keratoplasty (DSEK). Visual acuities had ranged from 20/100 to 20/400 in both eyes. Visual acuity in the right eye just before surgery was 20/400. Intraocular pressures were 22 mmHg in both eyes with functioning Ahmed glaucoma shunts. The stromal cyst was not suspected preoperatively and no clinical imaging was performed. STUDY METHODS: Histopathology including serial sections and immunohistochemistry. RESULTS: Histologic study demonstrated a trans-stromal corneal epithelial cyst without goblet cells that extended through a 500 µm gap in the donor tissue surface and edge. CONCLUSIONS: This large stromal cyst was an unusual complication of serial posterior lamellar keratoplasties and we postulate that multiple prior posterior lamellar grafts may have been a risk factor for this complication. Anterior segment imaging with either anterior segment optical coherence tomography or high-resolution ultrasound would likely have detected this stromal cyst.

Let-7 derived from endometrial extracellular vesicles is an important inducer of embryonic diapause in mice.

Embryonic diapause is a maternally controlled phenomenon. The molecule controlling the onset of the phenomenon is unknown. We demonstrated that overexpression of microRNA let-7a or incubation with let-7g-enriched extracellular vesicles from endometrial epithelial cells prolonged the in vitro survival of mouse blastocysts, which developed into live pups after having been transferred to foster mothers. Similar to in vivo dormant blastocysts, let-7-induced dormant blastocysts exhibited low level of proliferation, apoptosis, and nutrient metabolism. Let-7 suppressed c-myc/mTORC1 and mTORC2 signaling to induce embryonic diapause. It also inhibited ODC1 expression reducing biosynthesis of polyamines, which are known to reactivate dormant embryos. Furthermore, the overexpression of let-7 blocked trophoblast differentiation and implantation potential of human embryo surrogates, and prolonged survival of human blastocysts in vitro, supporting the idea that embryonic diapause was an evolutionary conserved phenomenon. In conclusion, let-7 is the main factor inducing embryonic diapause.

Shared and persistent asymptomatic cutaneous human papillomavirus infections in healthy skin.

Cutaneous human papillomavirus (HPV) types are commonly found in normal skin, and some of them have been suspected to play a role in the development of non-melanoma skin cancer. This present study is divided into three sections, the aims of this study were to examine if certain HPV-types persist over time and if HPV-types are shared within families. From the first part of the study, swab samples from foreheads were collected for three longitudinal studies from one family with a newborn baby. Five specific HPV-types were isolated from the family with a newborn, with HPV-5 and FA67 being found at various time points and prevalence rates in all four members of the family. Part 2 consisted of a followed up study from two families with a 6 years interval. Six of the family members were found to have at least one of the HPV-types identified in the family 6 years earlier. Many of the HPV-types identified were shared within the families studied. Part 3 of this study involved weekly samples from four healthy females for 4 months. Among the four healthy individuals, 11%, 65%, and 56% of the weekly samples were HPV-DNA positive with one individual HPV-negative. All specimens were tested for HPV-DNA by PCR using the broad range HPV-type primer pair FAP59/64. The positive samples were HPV-type determined by cloning and sequencing. Specific cutaneous HPV-types persist over long periods of time in healthy skin in most individuals investigated and certain HPVs are shared between family members.

The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon.

OBJECTIVES: Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual's tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect. METHODS: Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay. RESULTS: The relative quantification (condition/control) of WST-1 assay on day seven revealed a significant decrease in tenocyte proliferation in small-diameter culture wells (96 and 24 wells) due to the gelling effect. PRFM in large-diameter culture wells (12 and six wells) and co-culture systems induced a significant increase in tenocyte proliferation compared with the control group. The gelling effect of PRFM was avoided by the co-culture device. CONCLUSION: When PRFM and tenocytes are cultured in small-diameter culture wells, the gelling effect will occur and make screening of personalized best-fit PRFM difficult. This effect can be avoided with the co-culture device.Cite this article: C-H. Chiu, P. Chen, W-L. Yeh, A. C-Y. Chen, Y-S. Chan, K-Y. Hsu, K-F. Lei. The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon. Bone Joint Res 2019;8:216-223. DOI: 10.1302/2046-3758.85.BJR-2018-0258.R1.

Autofluorescence reveals menstrual phase in the endometrium.

OBJECTIVE: Tissue contains fluorophores that autofluoresce without additional dye or photosensitizer with the appropriate light excitation. This technique has been widely applied for discrimination between normal and precancerous tissue. The aim of this study was to explore the capability and reliability of autofluorescence phase determination in samples of human endometrium. METHODS: A total of 70 measurement sites from 31 endometrial tissue samples from hysterectomy were enrolled. Xenon light (330 nm) was directed at the endometrial tissue and the resultant autofluorescence intensity recorded. Spectra were then grouped according to the proliferative and secretory phase, with multivariant analysis, partial least square (PLS) and analysis of variance (ANOVA) used for evaluation of the statistical significance of phase determination. RESULTS: Both proliferative and secretory autofluorescence spectra showed a similar characteristic triple-peak curve shape pattern, however, each of the intensities at the three peaks between the two phases varied markedly (p < 0.01). PLS analysis confirmed that collagen, NADH and FAD autofluorescence were the principle determinants of endometrial spectrum; the sensitivity and specificity of phase determination by autofluorescent was 100% and 97%, respectively. CONCLUSION: Autofluorescence measurement provides real-time information on endometrial phase status and, based on our results, it appears reasonable to suggest that it may be promising as a clinical tool for prompt phase interpretation.

Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts.

The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.

Effects of hyperbaric oxygen and platelet derived growth factor on medial collateral ligament fibroblasts.

PURPOSE: This study investigated hyperbaric oxygen (HBO2) and platelet-derived growth factor-BB (PDGF-BB) to determine their combined effects on fibroblasts from rabbit medial collateral ligament (MCL). METHOD: Cells were divided into four groups: (I) Control, (II) HBO2 treatment, (III) PDGF-BB treatment and (IV) HBO2 combined with PDGF-BB treatment. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber for 120 minutes per 48 hours. Measurement of cell growth was based on increase in cell number. Cell cycle modulations were analyzed by fluorescence-activated cell sorter (FACS). Quantity of Type I and Type III collagen was determined by western blotting and image analyzer. RESULTS: Treatment doses of HBO2 alone or PDGF-bb alone dependently increased cell growth. A combination of HBO2 treatment plus PDGF-bb treatment had an additive effect on cell growth in comparison with HBO2 treatment alone or PDGF-bb treatment alone. FACS analysis revealed that HBO2 alone, PDGF-bb alone and PDGF-bb plus HBO2 treatment increase the percentage of cells accumulated in S-phase. Western blotting analysis revealed that Type III collagen content was decreased significantly after HBO2 treatment alone or HBO2 plus PDGF-bb treatment but not in PDGF-bb treatment alone. In contrast, although Type I collagen content was increased after HBO2 treatment, the increase in Type I collagen (increase /original) was not statistically significant. CONCLUSION: HBO2 or HBO2 plus PDGF-bb treatment decreases the Type III collagen/Type I collagen content, which could result in mechanically stronger collagen fibrils. We propose HBO2 therapy as a potentially effective treatment for MCL healing.

Estimating active layer thickness and volumetric water content from ground penetrating radar measurements in Barrow, Alaska.

Ground penetrating radar (GPR) has emerged as an effective tool for estimating active layer thickness (ALT) and volumetric water content (VWC) within the active layer. In August 2013, we conducted a series of GPR and probing surveys using a 500 MHz antenna and metallic probe around Barrow, Alaska. We collected about 15 km of GPR data and 1.5 km of probing data. Here, we describe the GPR data processing workflow from raw GPR data to the estimated ALT and VWC. We include the corresponding uncertainties for each measured and estimated parameter. The estimated average GPR-derived ALT was 41 cm, with a standard deviation of 9 cm. The average probed ALT was 40 cm, with a standard deviation of 12 cm. The average GPR-derived VWC was 0.65, with a standard deviation of 0.14.

Human breast cancer cells and normal mammary epithelial cells: retinol metabolism and growth inhibition by the retinol metabolite 4-oxoretinol.

To understand the signaling and growth-inhibitory effects of retinoids, we have examined the metabolism of [3H]retinol in a number of estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) human breast cancer cell lines. We have also assayed the metabolism of [3H]retinol in normal human mammary epithelial cells. The ER+ breast cancer cell lines MCF-7 and T47D produce [3H]4-oxoretinol from [3H]retinol; the production of [3H]4-oxoretinol is increased by initial culture in the presence of nonradiolabeled retinoic acid (RA) or N-(4-hydroxyphenyl)retinamide, indicating that these drugs enhance [3H]retinol metabolism to [3H]4-oxoretinol. No metabolism of [3H]retinol to [3H]RA in these ER+ tumor lines was detected. ER- breast cancer lines MDA-MB-231, MDA-MB-468, and BT20 do not metabolize [3H]retinol to [3H]4-oxoretinol. In the ER- tumor lines, most of the [3H]retinol remains unmetabolized during the 24-h culture period; MDA-MB-468 and BT20 metabolize some [3H]retinol to [3H]RA. Unlike the majority of the tumor lines, the normal human breast epithelial cell strains AD074 and MCF10A rapidly metabolize [3H]retinol to [3H]retinyl esters. No detectable [3H]RA is produced from [3H]retinol in AD074 and MCF10A cells. Thus, the normal breast epithelial strains, the ER+ tumor lines and the ER- tumor lines differ greatly in their pathways of [3H]retinol metabolism. The levels of cellular retinol binding protein-I mRNA expression are not correlated with the levels or types of various retinol metabolites. Whereas the normal breast epithelial cells and the ER+ tumor lines are growth inhibited by RA, N-(4-hydroxyphenyl)retinamide, and 4-oxoretinol, only the 4-oxoretinol is growth inhibitory in the ER- tumor lines. The cellular retinoic acid-binding protein II mRNA levels are not correlated with the growth inhibition by RA or 4-oxoretinol in the normal and tumor lines.

Expression of the cAMP response element binding protein (CREB) in hippocampus produces an antidepressant effect.

BACKGROUND: Recent studies have demonstrated that chronic antidepressant treatment increases the expression of the cyclic amp (cAMP) response element binding protein (CREB) in rat hippocampus. The study presented here was conducted to determine if CREB is a relevant target that produces an antidepressant-like effect. METHODS: We employed the herpes simplex virus (HSV)-mediated gene transfer technique to overexpress CREB and determined its effect on the learned helplessness and forced swim tests, two established models used for pharmacological screening of antidepressant drugs. RESULTS: In the learned helplessness model, rats that received bilateral microinjection of HSV-CREB into the dentate gyrus showed significantly fewer escape failures in the subsequent conditioned avoidance test than those injected with control vector (HSV-LacZ). In contrast, microinjection of HSV-CREB in either the CA1 pyramidal cell layer of hippocampus or the prefrontal cortex did not produce an antidepressant response. In the forced swim test, CREB expression in the dentate gyrus resulted in a significantly shorter immobility time than those injected with HSV-LacZ. CONCLUSIONS: These results demonstrate that over-expression of CREB in hippocampus results in an antidepressant effect and suggest that CREB may serve as a potential molecular target for novel therapeutic agents.

Lack of evidence for close linkage of the glutamate GluR6 receptor gene with schizophrenia.

OBJECTIVE: Previous research has consistently implicated genetic factors in the pathogenesis of schizophrenia. It has been hypothesized that an abnormality in glutamatergic function is of etiologic importance in schizophrenia, and therefore the glutamate receptor family of genes are potential susceptibility loci for schizophrenia. To test this hypothesis the authors sought to detect linkage between the GluR6 glutamate receptor gene and schizophrenia. METHOD: Twenty-three English and Icelandic families containing multiple cases of schizophrenia were genotyped with a microsatellite trinucleotide repeat polymorphism localized at the GluR6 glutamate receptor locus. Lod scores, model-free linkage analysis, and extended relative pair analysis were used to test for linkage. RESULTS: No statistically significant evidence of linkage between GluR6 and schizophrenia was found. CONCLUSIONS: The results do not support the hypothesis that GluR6 allelic variants provide a major gene contribution to the etiology of schizophrenia in a large proportion of these pedigrees.