Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines

Background Fas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL) during luteal regression. In certain cell-types, sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically, keratin 8/18 (K8/K18) filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface. Methods Here, Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5, postovulation) and late stages (Days 16–18, postovulation) of CL function, and the relationship between Fas expression, K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated. Results Both total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P0.05, n=4 CL/stage), despite evidence these conditions increased Fas expression on HepG2 cells (P0.05) or stage of CL (P\u3e0.05, n= 4 CL/stage) on this outcome. Conclusion In conclusion, we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling, however, remain a possibility. Importantly, the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum, particularly during early luteal development

Chemokines in the corpus luteum: Implications of leukocyte chemotaxis

Chemokines are small molecular weight peptides responsible for adhesion, activation, and recruitment of leukocytes into tissues. Leukocytes are thought to influence follicular atresia, ovulation, and luteal function. Many studies in recent years have focused attention on the characterization of leukocyte populations within the ovary, the importance of leukocyte-ovarian cell interactions, and more recently, the mechanisms of ovarian leukocyte recruitment. Information about the role of chemokines and leukocyte trafficking (chemotaxis) during ovarian function is important to understanding paracrine-autocrine relationships shared between reproductive and immune systems. Recent advances regarding chemokine expression and leukocyte accumulation within the ovulatory follicle and the corpus luteum are the subject of this mini-review

Evaluating the impact of the hexosamine biosynthesis pathway and O-GlcNAcylation on glucose metabolism in bovine granulosa cells

Granulosa cells (GCs) of ovarian follicles prefer glucose as a metabolic substrate for growth and maturation. Disruption of glucose utilization via the hexosamine biosynthesis pathway (HBP) impairs O-linked N-acetylglucosaminylation (O-GlcNAcylation) and inhibits proliferation of bovine GCs of both small (3–5 mm) and large (\u3e8.5 mm) antral follicles. Knowing that 2–5% of all glucose in cells is utilized via the HBP, the aim of this study was to characterize glucose metabolism in bovine GCs and determine the impact of the HBP and O-GlcNAcylation on metabolic activity. The GCs were initially cultured in serum-containing medium to confluency and then sub-cultured in serum-free medium in 96 well plates (n = 10 ovary pairs). The cells were exposed to vehicle and inhibitors of the HBP and O-GlcNAcylation for 24 h. Extracellular acidification rate (ECAR; an indicator of glycolysis) and oxygen consumption rate (OCR; an indicator of oxidative phosphorylation) of the GCs were measured using a Seahorse xFe96 Analyzer, including the implementation of glycolytic and mitochondrial stress tests. GCs from small antral follicles exhibited overall greater metabolic activity than GCs from large antral follicles as evidenced by increased ECAR and OCR. Inhibition of the HBP and O-GlcNAcylation had no effect on the metabolic activity of GCs from either type of follicle. The glycolytic stress test indicated that GCs from both types of follicles possessed additional glycolytic capacity; but again, inhibition of the HBP and O-GlcNAcylation did not affect this. Interestingly, inhibition of cellular respiration by 2-Deoxy-D-glucose impaired OCR only in GCs from small antral follicles, but exposure to the mitochondrial stress test had no effect. Conversely, in GCs from large antral follicles, oxidative metabolism was impaired by the mitochondrial stress test and was accompanied by a concomitant increase in glycolytic metabolism. Immunodetection of glycolytic enzymes revealed that phosphofructokinase expression is increased in GCs of small antral follicles compared to large follicles. Inhibition of O-GlcNAcylation impaired the expression of hexokinase only in GCs of small antral follicles. Inhibition of O-GlcNAcylation also impaired the expression of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in GCs of both types of follicles, but had no effect on the expression of lactate dehydrogenase. The results indicate that GCs of small antral follicles possess greater aerobic glycolytic capacity than GCs from large antral follicles; but disruption of the HBP and O-GlcNAcylation has little to no impact on metabolic activity

Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells

Background: Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS). Methods: The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey’s post-test for multiple comparisons; differences among means were considered statistically significant at P \u3c 0.05. Results: Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (\u3c25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (\u3e 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P \u3c 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis. Conclusions: The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention

Global Trends in the Use of Insecticides to Control Vector-Borne Diseases

Background: Data on insecticide use for vector control are essential for guiding pesticide management systems on judicious and appropriate use, resistance management, and reduction of risks to human health and the environment

International Comparison of Vocational Rehabilitation for Persons With Spinal Cord Injury:Systems, Practices, and Barriers

Background: Employment rates among people with spinal cord injury or spinal cord disease (SCI/D) show considerable variation across countries. One factor to explain this variation is differences in vocational rehabilitation (VR) systems. International comparative studies on VR however are nonexistent. Objectives: To describe and compare VR systems and practices and barriers for return to work in the rehabilitation of persons with SCI/D in multiple countries. Methods: A survey including clinical case examples was developed and completed by medical and VR experts from SCI/D rehabilitation centers in seven countries between April and August 2017. Results: Location (rehabilitation center vs community), timing (around admission, toward discharge, or after discharge from clinical rehabilitation), and funding (eg, insurance, rehabilitation center, employer, or community) of VR practices differ. Social security services vary greatly. The age and preinjury occupation of the patient influences the content of VR in some countries. Barriers encountered during VR were similar. No participant mentioned lack of interest in VR among team members as a barrier, but all mentioned lack of education of the team on VR as a barrier. Other frequently mentioned barriers were fatigue of the patient (86%), lack of confidence of the patient in his/her ability to work (86%), a gap in the team's knowledge of business/legal aspects (86%), and inadequate transportation/accessibility (86%). Conclusion: VR systems and practices, but not barriers, differ among centers. The variability in VR systems and social security services should be considered when comparing VR study results

Ecology: a prerequisite for malaria elimination and eradication

* Existing front-line vector control measures, such as insecticide-treated nets and residual sprays, cannot break the transmission cycle of Plasmodium falciparum in the most intensely endemic parts of Africa and the Pacific * The goal of malaria eradication will require urgent strategic investment into understanding the ecology and evolution of the mosquito vectors that transmit malaria * Priority areas will include understanding aspects of the mosquito life cycle beyond the blood feeding processes which directly mediate malaria transmission * Global commitment to malaria eradication necessitates a corresponding long-term commitment to vector ecolog

Recruitment and retention of participants from socioeconomically deprived communities : lessons from the Awareness and Beliefs About Cancer (ABACus3) randomised controlled trial

Background Recruitment of research participants poses challenges in socioeconomically deprived areas. The Awareness and Beliefs About Cancer (ABACus) phase 3 Randomised Control Trial recruited adult participants from socioeconomically deprived areas using a combined healthcare/community engagement model. We report the strategies used to successfully recruit and retain our trial participant sample. Methods Community and healthcare settings in areas of high socioeconomic deprivation were identified by lay advisors who recruited participants opportunistically or by appointment. Follow-up was done by telephone or post at 2-weeks and 6-months after recruitment, and all participants were offered financial incentives. Qualitative interviews were conducted with lay advisors regarding their experience and reflections. Results The lay advisors identified and contacted 107 potential recruitment venues across South and West Yorkshire and South East Wales of which 41.1% (n = 42) were opened for recruitment. A total of 234 participants were recruited, with 91% (n = 212) retention at 2-weeks and 85% (n = 199) at 6-months. Community settings yielded 75% (n = 176) of participants. Participants had a mean age of 61.3 years and 63.3% (n = 148) were female, with 66% (n = 154) resident in the most deprived geographical areas. Lay advisors described recruitment as intensive, although engaging participants was easier in community settings. Conclusions The ABACus3 trial achieved recruitment and high retention with a population that is often “hard to reach” or entirely missed in health research. Strategies were specifically tailored to engage the venues and adult residents of highly deprived areas. Future studies recruiting adults living in the most deprived areas might benefit from community recruitment and from collaborating with local gatekeepers who are key to engagement. This study adheres to CONSORT guidelines. Trial registration Retrospectively registered with ISRCTN (http://www.isrctn.com/ISRCTN16872545) on 12.01.2018

Supporting physical activity engagement in people with Huntington's disease (ENGAGEHD): study protocol for a randomized controlled feasibility trial.

Background: Huntington’s disease (HD) is a complex, single-gene inherited neurodegenerative condition resulting in symptoms that occur across a wide range of neurological domains, including cognitive, behavioral and motor. The benefits of regular physical activity for people with HD are widely recognized. However, a number of factors can prohibit sustained exercise and activity. The purpose of this trial is to explore the feasibility, acceptability and effectiveness of a physical activity intervention program targeted for people with early- to mid-stage HD. Methods/Design: The proposed trial is a single blind, multisite, exploratory, randomized controlled feasibility trial of a physical activity intervention. A total of 62 participants with genetically confirmed HD will be recruited. Each participant will be involved in the trial for 26 weeks. Participants will be randomized immediately following the baseline assessment into either a physical activity intervention or a social contact control intervention. The physical activity intervention is framed around self-determination theory placed within a broader behaviour change wheel framework. An HD-specific workbook and individual goal setting will be utilized over six 1:1 sessions, with interim telephone calls. All participants will be reassessed at 16 weeks following the baseline assessment, and then again at a final follow-up assessment 26 weeks later. At the end of the study, all participants will be offered a brief version of the alternative intervention, with one home visit and one follow-up telephone call. Discussion: Engaging and supporting people with HD in a regular physical activity program raises a number of challenges. The physical activity intervention and the comparator social interaction intervention have been developed following consultation with people with HD and their families. Each are individually tailored and determined on individual needs and goals. The results from this trial will provide guidance for the development of definitive trials. Trial registration: The trial was registered with ISRCTN (http://www.isrctn.com/ISRCTN65378754) on 13 March 2014