Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

Design,pharmacology, and NMR structure of a minimized cystine knot with agouti-related protein activity

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The NMR structure of this active domain was recently determined and suggested that melanocortin receptor contacts were made primarily by two loops presented by a well-structured cystine knot domain within AGRP(87-132) [McNulty et al. (2001) Biochemistry 40, 15520-15527]. This hypothesis is tested here with NMR structure and activity studies of a 34-residue AGRP analogue designed to contain only the cystine knot domain. The designed miniprotein folds to a homogeneous product, retains the desired cystine knot architecture, functions as an antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine knot region possesses the melanocortin receptor contact points. Moreover, this potent AGRP analogue is synthetically accessible, may serve in the development of therapeutics for the treatment of diseases related to energy balance. and may also find use as a new reagent for probing melanocortin receptor structure and function.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Age- and Gender-related Disparities in Primary Percutaneous Coronary Interventions for Acute ST-segment elevation Myocardial Infarction

Background

Previous analyses reported age- and gender-related differences in the provision of cardiac care. The objective of the study was to compare circadian disparities in the delivery of primary percutaneous coronary intervention (PCI) for acute myocardial infarction (AMI) according to the patient’s age and gender.

Methods

We investigated patients included into the Acute Myocardial Infarction in Switzerland (AMIS) registry presenting to one of 11 centers in Switzerland providing primary PCI around the clock, and stratified patients according to gender and age.

Findings

A total of 4723 patients presented with AMI between 2005 and 2010; 1319 (28%) were women and 2172 (54%) were ≥65 years of age. More than 90% of patients <65 years of age underwent primary PCI without differences between gender. Elderly patients and particularly women were at increased risk of being withheld primary PCI (males adj. HR 4.91, 95% CI 3.93–6.13; females adj. HR 9.31, 95% CI 7.37–11.75) as compared to males <65 years of age. An increased risk of a delay in door-to-balloon time >90 minutes was found in elderly males (adj HR 1.66 (95% CI 1.40–1.95), p<0.001) and females (adj HR 1.57 (95% CI 1.27–1.93), p<0.001), as well as in females <65 years (adj HR 1.47 (95% CI 1.13–1.91), p = 0.004) as compared to males <65 years of age, with significant differences in circadian patterns during on- and off-duty hours.

Conclusions

In a cohort of patients with AMI in Switzerland, we observed discrimination of elderly patients and females in the circadian provision of primary PCI.     

Innovation in mental health services: what are the key components of success?

Background

Service development innovation in health technology and practice is viewed as a pressing need within the field of mental health yet is relatively poorly understood. Macro-level theories have been criticised for their limited explanatory power and they may not be appropriate for understanding local and fine-grained uncertainties of services and barriers to the sustainability of change. This study aimed to identify contextual influences inhibiting or promoting the acceptance and integration of innovations in mental health services in both National Health Service (NHS) and community settings.

Methods

A comparative study using qualitative and case study data collection methods, including semi-structured interviews with key stakeholders and follow-up telephone interviews over a one-year period. The analysis was informed by learning organisation theory. Drawn from 11 mental health innovation projects within community, voluntary and NHS settings, 65 participants were recruited including service users, commissioners, health and non-health professionals, managers, and caregivers. The methods deployed in this evaluation focused on process-outcome links within and between the 11 projects.

Results

Key barriers to innovation included resistance from corporate departments and middle management, complexity of the innovation, and the availability and access to resources on a prospective basis within the host organisation. The results informed the construction of a proposed model of innovation implementation within mental health services. The main components of which are context, process, and outcomes.

Conclusions

The study produced a model of conducive and impeding factors drawn from the composite picture of 11 innovative mental health projects, and this is discussed in light of relevant literature. The model provides a rich agenda to consider for services wanting to innovate or adopt innovations from elsewhere. The evaluation suggested the importance of studying innovation with a focus on context, process, and outcomes.

     

Reaching Mothers and Babies with Early Postnatal Home Visits: The Implementation Realities of Achieving High Coverage in Large-Scale Programs

Background

Nearly half of births in low-income countries occur without a skilled attendant, and even fewer mothers and babies have postnatal contact with providers who can deliver preventive or curative services that save lives. Community-based maternal and newborn care programs with postnatal home visits have been tested in Bangladesh, Malawi, and Nepal. This paper examines coverage and content of home visits in pilot areas and factors associated with receipt of postnatal visits.

Methods

Using data from cross-sectional surveys of women with live births (Bangladesh 398, Malawi: 900, Nepal: 615), generalized linear models were used to assess the strength of association between three factors - receipt of home visits during pregnancy, birth place, birth notification - and receipt of home visits within three days after birth. Meta-analytic techniques were used to generate pooled relative risks for each factor adjusting for other independent variables, maternal age, and education.

Findings

The proportion of mothers and newborns receiving home visits within three days after birth was 57% in Bangladesh, 11% in Malawi, and 50% in Nepal. Mothers and newborns were more likely to receive a postnatal home visit within three days if the mother received at least one home visit during pregnancy (OR2.18, CI1.46–3.25), the birth occurred outside a facility (OR1.48, CI1.28–1.73), and the mother reported a CHW was notified of the birth (OR2.66, CI1.40–5.08). Checking the cord was the most frequently reported action; most mothers reported at least one action for newborns.

Conclusions

Reaching mothers and babies with home visits during pregnancy and within three days after birth is achievable using existing community health systems if workers are available; linked to communities; and receive training, supplies, and supervision. In all settings, programs must evaluate what community delivery systems can handle and how to best utilize them to improve postnatal care access.     

Unc45b is essential for early myofibrillogenesis and costamere formation in zebrafish

Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.     

A genetic technique to identify the diet of cownose rays,Rhinoptera bonasus: analysis of shellfish prey items from North Carolina and Virginia

Cownose rays are implicated in the consumption of commercially important shellfish on the U.S. East Coast. We tested this assumption by developing a molecular technique for species identification from cownose ray gut contents. Digestive tracts sampled from 33 rays in Pamlico Sound, NC and Chesapeake Bay, VA contained pieces of partially-digested tissue, well-digested tissue, fluid, and minute shell fragments which made visual identification to the species level nearly impossible. We sequenced the cytochrome oxidase subunit I (COI) for seven locally acquired bivalve species, chosen for their commercial and ecological importance in NC and VA. Sequences were used to design species-specific primers for each bivalve species to amplify polymerase chain reaction (PCR) products. We designed primers such that PCR products were sufficiently different in size to be distinguishable from one another when resolved on an agarose gel, and multiplexing of several species in one reaction was possible. Digestive tract sample testing revealed that cownose rays in Chesapeake Bay ate stout tagelus and soft shell clams. There was no evidence of the rays in the study consuming commercially important oysters, hard clams, and bay scallops. Further sampling over an extended period of time and additional locations is required to confirm these results. Our diagnostic tests could easily be expanded to elucidate the impact of cownose ray predation on prey populations.     

Mmp25β facilitates elongation of sensory neurons during zebrafish development

Matrix metalloproteinases (MMPs) are a large and complex family of zinc‐dependent endoproteinases widely recognized for their roles in remodeling the extracellular matrix (ECM) during embryonic development, wound healing, and tissue homeostasis. Their misregulation is central to many pathologies, and they have therefore been the focus of biomedical research for decades. These proteases have also recently emerged as mediators of neural development and synaptic plasticity in vertebrates, however, understanding of the mechanistic basis of these roles and the molecular identities of the MMPs involved remains far from complete. We have identified a zebrafish orthologue of mmp25 (a.k.a. leukolysin; MT6‐MMP), a membrane‐type, furin‐activated MMP associated with leukocytes and invasive carcinomas, but which we find is expressed by a subset of the sensory neurons during normal embryonic development. We detect high levels of Mmp25β expression in the trigeminal, craniofacial, and posterior lateral line ganglia in the hindbrain, and in Rohon‐Beard cells in the dorsal neural tube during the first 48 h of embryonic development. Knockdown of Mmp25β expression with morpholino oligonucleotides results in larvae that are uncoordinated and insensitive to touch, and which exhibit defects in the development of sensory neural structures. Using in vivo zymography, we observe that Mmp25β morphant embryos show reduced Type IV collagen degradation in regions of the head traversed by elongating axons emanating from the trigeminal ganglion, suggesting that Mmp25β may play a pivotal role in mediating ECM remodeling in the vicinity of these elongating axons. genesis 52:833–848, 2014. © 2014 Wiley Periodicals, Inc.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.