The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation

Background

Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/Principal Findings

To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/Significance

We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.     

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.     

Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis

In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung—common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.Severe sepsis is a common acute illness in intensive care units (ICUs)¹ and is associated with high mortality rates and chronic morbidity. When it is associated with hypotension (termed septic shock), the mortality rate is very high (50% to 80%). Cardiovascular dysfunction during sepsis is multifactorial and often associated with minimal loss of myocardial tissue, but with the release of myocardial-specific markers such as troponins. A key unmet clinical need is the availability of a biomarker that predicts myocardial dysfunction early, monitors response to treatment, and thus identifies a cohort of patients at higher risk of septic shock to aid in targeted interventions and improve outcome (1).In the present study, we used proteomics for biomarker discovery. Over the past decade, the field of proteomics has made impressive progress. Plasma and serum, however, are the most complex proteomes of the human body (2), and less abundant proteins tend to be missed in untargeted proteomics analyses of body fluids (3). Thus, we pursued an alternative strategy: the application of proteomics to diseased tissue (4), in which the potential biomarkers are less dilute and have a less uncertain cellular origin (5–7). We employed a solubility-based protein-subfractionation methodology to analyze inflammatory proteins that are retained with sepsis tissue. This innovative proteomics approach shall reveal inflammatory molecules that reside and persist within inflamed tissue. We hypothesized that proteins that accumulate in the susceptible tissues are more likely to be biomarker candidates for organ dysfunction than proteins that just circulate in plasma or serum. We then validated our proteomics findings in the preclinical model using samples from sepsis patients admitted to ICUs.     

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Marine foraging ecology influences mercury bioaccumulation in deep-diving northern elephant seals

Mercury contamination of oceans is prevalent worldwide and methylmercury concentrations in the mesopelagic zone (200–1000 m) are increasing more rapidly than in surface waters. Yet mercury bioaccumulation in mesopelagic predators has been understudied. Northern elephant seals (Mirounga angustirostris) biannually travel thousands of kilometres to forage within coastal and open-ocean regions of the northeast Pacific Ocean. We coupled satellite telemetry, diving behaviour and stable isotopes (carbon and nitrogen) from 77 adult females, and showed that variability among individuals in foraging location, diving depth and δ¹³C values were correlated with mercury concentrations in blood and muscle. We identified three clusters of foraging strategies, and these resulted in substantially different mercury concentrations: (i) deeper-diving and offshore-foraging seals had the greatest mercury concentrations, (ii) shallower-diving and offshore-foraging seals had intermediate levels, and (iii) coastal and more northerly foraging seals had the lowest mercury concentrations. Additionally, mercury concentrations were lower at the end of the seven-month-long foraging trip (n = 31) than after the two-month- long post-breeding trip (n = 46). Our results indicate that foraging behaviour influences mercury exposure and mesopelagic predators foraging in the northeast Pacific Ocean may be at high risk for mercury bioaccumulation.     

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg²⁺ ions and other 5′-domain proteins. Equilibrium titrations of Cy3-labeled 5′-domain RNA with Cy5-labeled protein S4 showed that Mg²⁺ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg²⁺ ions alone.     

Relationship of the Perceived Social and Physical Environment with Mental Health-Related Quality of Life in Middle-Aged and Older Adults: Mediating Effects of Physical Activity

Background

Mental health conditions are among the leading non-fatal diseases in middle-aged and older adults in Australia. Proximal and distal social environmental factors and physical environmental factors have been associated with mental health, but the underlying mechanisms explaining these associations remain unclear. The study objective was to examine the contribution of different types of physical activity in mediating the relationship of social and physical environmental factors with mental health-related quality of life in middle-aged and older adults.

Methods

Baseline data from the Wellbeing, Eating and Exercise for a Long Life (WELL) study were used. WELL is a prospective cohort study, conducted in Victoria, Australia. Baseline data collection took place in 2010. In total, 3,965 middle-aged and older adults (55–65 years, 47.4% males) completed the SF-36 Health Survey, the International Physical Activity Questionnaire, and a questionnaire on socio-demographic, social and physical environmental attributes. Mediation analyses were conducted using the MacKinnon product-of-coefficients test.

Results

Personal safety, the neighbourhood physical activity environment, social support for physical activity from family or friends, and neighbourhood social cohesion were positively associated with mental health-related quality of life. Active transportation and leisure-time physical activity mediated 32.9% of the association between social support for physical activity from family or friends and mental health-related quality of life. These physical activity behaviours also mediated 11.0%, 3.4% and 2.3% respectively, of the relationship between the neighbourhood physical activity environment, personal safety and neighbourhood social cohesion and mental health-related quality of life.

Conclusions

If these results are replicated in future longitudinal studies, tailored interventions to improve mental health-related quality of life in middle-aged and older adults should use a combined strategy, focusing on increasing physical activity as well as social and physical environmental attributes.