Calcium alginate microencapsulation of ovarian follicles impacts FSH delivery and follicle morphology

BACKGROUND: We have previously shown that suspension culture prevents follicle flattening and maintains three-dimensional follicle architecture better than culture on flat plates. However, many of the follicles cultured in suspension do eventually rupture, as basement membrane integrity is lost and the three-dimensional structure of the follicle is altered. Therefore, the objective of this study is to support three-dimensional follicle architecture during in vitro growth of ovarian follicles through encapsulation in calcium alginate, while maintaining responsiveness to FSH stimulation. METHODS: Preantral follicles (150 – 160 micrometers in diameter) were isolated from the ovaries of juvenile rats and grown in culture tubes or encapsulated in calcium alginate and grown in culture tubes. Previous studies revealed that follicles maintained structural integrity but did not grow as well when encapsulated in calcium alginate. In these studies, we evaluated the effect of calcium alginate on FSH-stimulated follicle growth, survival, and morphology in suspension culture. Follicles were grown under 5 culture conditions: 1) not encapsulated; with FSH in the medium, 2) encapsulated in the absence of FSH, grown in medium without FSH, 3) encapsulated with calcium alginate containing FSH but grown in medium without FSH, 4) encapsulated without FSH but grown in medium containing FSH and 5) encapsulated with calcium alginate containing FSH and in medium containing FSH. To assess growth rates, follicles were cultured for 72 hours and analyzed for follicle size increase and DNA content. Survival analysis for encapsulated and unencapsulated follicles was performed by constructing a Kaplan Meier survival curve of daily observations of intact follicle survival. Three-dimensional architecture was assessed histologically and by analysis of the pattern of connexin 43 expression in the cultured follicles. RESULTS: In the absence of FSH, follicle diameter increased by only 6.4%. When FSH was included in the alginate bead alone or the media alone, the follicle diameter increased by 13.5% and 19.9% respectively. This was greater than follicles cultured in the absence of FSH (p < 0.05), but less than that of the FSH-treated unencapsulated follicles (p < 0.05). However, when follicles were cultured with FSH included in both the media and the bead, a 32.6% increase in follicle diameter was observed, statistically no different than the growth rate of the unencapsulated follicles grown with FSH. CONCLUSION: Microencapsulation supports three-dimensional follicle growth, but may limit access to hormones in the medium resulting in altered development compared to unencapsulated follicles. Inclusion of FSH in the alginate bead restores the follicle growth response to FSH, while also providing a scaffold of support for three-dimensional growth. The application of tissue engineering principles to the problems of follicle culture in vitro may provide advances applicable to fertility preservation in women and endangered species

Activation of the LH receptor up regulates the type 2 adiponectin receptor in human granulosa cells

PURPOSE: Adiponectin is a predominantly adipocyte-derived hormone which influences insulin sensitivity and energy homeostasis through at least two receptors, AdipoR1 and AdipoR2. In animal models, adiponectin may regulate ovarian steroidogenesis, folliculogenesis, and ovulation. The receptors AdipoR1 and AdipoR2 are present in the human ovary, but their regulation is unknown. In these studies, we determined the effects of LH receptor activation on the expression and function of the two adiponectin receptors in human granulosa cells. METHODS: Granulosa cells were obtained at the time of oocyte retrieval in women undergoing in vitro fertilization (IVF). Cells were isolated and cultured for 48 h in DMEM/F12 medium with 5 % FBS and 50 ug/ml gentamicin. Medium was changed to low serum for 12 h and cells were treated with hCG (100 ng/ml), forskolin (30 μMol/L), or FSH (1 IU/ml) for 24 h for mRNA experiments. mRNA was isolated and RT PCR was performed using Taqman assays and quantification with the delta delta CT method. For immunocytochemistry, cells were grown on chamber slides and treated with hCG for 1 to 24 h and fixed with acetone. ICC was performed with polyclonal rabbit primary antibodies followed by alexa fluor goat anti-rabbit antibody and imaging with a fluorescence microscope and Zeiss software analysis. 3β-hydroxysteroid dehydrogenase (3βHSD) enzyme activity was determined by measuring the progesterone produced when cells were provided with an excess of 22-hydroxy-cholesterol as substrate following an incubation with hCG (1 IU/ml) and/or adiponectin (10 ng/ml). Progesterone content in the media was determined by ELISA. RESULTS: Messenger RNA for the two Adiponectin receptors is differentially regulated by activation of LHR with hCG treatment. AdipoR2 was increased nearly 4-fold (p < 0.05), whereas AdipoR1 expression was not changed by hCG treatment. Treatment with either FSH or forskolin (an activator of cAMP) had similar effects. Basal AdipoR2 protein was fairly low in granulosa cells in culture however treatment of cells with hCG resulted in a discernible increase in immunodetectable cytoplasmic protein as early as 6 h after treatment and was maintained for at least 24 h. The number of cells positive for AdipoR2 at 6 h increased from a basal of 20 % to almost 60 % (p < 0.05). Adiponectin treatment of hCG-primed cells resulted in increased 3βHSD activity by approximately 60 % over hCG alone and more than 3-fold over basal levels. CONCLUSIONS: AdipoR2 is regulated by the LH receptor function via a cAMP dependant mechanism. Increased expression of adipoR2 prior to and following ovulation may contribute to enhanced 3βHSD activity and increased progesterone secretion by the corpus luteum of the ovary. Dysregulation of adiponectin that may occur with PCOS may impair normal progesterone production

The Australia Telescope 20GHz (AT20G) Survey: analysis of the extragalactic source sample

The Australia Telescope 20 GHz (AT20G) survey is a blind survey of the whole Southern sky at 20 GHz with follow-up observations at 4.8, 8.6, and 20 GHz carried out with the Australia Telescope Compact Array (ATCA). In this paper we present an analysis of radio spectral properties in total intensity and polarisation, sizes, optical identifications, and redshifts of the sample of the 5808 extragalactic sources in the survey catalogue of confirmed sources over the whole Southern sky excluding the strip at Galactic latitude |b|<1.5deg. The sample has a flux density limit of 40 mJy. Completeness has been measured as a function of scan region and flux density. Averaging over the whole survey area the follow-up survey is 78% complete above 50mJy and 93% complete above 100mJy. 3332 sources with declination <-15deg have good quality almost simultaneous observations at 4.8, 8.6, and 20GHz. The spectral analysis shows that the sample is dominated by flat-spectrum sources. The fraction of flat-spectrum sources decreases from 81% for 20GHz flux densities S>500mJy, to 60% for S<100mJy. There is also a clear spectral steepening at higher frequencies with the median spectral index decreasing from -0.16 between 4.8 and 8.6GHz to -0.28 between 8.6 and 20GHz. Simultaneous observations in polarisation are available for all the sources at all the frequencies. 768 sources have a good quality detection of polarised flux density at 20GHz; 467 of them were also detected in polarisation at 4.8 and/or at 8.6GHz so that it has been possible to compare the spectral behaviour in total intensity and polarisation. We have found that the polarised fraction increases slightly with frequency and decreases with flux density. Cross matches and comparisons have been made with other catalogues at lower radio frequencies, and in the optical, X-ray and gamma-ray bands. Redshift estimates are available for 825 sources.Comment: 15 pages, 16 figures, accepted for publication in MNRA

Quantity of alcohol drinking positively correlates with serum levels of endotoxin and markers of monocyte activation

It is unknown if LPS (lipopolysaccharides) and markers of immune activation, soluble CD14 (sCD14) and CD163 (sCD163) are associated with the quantity of alcohol consumption. 148 subjects were enrolled (97 excessive drinkers (ED) and 51 controls). Time Line Follow-Back questionnaire was used to quantify the amount of alcohol consumed. Serum LPS, sCD14, and sCD163 were measured. Peripheral blood mononuclear cells (PBMCs) were also isolated. Compared to controls, ED had higher total drinks in the past 30 days, higher levels of LPS, sCD14 and sCD163. The levels of serum LPS, sCD14, and sCD163 were higher among ED with recent alcohol consumption (last drink <10 days before enrollment) compared to those without recent drinking. Similar bacterial genome copy numbers were detected in control and ED groups. We found that ethanol primed PBMCs for LPS-induced inflammatory responses. A positive correlation between serum LPS, sCD14, sCD163 and the quantity of alcohol drinking was observed after adjusting for covariates and that abstinence was associated with decline in the levels of LPS, sCD14 and sCd163. We found an increase in the levels of LPS and markers of monocyte activations in ED. Further studies are needed to determine whether these can be used as the biomarkers for excessive alcohol use

Sex differences and drug dose influence the role of the alpha 7 nicotinic acetylcholine receptor in the mouse dextran sodium sulfate-induced colitis model

Introduction: alpha 7 nicotinic acetylcholine receptors (nAChRs) play an important role in vagus nerve-based cholinergic anti-inflammatory effects. This study was designed to assess the role of alpha 7 nAChRs in dextran sodium sulfate (DSS)-induced colitis in male and female mouse. We first compared disease activity and pathogenesis of colitis in alpha 7 knockout and wild-type mice. We then evaluated the effect of several alpha 7 direct and indirect agonists on the severity of disease in the DSS-induced colitis. Methods: Male and female adult mice were administered 2.5% DSS solution freely in the drinking water for 7 consecutive days and the colitis severity (disease activity index) was evaluated as well as colon length, colon histology, and levels of tumor necrosis factor-alpha colonic levels. Results: Male, but not female, alpha 7 knockout mice displayed a significantly increased colitis severity and higher tumor necrosis factor-alpha levels as compared with their littermate wild-type mice. Moreover, pretreatment with selective alpha 7 ligands PHA-543613, choline, and PNU-120596 decreased colitis severity in male but not female mice. The anti-colitis effects of these alpha 7 compounds dissipated when administered at higher doses. Conclusions: Our results suggest the presence of a alpha 7-dependent anti-colitis endogenous tone in male mice. Finally, our results show for the first time that female mice are less sensitive to the anticolitis activity of alpha 7 agonists. Ovarian hormones may play a key role in the sex difference effect of alpha 7 nAChRs modulation of colitis in the mouse. Implications: Our collective results suggest that targeting alpha 7 nAChRs could represent a viable therapeutic approach for intestinal inflammation diseases such as ulcerative colitis with the consideration of sex differences.United States Department of Health & Human Services National Institutes of Health (NIH) - USA - DA-019377 - DK046367United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - R01DA03697

Children\u27s experiences of companion animal maltreatment in households characterized by intimate partner violence

Cruelty toward companion animals is a well-documented, coercive tactic used by abusive partners to intimidate and control their intimate partners. Experiences of co-occurring violence are common for children living in families with intimate partner violence (IPV) and surveys show that more than half are also exposed to abuse of their pets. Given children\u27s relationships with their pets, witnessing such abuse may be traumatic for them. Yet little is known about the prevalence and significance of this issue for children. The present study examines the experiences of children in families with co-occurring pet abuse and IPV. Using qualitative methods, 58 children ages 7–12 who were exposed to IPV were asked to describe their experiences of threats to and harm of their companion animals. Following the interviews, template analysis was employed to systematically develop codes and themes. Coding reliability was assessed using Randolph\u27s free-marginal multirater kappa (kfree = .90). Five themes emerged from the qualitative data, the most common being children\u27s exposure to pet abuse as a power and control tactic against their mother in the context of IPV. Other themes were animal maltreatment to discipline or punish the pet, animal cruelty by a sibling, children intervening to prevent pet abuse, and children intervening to protect the pet during a violent episode. Results indicate that children\u27s experiences of pet abuse are multifaceted, potentially traumatic, and may involve multiple family members with diverse motives

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affectcartilage development

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the roleof Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre,Osx1-Cre, and Col2a1-Cre drivers, respectively. Wild-type and conditional knockout mice were phenotypically assessed by grossmorphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated usingRNA-sequencing, histologic evaluation, and western blotting. Aged mice with Ezh2 deficiency were also evaluated for prematuredevelopment of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age, although caused no other gross developmentaleffects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3lysine 27 (H3K27me3) and altered differentiation in vitro. RNA-seq analysis revealed enrichment of an osteogenic gene expressionprofile in Ezh2 deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes withoutinducing excessive hypertrophy or premature osteoarthritis in vivo. In summary, loss of Ezh2 reduced H3K27me3 levels, increased expression of osteogenic genes in chondrocytes, and resulted ina transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondralossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage-commitment

The Australia Telescope 20 GHz Survey: The Source Catalogue

We present the full source catalogue from the Australia Telescope 20 GHz (AT20G) Survey. The AT20G is a blind radio survey carried out at 20 GHz with the Australia Telescope Compact Array (ATCA) from 2004 to 2008, and covers the whole sky south of declination 0 deg. The AT20G source catalogue presented here is an order of magnitude larger than any previous catalogue of high-frequency radio sources, and includes 5890 sources above a 20 GHz flux-density limit of 40 mJy. All AT20G sources have total intensity and polarisation measured at 20 GHz, and most sources south of declination -15 deg also have near-simultaneous flux-density measurements at 5 and 8 GHz. A total of 1559 sources were detected in polarised total intensity at one or more of the three frequencies. We detect a small but significant population of non-thermal sources that are either undetected or have only weak detections in low-frequency catalogues. We introduce the term Ultra-Inverted Spectrum (UIS) to describe these radio sources, which have a spectral index alpha(5, 20) > +0.7 and which constitute roughly 1.2 per cent of the AT20G sample. The 20 GHz flux densities measured for the strongest AT20G sources are in excellent agreement with the WMAP 5-year source catalogue of Wright et al. (2009), and we find that the WMAP source catalogue is close to complete for sources stronger than 1.5 Jy at 23 GHz.Comment: 21 pages, accepted for publication in MNRA

The association between family and community social capital and health risk behaviours in young people: an integrative review

Background: Health risk behaviours known to result in poorer outcomes in adulthood are generally established in late childhood and adolescence. These ‘risky’ behaviours include smoking, alcohol and illicit drug use and sexual risk taking. While the role of social capital in the establishment of health risk behaviours in young people has been explored, to date, no attempt has been made to consolidate the evidence in the form of a review. Thus, this integrative review was undertaken to identify and synthesise research findings on the role and impact of family and community social capital on health risk behaviours in young people and provide a consolidated evidence base to inform multi-sectorial policy and practice.&lt;p&gt;&lt;/p&gt; Methods: Key electronic databases were searched (i.e. ASSIA, CINAHL, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, Embase, Medline, PsycINFO, Sociological Abstracts) for relevant studies and this was complemented by hand searching. Inclusion/exclusion criteria were applied and data was extracted from the included studies. Heterogeneity in study design and the outcomes assessed precluded meta-analysis/meta-synthesis; the results are therefore presented in narrative form.&lt;p&gt;&lt;/p&gt; Results: Thirty-four papers satisfied the review inclusion criteria; most were cross-sectional surveys. The majority of the studies were conducted in North America (n=25), with three being conducted in the UK. Sample sizes ranged from 61 to 98,340. The synthesised evidence demonstrates that social capital is an important construct for understanding the establishment of health risk behaviours in young people. The different elements of family and community social capital varied in terms of their saliency within each behavioural domain, with positive parent–child relations, parental monitoring, religiosity and school quality being particularly important in reducing risk.&lt;p&gt;&lt;/p&gt; Conclusions: This review is the first to systematically synthesise research findings about the association between social capital and health risk behaviours in young people. While providing evidence that may inform the development of interventions framed around social capital, the review also highlights key areas where further research is required to provide a fuller account of the nature and role of social capital in influencing the uptake of health risk behaviours.&lt;p&gt;&lt;/p&gt

Cardiopulmonary toxicity of peat wildfire particulate matter and the predictive utility of precision cut lung slices

BackgroundEmissions from a large peat fire in North Carolina in 2008 were associated with increased hospital admissions for asthma and the rate of heart failure in the exposed population. Peat fires often produce larger amounts of smoke and last longer than forest fires, however few studies have reported on their toxicity. Moreover, reliable alternatives to traditional animal toxicity testing are needed to reduce the number of animals required for hazard identification and risk assessments.MethodsSize-fractionated particulate matter (PM; ultrafine, fine, and coarse) were obtained from the peat fire while smoldering (ENCF-1) or when nearly extinguished (ENCF-4). Extracted samples were analyzed for chemical constituents and endotoxin content. Female CD-1 mice were exposed via oropharyngeal aspiration to 100μg/mouse, and assessed for relative changes in lung and systemic markers of injury and inflammation. At 24h post-exposure, hearts were removed for ex vivo functional assessments and ischemic challenge. Lastly, 8mm diameter lung slices from CD-1 mice were exposed (11μg) ± co-treatment of PM with polymyxin B (PMB), an endotoxin-binding compound.ResultsOn an equi-mass basis, coarse ENCF-1PM had the highest endotoxin content and elicited the greatest pro-inflammatory responses in the mice including: increases in bronchoalveolar lavage fluid protein, cytokines (IL-6, TNF-α, and MIP-2), neutrophils and intracellular reactive oxygen species (ROS) production. Exposure to fine or ultrafine particles from either period failed to elicit significant lung or systemic effects. In contrast, mice exposed to ENCF-1 ultrafine PM developed significantly decreased cardiac function and greater post-ischemia-associated myocardial infarction. Finally, similar exposures to mouse lung slices induced comparable patterns of cytokine production; and these responses were significantly attenuated by PMB.ConclusionsThe findings suggest that exposure to coarse PM collected during a peat fire causes greater lung inflammation in association with endotoxin and ROS, whereas the ultrafine PM preferentially affected cardiac responses. In addition, lung tissue slices were shown to be a predictive, alternative assay to assess pro-inflammatory effects of PM of differing size and composition. Importantly, these toxicological findings were consistent with the cardiopulmonary health effects noted in epidemiologic reports from exposed populations