Investigation of mechanisms for restricting the activity of cyclic-AMP dependent protein kinase

Cyclic AMP (cAMP) is an ancient second messenger that is essential for many cellular processes including synaptic plasticity and control of heart rate and contractility. Cyclic AMP-dependent protein kinase (PKA) is the major intracellular receptor for cAMP. PKA consists of dimeric regulatory (R) subunits that bind and inhibit catalytic (C) subunits. PKA is activated upon binding of cAMP to the R subunits, which leads to the release of C subunits, and phosphorylation of intracellular protein substrates. An enduring challenge in cAMP research is to understand how PKA activity is directed to specific substrates, as the C subunits exhibit only limited substrate specificity in vitro. Elevations of cAMP are controlled in both space and time in the cell. This is achieved by the co-localization of enzymes for both the synthesis (cyclases) and breakdown (phosphodiesterases) of cAMP. Anchoring proteins are also essential for directing PKA to substrates in their immediate vicinity. However, a mechanism is yet to be established to explain how the activity of the C subunit of PKA is restrained following its dissociation from R subunits. This thesis details three parallel investigations that apply novel approaches with the shared aim of understanding how C subunit restraint is achieved. First, using quantitative immunoblotting in conjunction with purified PKA subunits, I investigated PKA subunit stoichiometry, finding that PKA R subunits typically outnumber C subunits by ~15-fold. Second, I developed a novel approach for monitoring R subunit isoform-specific association with C subunits in cells, with temporal precision. Comparative experiments using this approach and measurements with a fluorescent reporter of PKA activity show that only a small portion of C subunits need be dissociated to achieve high PKA activity. Third, I applied and developed a novel cross-linking coupled to mass spectrometry (XL-MS) protocol for analysis of the structure of PKA complexes. Insights include the likely orientation of PKA complexes that contain type II R (RII) subunits towards the membrane, and identification of a possible conformational change in PKA upon binding an anchoring protein. Together these experiments illuminate several aspects of PKA to show how the activity of this critical signalling enzyme is restrained within cells

Progression of Neuropsychiatric Symptoms over Time in an Incident Parkinson's Disease Cohort (ICICLE-PD).

BACKGROUND: Cross-sectional studies have identified that the prevalence of neuropsychiatric symptoms (NPS) in Parkinson's disease (PD) ranges from 70-89%. However, there are few longitudinal studies determining the impact of NPS on quality of life (QoL) in PD patients and their caregivers. We seek to determine the progression of NPS in early PD. METHODS: Newly diagnosed idiopathic PD cases (n = 212) and age-matched controls (n = 99) were recruited into a longitudinal study. NPS were assessed using the Neuropsychiatric Inventory with Caregiver Distress scale (NPI-D). Further neuropsychological and clinical assessments were completed by participants, with reassessment at 18 and 36 months. Linear mixed-effects modelling determined factors associated with NPI-D and QoL over 36 months. RESULTS: Depression, anxiety, apathy and hallucinations were more frequent in PD than controls at all time points (p < 0.05). Higher motor severity at baseline was associated with worsening NPI-D scores over time (β = 0.1, p < 0.05), but not cognition. A higher NPI total score was associated with poorer QoL at any time point (β = 0.3, p < 0.001), but not changed in QoL scores. CONCLUSION: NPS are significantly associated with poorer QoL, even in early PD. Screening for NPS from diagnosis may allow efficient delivery of better support and treatment to patients and their families

Review: The spectrum of clinical features seen with alpha synuclein pathology.

It has been recognized for many years that a number of chronic neurodegenerative diseases of the CNS are characterized by the development of intracellular inclusion bodies, but it is only relatively recently that the core proteins defining these pathologies have been defined. One such protein is alpha synuclein, that was found to be the main component of Lewy bodies in the late 1990s, and this discovery reinforced the emerging view that alpha synuclein was intimately linked to diseases characterized by this type of pathology--namely Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Furthermore at around this time, this same protein was also found within the glial inclusion bodies of patients dying with multiple system atrophy (MSA). These three disorders constitute the majority of patients with an 'alpha synucleinopathy', although there are a number of rarer conditions that can also cause this pathology including inherited metabolic disorders such as Gaucher's disease (GD). In this review, we will concentrate on PD, the commonest alpha synucleinopathy, and its associated dementia (PDD), as well as discussing DLB and MSA and will highlight how the clinical features of these conditions vary as a function of pathology.Our own work on the natural history of Parkinson’s disease has been supported by the Wellcome Trust; MRC; Patrick Berthoud Trust; Cure Parkinson’s Trust; Parkinson’s UK and the Cambridge NIHR Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1111/nan.1230

Self-Swabbing for Virological Confirmation of Influenza-Like Illness Among an Internet-Based Cohort in the UK During the 2014-2015 Flu Season: Pilot Study

BACKGROUND: Routine influenza surveillance, based on laboratory confirmation of viral infection, often fails to estimate the true burden of influenza-like illness (ILI) in the community because those with ILI often manage their own symptoms without visiting a health professional. Internet-based surveillance can complement this traditional surveillance by measuring symptoms and health behavior of a population with minimal time delay. Flusurvey, the UK's largest crowd-sourced platform for surveillance of influenza, collects routine data on more than 6000 voluntary participants and offers real-time estimates of ILI circulation. However, one criticism of this method of surveillance is that it is only able to assess ILI, rather than virologically confirmed influenza. OBJECTIVE: We designed a pilot study to see if it was feasible to ask individuals from the Flusurvey platform to perform a self-swabbing task and to assess whether they were able to collect samples with a suitable viral content to detect an influenza virus in the laboratory. METHODS: Virological swabbing kits were sent to pilot study participants, who then monitored their ILI symptoms over the influenza season (2014-2015) through the Flusurvey platform. If they reported ILI, they were asked to undertake self-swabbing and return the swabs to a Public Health England laboratory for multiplex respiratory virus polymerase chain reaction testing. RESULTS: A total of 700 swab kits were distributed at the start of the study; from these, 66 participants met the definition for ILI and were asked to return samples. In all, 51 samples were received in the laboratory, 18 of which tested positive for a viral cause of ILI (35%). CONCLUSIONS: This demonstrated proof of concept that it is possible to apply self-swabbing for virological laboratory testing to an online cohort study. This pilot does not have significant numbers to validate whether Flusurvey surveillance accurately reflects influenza infection in the community, but highlights that the methodology is feasible. Self-swabbing could be expanded to larger online surveillance activities, such as during the initial stages of a pandemic, to understand community transmission or to better assess interseasonal activity

Molecular evolution: sex accelerates adaptation

An analysis confirms the long-standing theory that sex increases the rate of adaptive evolution by accelerating the speed at which beneficial mutations sweep through sexual, as opposed to asexual, populations

"After my husband's circumcision, I know that I am safe from diseases": Women's Attitudes and Risk Perceptions Towards Male Circumcision in Iringa, Tanzania.

While male circumcision reduces the risk of female-to-male HIV transmission and certain sexually transmitted infections (STIs), there is little evidence that circumcision provides women with direct protection against HIV. This study used qualitative methods to assess women's perceptions of male circumcision in Iringa, Tanzania. Women in this study had strong preferences for circumcised men because of the low risk perception of HIV with circumcised men, social norms favoring circumcised men, and perceived increased sexual desirability of circumcised men. The health benefits of male circumcision were generally overstated; many respondents falsely believed that women are also directly protected against HIV and that the risk of all STIs is greatly reduced or eliminated in circumcised men. Efforts to engage women about the risks and limitations of male circumcision, in addition to the benefits, should be expanded so that women can accurately assess their risk of HIV or STIs during sexual intercourse with circumcised men

WESTT (Workload, Error, Situational Awareness, Time and Teamwork): An analytical prototyping system for command and control

Modern developments in the use of information technology within command and control allow unprecedented scope for flexibility in the way teams deal with tasks. These developments, together with the increased recognition of the importance of knowledge management within teams present difficulties for the analyst in terms of evaluating the impacts of changes to task composition or team membership. In this paper an approach to this problem is presented that represents team behaviour in terms of three linked networks (representing task, social network structure and knowledge) within the integrative WESTT software tool. In addition, by automating analyses of workload and error based on the same data that generate the networks, WESTT allows the user to engage in the process of rapid and iterative “analytical prototyping”. For purposes of illustration an example of the use of this technique with regard to a simple tactical vignette is presented

Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity

Spin-enhanced nanodiamond biosensing for ultrasensitive diagnostics

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness, low cost and selective manipulation of their emission. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity and frequency-domain analysis to separate the signal from background autofluorescence, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10−19 molar for a biotin–avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations

Misaligned spin and orbital axes cause the anomalous precession of DI Herculis

The orbits of binary stars precess as a result of general relativistic effects, forces arising from the asphericity of the stars, and forces from additional stars or planets in the system. For most binaries, the theoretical and observed precession rates are in agreement. One system, however -- DI Herculis -- has resisted explanation for 30 years. The observed precession rate is a factor of four slower than the theoretical rate, a disagreement that once was interpreted as evidence for a failure of general relativity. Among the contemporary explanations are the existence of a circumbinary planet and a large tilt of the stellar spin axes with respect to the orbit. Here we report that both stars of DI Herculis rotate with their spin axes nearly perpendicular to the orbital axis (contrary to the usual assumption for close binary stars). The rotationally induced stellar oblateness causes precession in the direction opposite to that of relativistic precession, thereby reconciling the theoretical and observed rates.Comment: Nature, in press [11 pg