A model for agonism and antagonism in an ancient and ubiquitous cAMP-binding domain

The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.     

Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein

Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)(35) mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.     

Early Depression Screening Is Feasible in Hospitalized Stroke Patients

Background and Purpose

Post-stroke depression (PSD) is common but is not routinely assessed for in hospitalized patients. As a Comprehensive Stroke Center, we screen all stroke inpatients for depression, though the feasibility of early screening has not been established. We assessed the hypothesis that early depression screening in stroke patients is feasible. We also explored patient level factors associated with being screened for PSD and the presence of early PSD.

Methods

The medical records of all patients admitted with ischemic stroke (IS) or intracerebral hemorrhage (ICH) between 01/02/13 and 15/04/13 were reviewed. A depression screen, modified from the Patient Health Questionnaire-9, was administered (maximum score 27, higher scores indicating worse depression). Patients were eligible if they did not have a medical condition precluding screening. Feasibility was defined as screening 75% of all eligible patients.

Results

Of 303 IS and ICH inpatients, 70% (211) were eligible for screening, and 75% (158) of all eligible patients were screened. More than one-third of all patients screened positive for depression (score > 4). Women (OR 2.06, 95% CI 1.06–4.01) and younger patients (OR 0.97, 95% CI 0.96–0.99) were more likely to screen positive. Screening positive was not associated with poor discharge/day 7 outcome (mRS > 3; OR 1.45, 95% CI 0.74–2.83).

Conclusions

Screening stroke inpatients for depression is feasible and early depression after stroke is common. Women and younger patients are more likely to experience early PSD. Our results provide preliminary evidence supporting continued screening for depression in hospitalized stroke patients.     

Pleural Fluid Adenosine Deaminase (Pfada) in the Diagnosis of Tuberculous Effusions in a Low Incidence Population

IntroductionPrevious studies have assessed the diagnostic ability of pleural fluid adenosine deaminase (pfADA) in detecting tuberculous pleural effusions, with good specificity and sensitivity reported. However, in North Western Europe pfADA is not routinely used in the investigation of a patient with an undiagnosed pleural effusion, mainly due to a lack of evidence as to its utility in populations with low mycobacterium tuberculosis (mTB) incidence.MethodsPatients presenting with an undiagnosed pleural effusion to a tertiary pleural centre in South-West England over a 3 year period, were prospectively recruited to a pleural biomarker study. Pleural fluid from consecutive patients with robust 12-month follow up data and confirmed diagnosis were sent for pfADA analysis.ResultsOf 338 patients enrolled, 7 had confirmed tuberculous pleural effusion (2%). All mTB effusions were lymphocyte predominant with a median pfADA of 72.0 IU/L (range- 26.7 to 91.5) compared to a population median of 12.0 IU/L (range- 0.3 to 568.4). The optimal pfADA cut off was 35 IU/L, which had a negative predictive value (NPV) of 99.7% (95% CI; 98.2-99.9%) for the exclusion of mTB, and sensitivity of 85.7% (95% CI; 42.2-97.6%) with an area under the curve of 0.88 (95% CI; 0.732–1.000).DiscussionThis is the first study examining the diagnostic utility of pfADA in a low mTB incidence area. The chance of an effusion with a pfADA under 35 IU/L being of tuberculous aetiology was negligible. A pfADA of over 35 IU/L in lymphocyte-predominant pleural fluid gives a strong suspicion of mTB.     

Metagenomic Surveys of Gut Microbiota

Gut microbiota of higher vertebrates is host-specific. The number and diversity of the organisms residing within the gut ecosystem are defined by physiological and environmental factors,such as host genotype, habitat, and diet. Recently, culture-independent sequencing techniques have added a new dimension to the study of gut microbiota and the challenge to analyze the large volume of sequencing data is increasingly addressed by the development of novel computational tools and methods. Interestingly, gut microbiota maintains a constant relative abundance at operational taxonomic unit(OTU) levels and altered bacterial abundance has been associated with complex diseases such as symptomatic atherosclerosis, type 2 diabetes, obesity, and colorectal cancer. Therefore, the study of gut microbial population has emerged as an important field of research in order to ultimately achieve better health. In addition, there is a spontaneous, non-linear, and dynamic interaction among different bacterial species residing in the gut. Thus, predicting the influence of perturbed microbe–microbe interaction network on health can aid in developing novel therapeutics. Here, we summarize the population abundance of gut microbiota and its variation in different clinical states,computational tools available to analyze the pyrosequencing data, and gut microbe–microbe interaction networks.     

Aberrant Fat Metabolism in Caenorhabditis elegans Mutants with Defects in the Defecation Motor Program

The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisin-family, Ca²⁺-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steady-state levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status.     

Improving Health-Related Quality of Life among People Living with HIV: Results from an Impact Evaluation of a Food Assistance Program in Uganda

Introduction

Widespread food insecurity in Africa continues to compromise an effective response to the AIDS epidemic. Health-related quality of life (HRQoL) is a comprehensive indicator of physical, mental, and social well-being that is associated with food insecurity and increasingly used to assess the well-being of people living with HIV/AIDS (PLHIV). We examined the impact of a food assistance intervention, previously shown to have reduced household food insecurity and improved nutritional status, on HRQoL of PLHIV.

Methods

We capitalized on an existing intervention targeting antiretroviral therapy (ART)- naïve PLHIV in Uganda, and conducted a prospective impact evaluation including a treatment and a comparison group. Data analyzed included 640 participants from two districts (318 in the intervention district) interviewed in both clinic and household settings at baseline and again approximately one year later. The main outcomes considered were physical and mental health dimensions of HRQoL, and other outcomes included self- and healthcare provider-reported symptoms. We utilized difference-in-difference propensity score matching methodologies to infer causality and examine program impacts.

Results

Over 12 months, food assistance significantly increased physical health scores (PHS) by 2.85 (P < .01) or approximately 0.35 SD, and reduced substantially the number of self- and healthcare provider-reported HIV-related symptoms by 3.83 and 2.68, respectively (P < .01). There was no significant impact, however, on mental health scores (MHS).

Conclusions

This study demonstrates the potential importance for HRQoL of including food assistance programming as part of the standard of care for PLHIV in areas of widespread food insecurity.     

Comparative Effectiveness of Hepatic Artery Based Therapies for Unresectable Colorectal Liver Metastases: A Meta-Analysis

BackgroundPatients with unresectable Colorectal Liver Metastases (CRLM) are increasingly being managed using Hepatic Artery Based Therapies (HAT), including Hepatic Arterial Infusion (HAI), Radioembolization (RE), and Transcatheter Arterial Chemoembolization (TACE). Limited data is available on the comparative effectiveness of these options. We hypothesized that outcomes in terms of survival and toxicity were equivalent across the three strategies.MethodsA meta-analysis was performed using a prospectively registered search strategy at PROSPERO (CRD42013003861) that utilized studies from PubMed (2003–2013). Primary outcome was median overall survival (OS). Secondary outcomes were treatment toxicity, tumor response, and conversion of the tumor to resectable. Additional covariates included prior or concurrent systemic therapy.ResultsOf 491 studies screened, 90 were selected for analyses—52 (n = 3,000 patients) HAI, 24 (n = 1,268) RE, 14 (n = 1,038) TACE. The median OS (95% CI) for patients receiving HAT in the first-line were RE 29.4 vs. HAI 21.4 vs. TACE 15.2 months (p = 0.97, 0.69 respectively). For patients failing at least one line of prior systemic therapy, the survival outcomes were TACE 21.3 (20.6–22.4) months vs. HAI 13.2 (12.2–14.2) months vs. RE 10.7 (9.5–12.0). Grade 3–4 toxicity for HAT alone was 40% in the HAI group, 19% in the RE group, and 18% in the TACE groups, which was increased with the addition of systemic chemotherapy. Level 1 evidence was available in 5 studies for HAI, 2 studies for RE and 1 for TACE.ConclusionHAI, RE, and TACE are equally effective in patients with unresectable CRLM with marginal differences in survival.