18†Engaging with young people to improve research, services and workforce development: eye-YPAG and 'visually' workshops.

Involving children and young people (CYP) in service and research design improves quality and accessibility. Running events in schools to invite CYP to volunteer and explore careers in the NHS may contribute to uptake of training posts and developing the NHS workforce.Here we evaluate two activities with CYP, our Young Person's Advisory Group for research (eye-YPAG) and our workshop for secondary schools, 'visually'.We evaluated eye-YPAG in focus groups and online surveys with group members, parents/carers, researchers, facilitators and funders. We conducted thematic analysis and descriptive statistics. To evaluate 'visually', we monitored the numbers of workshops and young people applying for volunteering roles. We asked those who started working with us about their experience.eye-YPAG members valued social and creative aspects as well as learning about research and developing skills and confidence. Researchers reported that CYP gave novel suggestions, modifying research plans, and that their different perspective was helpful in making research more relevant for children and families.Over 6 months, we held 15 'visually' workshops in secondary schools. Ninety students applied for volunteering roles, and 20 have completed the Human Resources onboarding process. Young volunteers report that this work has increased their confidence and that they have gained insights into how a hospital works. One is considering training to become an orthoptist.Both eye-YPAG and 'visually' are available to all eye researchers and units in the UK and can facilitate outreach activities.

Mediating effects of inequitable gender norms on intimate partner violence and contraceptive use in a cluster randomized control trial in Niger: A causal inference mediation analysis.

Background: Gender inequity, a deeply-rooted driver of poor health globally, is expressed in society through gender norms, the unspoken rules that govern gender-related roles and behavior. The development of public health interventions focused on promoting equitable gender norms are gaining momentum internationally, but there remain critical gaps in the evidence about how these interventions are working to change behavioral outcomes. Methods: A four-arm cluster randomized control trial (cRCT) was conducted to evaluate the effects of the Reaching Married Adolescents in Niger (RMA) intervention on modern contraceptive use and intimate partner violence (IPV) among married adolescent girls and their husbands in Dosso, Niger (T1: 1042 dyads; 24 mos. follow-up: 737 dyads, 2016-2019). This study seeks to understand if changes in perceived inequitable gender norms among husbands are the mechanism behind effects on modern contraceptive use and IPV. We estimated natural direct and indirect effects via these gender norms using inverse odds ratio weighting. An intention-to-treat approach and a difference-in-differences estimator in a hierarchical linear probability model was used to estimate prevalence differences, along with bootstrapping to estimate confidence intervals. Results: The total effects of the RMA small group intervention (Arm 2) is estimated to be an 8% reduction in prevalence of IPV [95% CI: -0.18, 0.01]. For this arm, the natural indirect effect through gender inequitable social norms is associated with a 2% decrease (95% CI: -0.07, 0.12), accounting for 22.3% of this total effect, and the natural direct effect with a 6% decrease (95% CI: -0.20, -0.02) in IPV. Of the total effect of the RMA household visit intervention (Arm 1) on contraceptive use (20% increase), indirect effects via inequitable gender norms were associated with an 11% decrease (95% CI: -0.18, -0.01) and direct effects with a 32% increase (95% CI: 0.13, 0.44) in contraceptive use. For the combination arm, of the total effects on contraceptive use (19% increase), indirect effects were associated with a 9% decrease (95% CI: -0.20, 0.02) and direct effects with a 28% increase (95% CI: 0.12, 0.46). Conclusion: The present study contributes experimental evidence that the small group RMA intervention reduced IPV partially via reductions in perceived inequitable gender norms among husbands. Evidence also suggests that increases in perceived inequitable gender norms resulted in decreased contraceptive use among those receiving the household visit intervention component. Not only do these results open the "black box" around how the RMA small group intervention may create behavior change to help inform its future use, they provide evidence supporting behavior change theories and frameworks that postulate the importance of changing underlying social norms in order to reduce IPV and increase modern contraceptive use.

Population niche width is driven by within-individual niche expansion and individual specialization in introduced brook trout in mountain lakes.

The width of a population's resource-use niche is determined by individual diet breadth ("within-individual component") and the degree of niche partitioning between individuals ("between-individual component"). The balance between these two factors affects ecological stability and evolutionary trajectories, and may shift as ecological opportunity permits broader population niches. Lakes in California's Sierra Nevada Mountains vary in resource diversity for introduced brook trout (Salvelinus fontinalis) due to elevation, lake morphometry, and watershed features. We compared the relative contributions of within- and between-individual niche components to two measures of the dietary niches of thirteen populations of brook trout: prey taxonomic composition and prey size distribution. For both taxonomic and size diversity of fish diets, population niche width was positively related to both the within- and between-individual components. For taxonomic diversity, the two components increased in parallel, while for size diversity, the between-individual component became more important relative to the within-individual component in populations with the greatest niche widths. Our results support the Niche Variation Hypothesis that populations with broader niches are more heterogeneous among individuals and show that individual niche width and individual specialization can operate in parallel to expand the population niche.

Comparison of visual and computerised antenatal cardiotocography in the prevention of perinatal morbidity and mortality. A systematic review and meta-analysis.

INTRODUCTION: Antenatal cardiotocography (CTG) is used to monitor fetal well-being. There are two methods: visual (vCTG) or computerised (cCTG). An earlier Cochrane review compared the effects of both approaches on maternal and fetal outcomes. The objective of this systematic review was to update this search and identify studies not included in the Cochrane review. MATERIALS AND METHODS: MEDLINE, EMBASE, CINAHL and MIDIRS databases were searched up to February 2021. We included randomised controlled trials (RCT) and non-randomised studies (NRS) of pregnant women receiving antenatal CTG with comparison of cCTG to vCTG and clinical outcomes. The Cochrane Risk of Bias Tool and Joanna Briggs Institute Critical Appraisal Checklist were used for quality assessment. Data is presented as risk ratios with 95% confidence intervals and I2 is used as the statistical measure of heterogeneity. RESULTS: Three RCTs and three NRS were included. Meta-analysis of RCTs demonstrated a non-significant reduction in all-cause perinatal mortality (RR 0.23 [95%CI 0.04-1.30]), preventable perinatal mortality excluding congenital anomalies (RR 0.27 [95% CI 0.05-1.56]) and cesarean section (RR 0.91 [95%CI 0.68-1.22]). All RCTs included high-risk women and had a high risk of bias. There was one antenatal stillbirth across the three RCTs (n = 497). The NRS were at high-risk of bias and statistical analysis was not possible due to heterogeneity. Individual findings suggest reduced investigation and better prediction of neonatal outcomes with cCTG. CONCLUSIONS: There is a non-significant reduction in perinatal mortality with cCTG. Despite no clear reduction in perinatal mortality and morbidity with cCTG, it is objective and may reduce time spent in hospital and further investigations for women.

Investigating the electronic structure of confined multiexcitons with nonlinear spectroscopies.

Strong confinement in semiconductor quantum dots enables them to host multiple electron-hole pairs or excitons. The excitons in these materials are forced to interact, resulting in quantum-confined multiexcitons (MXs). The MXs are integral to the physics of the electronic properties of these materials and impact their key properties for applications such as gain and light emission. Despite their importance, the electronic structure of MX has yet to be fully characterized. MXs have a complex electronic structure arising from quantum many-body effects, which is challenging for both experiments and theory. Here, we report on the investigation of the electronic structure of MX in colloidal CdSe QDs using time-resolved photoluminescence, state-resolved pump-probe, and two-dimensional spectroscopies. The use of varying excitation energy and intensities enables the observation of many signals from biexcitons and triexcitons. The experiments enable the study of MX structures and dynamics on time scales spanning 6 orders of magnitude and directly reveal dynamics in the biexciton manifold. These results outline the limits of the simple concept of binding energy. The methods of investigations should be applicable to reveal complex many-body physics in other nanomaterials and low-dimensional materials of interest.

Combinatorial metabolism drives the naive to primed pluripotent chromatin landscape.

Since epigenetic modifications are a key driver for cellular differentiation, the regulation of these modifications is tightly controlled. Interestingly, recent studies have revealed metabolic regulation for epigenetic modifications in pluripotent cells. As metabolic differences are prominent between naive (pre-implantation) and primed (post-implantation) pluripotent cells, the epigenetic changes regulated by metabolites has become an interesting topic of analysis. In this review we discuss how combinatorial metabolic activities drive the developmental progression through early pluripotent stages.

Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency.

To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.

Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries exceed the considerable intrinsic regenerative capacity of skeletal muscle, resulting in permanent functional and cosmetic deficits. VML and VML-like injuries occur in military and civilian populations, due to trauma and surgery as well as due to a host of congenital and acquired diseases/syndromes. Current therapeutic options are limited, and new approaches are needed for a more complete functional regeneration of muscle. A potential solution is human hair-derived keratin (KN) biomaterials that may have significant potential for regenerative therapy. The goal of these studies was to evaluate the utility of keratin hydrogel formulations as a cell and/or growth factor delivery vehicle for functional muscle regeneration in a surgically created VML injury in the rat tibialis anterior (TA) muscle. VML injuries were treated with KN hydrogels in the absence and presence of skeletal muscle progenitor cells (MPCs), and/or insulin-like growth factor 1 (IGF-1), and/or basic fibroblast growth factor (bFGF). Controls included VML injuries with no repair (NR), and implantation of bladder acellular matrix (BAM, without cells). Initial studies conducted 8 weeks post-VML injury indicated that application of keratin hydrogels with growth factors (KN, KN+IGF-1, KN+bFGF, and KN+IGF-1+bFGF, n = 8 each) enabled a significantly greater functional recovery than NR (n = 7), BAM (n = 8), or the addition of MPCs to the keratin hydrogel (KN+MPC, KN+MPC+IGF-1, KN+MPC+bFGF, and KN+MPC+IGF-1+bFGF, n = 8 each) (p < 0.05). A second series of studies examined functional recovery for as many as 12 weeks post-VML injury after application of keratin hydrogels in the absence of cells. A significant time-dependent increase in functional recovery of the KN, KN+bFGF, and KN+IGF+bFGF groups was observed, relative to NR and BAM implantation, achieving as much as 90% of the maximum possible functional recovery. Histological findings from harvested tissue at 12 weeks post-VML injury documented significant increases in neo-muscle tissue formation in all keratin treatment groups as well as diminished fibrosis, in comparison to both BAM and NR. In conclusion, keratin hydrogel implantation promoted statistically significant and physiologically relevant improvements in functional outcomes post-VML injury to the rodent TA muscle.

WNT/ß-catenin signaling regulates mitochondrial activity to alter the oncogenic potential of melanoma in a PTEN-dependent manner.

Aberrant regulation of WNT/ß-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/ß-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/ß-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/ß-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/ß-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.

Drosophila melanogaster as a Model of Muscle Degeneration Disorders.

Drosophila melanogaster provides a powerful platform with which researchers can dissect complex genetic questions and biochemical pathways relevant to a vast array of human diseases and disorders. Of particular interest, much work has been done with flies to elucidate the molecular mechanisms underlying muscle degeneration diseases. The fly is particularly useful for modeling muscle degeneration disorders because there are no identified satellite muscle cells to repair adult muscle following injury. This allows for the identification of endogenous processes of muscle degeneration as discrete events, distinguishable from phenotypes due to the lack of stem cell-based regeneration. In this review, we will discuss the ways in which the fruit fly provides a powerful platform with which to study human muscle degeneration disorders.

Multi-stakeholder perspectives of locally commissioned enhanced optometric services.

OBJECTIVES: To explore views of all stakeholders (patients, optometrists, general practitioners (GPs), commissioners and ophthalmologists) regarding the operation of community-based enhanced optometric services. DESIGN: Qualitative study using mixed methods (patient satisfaction surveys, semi-structured telephone interviews and optometrist focus groups). SETTING: A minor eye conditions scheme (MECS) and glaucoma referral refinement scheme (GRRS) provided by accredited community optometrists. PARTICIPANTS: 189 patients, 25 community optometrists, 4 glaucoma specialist hospital optometrists (GRRS), 5 ophthalmologists, 6 GPs (MECS), 4 commissioners. RESULTS: Overall, 99% (GRRS) and 100% (MECS) patients were satisfied with their optometrists' examination. The vast majority rated the following as 'very good'; examination duration, optometrists' listening skills, explanations of tests and management, patient involvement in decision-making, treating the patient with care and concern. 99% of MECS patients would recommend the service. Manchester optometrists were enthusiastic about GRRS, feeling fortunate to practise in a 'pro-optometry' area. No major negatives were reported, although both schemes were limited to patients resident within certain postcode areas, and some inappropriate GP referrals occurred (MECS). Communication with hospitals was praised in GRRS but was variable, depending on hospital (MECS). Training for both schemes was valuable and appropriate but should be ongoing. MECS GPs were very supportive, reporting the scheme would reduce secondary care referral numbers, although some MECS patients were referred back to GPs for medication. Ophthalmologists (MECS and GRRS) expressed very positive views and widely acknowledged that these new care pathways would reduce unnecessary referrals and shorten patient waiting times. Commissioners felt both schemes met or exceeded expectations in terms of quality of care, allowing patients to be seen quicker and more efficiently. CONCLUSIONS: Locally commissioned schemes can be a positive experience for all involved. With appropriate training, clear referral pathways and good communication, community optometrists can offer high-quality services that are highly acceptable to patients, health professionals and commissioners.

Evaluation of a minor eye conditions scheme delivered by community optometrists.

BACKGROUND: The establishment of minor eye conditions schemes (MECS) within community optometric practices provides a mechanism for the timely assessment of patients presenting with a range of acute eye conditions. This has the potential to reduce waiting times and avoid unnecessary referrals to hospital eye services (HES). OBJECTIVE: To evaluate the clinical effectiveness, impact on hospital attendances and patient satisfaction with a minor eye service provided by community optometrists. METHODS: Activity and outcome data were collected for 12 months in the Lambeth and Lewisham MECS. A patient satisfaction questionnaire was given to patients at the end of their MECS appointment. A retrospective difference-in-differences analysis of hospital activity compared changes in the volume of referrals by general practitioners (GPs) from a period before (April 2011-March 2013) to after (April 2013-March 2015) the introduction of the scheme in Lambeth and Lewisham relative to a neighbouring area (Southwark) where the scheme had not been commissioned. Appropriateness of case management was assessed by consensus using clinical members of the research team. RESULTS: A total of 2123 patients accessed the scheme. Approximately two-thirds of patients (67.5%) were referred by their GP. The commonest reasons for patients attending for a MECS assessment were 'red eye' (36.7% of patients), 'painful white eye' (11.1%) and 'flashes and floaters' (10.2%). A total of 64.1% of patients were managed in optometric practice and 18.9% were referred to the HES; of these, 89.2% had been appropriately referred. First attendances to HES referred by GPs reduced by 26.8% (95% CI -40.5% to -13.1%) in Lambeth and Lewisham compared to Southwark. CONCLUSIONS: The Lambeth and Lewisham MECS demonstrates clinical effectiveness, reduction in hospital attendances and high patient satisfaction and represents a successful collaboration between commissioners, local HES units and primary healthcare providers.

Can you ever collect too many oocytes?

STUDY QUESTION: Does the chance of pregnancy keep improving with increasing number of oocytes, or can you collect too many? SUMMARY ANSWER: Clinical pregnancy (CP) and live birth (LB) rates per embryo transfer varied from 10.2 and 9.2% following one oocyte collected to 37.7 and 31.3% when >16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same with number of oocytes collected. WHAT IS KNOWN ALREADY: It has been suggested that if >15 oocytes are collected, the success rate for fresh embryo transfers decreases. As this is counterintuitive, as more oocytes should result in more embryos, with a better choice of quality embryos, we decided to analyse the recent experience in a busy IVF unit. STUDY DESIGN, SIZE DURATION: A retrospective analysis of clinical pregnancy and live birth outcome, with respect to number of oocytes collected at Monash IVF for the 2-year period between August 2010 and July 2012, where patients under the age of 45 years underwent a fresh embryo transfer. This included 7697 stimulated cycles for IVF and ICSI. PARTICIPANT/MATERIALS, SETTING, METHODS: Statistical analysis involved data tables and graphs comparing oocyte number with outcome. Results of women who had their first oocyte collection with an embryo transfer within the reference period were analysed by logistic regression analysis including other covariates that might influence pregnancy outcome. Analysis was also carried out of all the 7679 oocyte collections undertaken, resulting in fresh embryo transfers by generalized estimating equations to allow for the within subject correlation in outcomes for repeated treatments. MAIN RESULTS AND THE ROLE OF CHANCE: The number of oocytes collected varied from 1 to 48. Clinical pregnancy and live birth rates per embryo transfer varied from 10.2 and 9.2% when only one oocyte was collected to 37.7 and 31.3% when >16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same or with the number of oocytes collected. The percentage of women with embryos cryopreserved increased from under 20% with <4 oocytes collected to over 70% with >16 oocytes collected. There was a slight increase (from 18 to 22%) in oocyte immaturity and a more marked increase (from 0 to 3%) in cancelling fresh transfers to prevent Ovarian Hyperstimulation Syndrome (OHSS) with increase in number of oocytes collected above 16. The results of this study suggest that you cannot collect too many oocytes as both clinical pregnancy and live birth rates do not decrease with high numbers of oocytes collected. However, once >15 oocytes are collected, everything gets quite uncertain. LIMITATIONS, REASONS FOR CAUTION: As the data become sparse above 15 oocytes, we could not demonstrate a significant increase in pregnancy rates above this number. Larger studies would be required to answer the question whether there is a plateau, or rates continue to increase. The negative of aggressive stimulation to produce many oocytes is that the risk of OHSS increases, and this is the most serious complication of ovarian stimulation. STUDY FUNDING/COMPLETING OF INTERESTS: No funding was required. There is no conflict of interest, except that G.K., V.M. and C.M. are shareholders in Monash IVF Pty Ltd.

Widespread correction of central nervous system disease after intracranial gene therapy in a feline model of Sandhoff disease.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-ß-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

Imaging of frequent emergency department users with alcohol use disorders.

BACKGROUND: Patients with altered level of consciousness secondary to alcohol use disorders (AUDs) often undergo imaging in the emergency department (ED), although the frequency and yield of this practice over time are unknown. STUDY OBJECTIVES: We describe the use of imaging, the associated ionizing radiation exposure, cumulative costs, and identified acute and chronic injuries and abnormalities among frequent users of the ED with AUDs. METHODS: This is a retrospective case series of individuals identified through an administrative database having 10 or more annual ED visits in 2 consecutive years who were prospectively followed for a third year. International Classification of Diseases, 9(th) Revision, Clinical Modification and Current Procedural Terminology codes were used to select individuals with alcohol-related diagnoses, track imaging procedures, and calculate cost. Diagnoses, imaging results, and radiation exposure per computed tomography (CT) study were abstracted from the medical record. RESULTS: Fifty-one individuals met inclusion criteria and had a total of 1648 imaging studies over the 3-year period. Subjects had a median of 5 (interquartile range [IQR] 2-10) CT scans, 20 (IQR 10-33) radiographs, 28.3 mSv (IQR 8.97-61.71) ionizing radiation, 0.2% (IQR 0.07-0.4) attributable risk of cancer, and $2979 (IQR 1560-5440) in charges using a national rate. The incidence of acute fracture or intracranial head injury was 55%, and 39% of the cohort had a history of moderate or severe traumatic brain injury. CONCLUSION: The remarkable use of imaging and occurrence of injury among these highly vulnerable and frequently encountered individuals compels further study to refine clinical practices through the development of evidence-based, effective interventions.

MicroRNA regulation and role in stem cell maintenance, cardiac differentiation and hypertrophy.

There are currently 1527 known microRNAs (miRNAs) in human, each of which may regulate hundreds or thousands of target genes. miRNA expression levels vary between cell types; for example, miR- 302 and miR-290 families are highly enriched in embryonic stem cells, while miR-1 is a muscle specific miRNA. miRNA biosynthesis and function are highly regulated and this regulation may be cell type specific. The processing enzymes and factors that recognize features in sequence and secondary structure of the miRNA play key roles in regulating the production of mature miRNA. Mature miRNA enriched in stem cells control stem cell self-renewal as well as their differentiation. Though specific miRNAs have been shown to control differentiation towards various lineages such as neural or skin cells, some of the most well characterized miRNAs have been found in promoting the formation of cardiac cells. In addition, miRNAs also play a critical role in cardiomyocyte hypertrophy, especially in a pathological context. Such miRNAs are predicted to be therapeutic targets for treating cardiovascular diseases. In this review we will discuss how miRNAs act to maintain the stem cell state and also explore the current knowledge of the mechanisms that regulate miRNAs. Furthermore, we will discuss the emerging roles of miRNAs using cardiomyocyte differentiation and maturation as a paradigm. Emphasis will also be given on some of the less ventured areas such as the role of miRNAs in the physiological maturation of cardiomyocytes. These potentially beneficial miRNAs are likely to improve cardiac function in both in vivo and in vitro settings and thus provide additional strategy to treat heart diseases and more importantly serve as a good model for understanding cardiomyocyte maturation in vitro.

Defective protein kinase A and C pathways are common causes of disordered zona pellucida (ZP)--induced acrosome reaction in normozoospermic infertile men with normal sperm-ZP binding.

OBJECTIVE: To determine association between defective protein kinases C (PKC) and A (PKA) and disordered zona pellucida (ZP)-induced acrosome reaction (DZPIAR) in normozoospermic infertile men with normal sperm-ZP binding. DESIGN: Sperm from DZPIAR infertile men were treated without (control) or with (test) phorbol myristate acetate (PMA, PKC activator) or dibutyryl cyclic AMP (dbcAMP, PKA activator) under in vitro standard culture condition. The ZP-induced AR was assessed and compared between control and test. SETTING: Public and private hospital-based clinical assisted reproduction technology (ART) centers. PATIENT(S): A total of 51 DZPIAR infertile men were involved in this study. INTERVENTION(S): None. MAIN OUTCOMES MEASURE(S): Sperm-ZP binding and the ZP-induced IAR. RESULT(S): Both PMA and dbcAMP enhanced ZP-induced AR up to a normal level (≥25%) in some subjects with DZPIAR: 29 (57%) with PMA and 27 (53%) with dbcAMP. Overall 35 (69%) had the ZP-induced AR enhanced to normal by PMA or dbcAMP but 16 (31%) had little or no response to either agent. Fourteen men responded to the two activators differently: 8 effective only with PMA and 6 effective only with dbcAMP. CONCLUSION(S): Defective upstream of PKC and PKA pathways are highly associated with disordered ZPIAR in normozoospermic infertile men with normal sperm-ZP binding.

Generation and characterization of phage-GnRH chemical conjugates for potential use in cat and dog immunocontraception.

Overpopulation of cats and dogs is a serious worldwide problem that demands novel, safe and cost-effective solutions. The objective of this study was to generate and characterize phage-peptide conjugates with gonadotropin-releasing hormone (GnRH) for potential use as an immunocontraceptive. A filamentous phage vector f5-8 with wild-type phage coat proteins was used as a carrier for construction of chemical conjugates with GnRH, a peptide that acts as a master reproductive hormone. In such conjugates, the phage body plays the role of a carrier protein, while multiple copies of GnRH peptide stimulate production of neutralizing anti-GnRH antibodies potentially leading to contraceptive effects. To generate the constructs, four different GnRH-based peptides were synthesized and conjugated to phage particles in a two-step procedure: (i) peptides were reacted with phage to form a conjugate using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) and (ii) the conjugates were separated from remaining free peptides by dialysis. Formation and specificity of phage-GnRH conjugates were confirmed by three independent methods: spectrophotometry, electron microscopy and ELISA. When the conjugates were tested for interaction with sera collected from cats and dogs immunized with GnRH-based vaccines in independent studies, strong specific ELISA signals were obtained, suggesting the potential use of the conjugates for cat and dog immunosterilization. The ability of the conjugates to stimulate production of anti-GnRH antibodies in vivo was evaluated in mice. While optimization of dose, immunization route and adjuvant still requires investigation, our preliminary results demonstrated the presence of anti-GnRH antibodies in sera of mice immunized with such conjugates. Fertility trials in cats and dogs will be needed to evaluate contraceptive potentials of the phage-GnRH peptide chemical conjugates.

Quinn's advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium.

The aim of this study was to compare sperm velocity, hyperactivation, zona pellucida (ZP) binding and ZP-induced acrosome reaction (AR) between Quinn's advantage fertilization (QAF), human tubal fluid (HTF) and Ham's F10 media. Semen samples were obtained from normozoospermic men and motile spermatozoa were prepared by gradient centrifugation (PureSperm). Unfertilized oocytes from clinical IVF were used for spermatozoa-oocyte interaction tests. Sperm velocity and hyperactivation were assessed using a Hamilton-Thorn motility analyser. When media were supplemented with human albumin, sperm motility and velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF (42±22 versus 21±18, P<0.th001). Sperm velocity, hyperactivation and sperm binding were also significantly higher in QAF than Ham's F10 media. Supplementation of media with either human serum or human albumin showed no difference in effect on all sperm test results. In conclusion, QAF medium significantly enhances ZP-induced AR which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF.