A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods

Age related deviation of gait from normality in alkaptonuria.

Alkaptonuria is a rare metabolic disease leading to systemic changes including early and severe arthropathy which affects mobility. Due to unknown reasons, the onset of degenerative changes is delayed to around 30 years of age when both objective and subjective symptoms develop. In order to complement describing the structural changes in alkaptonuria with measures of movement function, clinical gait analysis was added to the list of assessments in 2013. The aim of this study was to describe the deviation of gait from normality as a function of age in patients with alkaptonuria. Three-dimensional movement of reflective markers attached to joints were captured during walking in 39 patients and 10 controls. Subsequent to processing the data to emphasise the shape of marker trajectories, the mean Movement Deviation Profile was generated for all participants. This single number measure gives the deviation of a patient’s gait from a distributed definition of gait normality. Results showed that gait deviation roughly follows a sigmoid profile with minimal increase of gait deviations in a younger patient group and an abrupt large increase around the second half of the 4th decade of life. Larger variations of gait deviations were found in the older group than in the younger group suggesting a complex interaction of multiple factors which determine gait function after symptoms manifest. Continued gait analysis of adults with AKU, extended to younger adults and children with AKU, is expected to complete understanding of both the natural history of alkaptonuria and how interventions can affect movement function

Distinct structural features of G protein-coupled receptor kinase 5 (GRK5) regulate its nuclear localization and DNA-binding ability

G protein-coupled receptor kinases (GRKs) act to desensitize G protein-coupled receptors (GPCRs). In addition to this role at the plasma membrane, a nuclear function for GRK5, a member of the GRK4 subfamily of GRKs, has been reported. GRK5 phosphorylates and promotes the nuclear export of the histone deacetylase, HDAC5. Here we demonstrate that the possession of a nuclear localization sequence (NLS) is a common feature of GRK4 subfamily members (GRKs 4, 5 and 6). However, the location of the NLS and the ability of these GRKs to bind DNA in vitro are different. The NLSs of GRK5 and 6 bind DNA in vitro, whilst the NLS of GRK4 does not. Using mutants of GRK5 we identify the regions of GRK5 required for DNA-binding in vitro and nuclear localization in cells. The DNA-binding ability of GRK5 requires both the NLS and an N-terminal calmodulin (CaM)-binding site. A functional nuclear export sequence (NES), required for CaM-dependent nuclear export of the kinase, is also identified. Based on our observations we propose a model to explain how nuclear localization of GRK5 may be regulated. Notably, the nuclear localization of GRK5 and 6 is differentially regulated. These results suggest subfamily specific nuclear functions for the GRK4 subfamily members. Identification of GRK specific small molecule inhibitors of nuclear localization and/or function for the GRK4 subfamily may thus be an achievable goal

Long term geological record of a global deep subsurface microbial habitat in sand injection complexes

Peer reviewedPublisher PD

Spontaneous charged lipid transfer between lipid vesicles

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures

Challenges of Loss to Follow-up in Tuberculosis Research.

In studies evaluating methods for diagnosing tuberculosis (TB), follow-up to verify the presence or absence of active TB is crucial and high dropout rates may significantly affect the validity of the results. In a study assessing the diagnostic performance of the QuantiFERON®-TB Gold In-Tube test in TB suspect children in Tanzania, factors influencing patient adherence to attend follow-up examinations and reasons for not attending were examined. In 160 children who attended and 102 children who did not attend scheduled 2-month follow-up baseline health characteristics, demographic data and risk factors for not attending follow-up were determined. Qualitative interviews were used to understand patient and caretakers reasons for not returning for scheduled follow-up. Being treated for active tb in the dots program (OR: 4.14; 95% CI:1.99-8.62;p-value<0.001) and receiving money for the bus fare (OR:129; 95% CI 16->100;P-value<0.001) were positive predictors for attending follow-up at 2 months, and 21/85(25%) of children not attending scheduled follow-up had died. Interviews revealed that limited financial resources, i.e. lack of money for transportation and poor communication, were related to non-adherence. Patients lost to follow-up is a potential problem for TB research. Receiving money for transportation to the hospital and communication is crucial for adherence to follow-up conducted at a study facility. Strategies to ensure follow-up should be part of any study protocol

High levels of untreated distress and fatigue in cancer patients

The purpose of the study was to assess a large representative sample of cancer patients on distress levels, common psychosocial problems, and awareness and use of psychosocial support services. A total of 3095 patients were assessed over a 4-week period with the Brief Symptom Inventory-18 (BSI-18), a common problems checklist, and on awareness and use of psychosocial resources. Full data was available on 2776 patients. On average, patients were 60 years old, Caucasian (78.3%), and middle class. Approximately, half were attending for follow-up care. Types of cancer varied, with the largest groups being breast (23.5%), prostate (16.9%), colorectal (7.5%), and lung (5.8%) cancer patients. Overall, 37.8% of all patients met criteria for general distress in the clinical range. A higher proportion of men met case criteria for somatisation, and more women for depression. There were no gender differences in anxiety or overall distress severity. Minority patients were more likely to be distressed, as were those with lower income, cancers other than prostate, and those currently on active treatment. Lung, pancreatic, head and neck, Hodgkin's disease, and brain cancer patients were the most distressed. Almost half of all patients who met distress criteria had not sought professional psychosocial support nor did they intend to in the future. In conclusion, distress is very common in cancer patients across diagnoses and across the disease trajectory. Many patients who report high levels of distress are not taking advantage of available supportive resources. Barriers to such use, and factors predicting distress and use of psychosocial care, require further exploration

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Garden and landscape-scale correlates of moths of differing conservation status: significant effects of urbanization and habitat diversity

Moths are abundant and ubiquitous in vegetated terrestrial environments and are pollinators, important herbivores of wild plants, and food for birds, bats and rodents. In recent years, many once abundant and widespread species have shown sharp declines that have been cited by some as indicative of a widespread insect biodiversity crisis. Likely causes of these declines include agricultural intensification, light pollution, climate change, and urbanization; however, the real underlying cause(s) is still open to conjecture. We used data collected from the citizen science Garden Moth Scheme (GMS) to explore the spatial association between the abundance of 195 widespread British species of moth, and garden habitat and landscape features, to see if spatial habitat and landscape associations varied for species of differing conservation status. We found that associations with habitat and landscape composition were species-specific, but that there were consistent trends in species richness and total moth abundance. Gardens with more diverse and extensive microhabitats were associated with higher species richness and moth abundance; gardens near to the coast were associated with higher richness and moth abundance; and gardens in more urbanized locations were associated with lower species richness and moth abundance. The same trends were also found for species classified as increasing, declining and vulnerable under IUCN (World Conservation Union) criteria

Ultrasound imaging of the carpal tunnel during median nerve compression

Median nerve (MN) compression is a recognized component of carpal tunnel syndrome (CTS). In order to document compressive changes in the MN during hand activity, the carpal tunnel was imaged with neuromuscular ultrasound (NMUS). Ten patients with CTS and five normal controls underwent NMUS of the MN at rest and during dynamic stress testing (DST). DST maneuvers involve sustained isometric flexion of the distal phalanges of the first three digits. During DST in the CTS patients, NMUS demonstrated MN compression between the contracting thenar muscles ventrally and the taut flexor tendons dorsally. The mean MN diameter decreased nearly 40%, with focal narrowing in the mid-distal carpal canal. Normal controls demonstrated no MN compression and a tendency towards MN enlargement, with an average diameter increase of 17%. Observing the pathologic mechanism of MN injury during common prehensile hand movements could help better understand how to treat and prevent CTS