Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.     

Evaluation of a Rapid Point of Care Test for Detecting Acute and Established HIV Infection,and Examining the Role of Study Quality on Diagnostic Accuracy: A Bayesian Meta-Analysis

Introduction

Fourth generation (Ag/Ab combination) point of care HIV tests like the FDA-approved Determine HIV1/2 Ag/Ab Combo test offer the promise of timely detection of acute HIV infection, relevant in the context of HIV control. However, a synthesis of their performance has not yet been done. In this meta-analysis we not only assessed device performance but also evaluated the role of study quality on diagnostic accuracy.

Methods

Two independent reviewers searched seven databases, including conferences and bibliographies, and independently extracted data from 17 studies. Study quality was assessed with QUADAS-2. Data on sensitivity and specificity (overall, antigen, and antibody) were pooled using a Bayesian hierarchical random effects meta-analysis model. Subgroups were analyzed by blood samples (serum/plasma vs. whole blood) and study designs (case-control vs. cross-sectional).

Results

The overall specificity of the Determine Combo test was 99.1%, 95% credible interval (CrI) [97.3–99.8]. The overall pooled sensitivity for the device was at 88.5%, 95% [80.1–93.4]. When the components of the test were analyzed separately, the pooled specificities were 99.7%, 95% CrI [96.8–100] and 99.6%, 95% CrI [99.0–99.8], for the antigen and antibody components, respectively. Pooled sensitivity of the antibody component was 97.3%, 95% CrI [60.7–99.9], and pooled sensitivity for the antigen component was found to be 12.3%, 95% (CrI) [1.1–44.2]. No significant differences were found between subgroups by blood sample or study design. However, it was noted that many studies restricted their study sample to p24 antigen or RNA positive specimens, which may have led to underestimation of overall test performance. Detection bias, selection (spectrum) bias, incorporation bias, and verification bias impaired study quality.

Conclusions

Although the specificity of all test components was high, antigenic sensitivity will merit from an improvement. Besides the accuracy of the device itself, study quality, also impacts the performance of the test. These factors must be kept in mind in future evaluations of an improved device, relevant for global scale up and implementation.     

Establishment and comparison of fibroblast cell lines from the medial collateral and anterior cruciate ligaments of the rabbit

Summary We have developed a procedure to explant fibroblasts from the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) of the rabbit knee, and have optimized conditions for maintaining them in culture. Maximal growth for both ACL and MCL cells was obtained with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 250 μM ascorbate. ACL and MCL fibroblasts displayed intrinsic differences in their responses to changes in culture parameters. Specifically, they displayed different growth responses when plated at different densities and responded to RPMI 1640 medium in very different ways. There were also biochemical differences between the cell types. Both cell types produced similar amounts of collagen in culture, but the ratio of type I to type III, the major collagen subtypes produced by these cells, were different. ACL fibroblasts produced 86.7% type I and 13.3% type III, and MCL fibroblasts produced 71.1% type I and 28.9% type III. In addition, total protein produced by ACL fibroblasts was higher than that produced by MCL cells. This confirms the suggestions of previous researchers that such differences might exist. This work was funded by a grant-in-aid from Medtronic of Canada, by an R&D Grant from the Alberta Ministry of Technology, Research and Telecommunications, and by the Alberta Heritage Foundation for Medical Research.     

Racialization: the genealogy and critique of a concept

Recently sociological analysis of what used to be identified as 'race' and 'race relations' has shifted to racism as an ideology and racialization as a process that ascribes physical and cultural differences to individuals and groups. While scholars have critically examined 'race' and 'race relations', the concept of racialization has received insufficient systematic attention. The purpose of this article is to trace the genealogy of concepts of racialization and deracialization and to demonstrate that the meaning of these designations has changed since their appearance in the late-nineteenth century to the emergence of racialization in contemporary debates on effects of racism; and to trace the different trajectories of racialization from the centre and from the periphery.     

Draft Genome Sequence of an Ammonia-Oxidizing Archaeon, “Candidatus Nitrosopumilus sediminis” AR2, from Svalbard in the Arctic Circle

Ammonia-oxidizing archaea (AOA) typically predominate over ammonia-oxidizing bacteria in marine sediments. We herein present the draft genome sequence of an ammonia-oxidizing archaeon, “Candidatus Nitrosopumilus sediminis” AR2, which was enriched in culture from a marine sediment obtained off Svalbard, within the Arctic Circle. The typical genes involved in archaeal ammonia oxidation and carbon fixation necessary for chemolithoautotrophic growth were observed. Interestingly, the AR2 genome sequence was revealed to possess, uniquely among cultivated AOA from marine environments, a capability for urea utilization.     

Novel roles for the E3 ubiquitin ligase atrophin-interacting protein 4 and signal transduction adaptor molecule 1 in G protein-coupled receptor signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.     

Setting sail for glucose homeostasis with the AKAP150‐PP2B‐anchor

EMBO J (2012) 31 20, 3991–4004 doi:10.1038/emboj.2012.244; published online August312012Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.Insulin secretion is a key component in the regulation of glucose homeostasis. The initiation of glucose-stimulated insulin secretion (GSIS) is coordinated by numerous protein phosphorylation and dephosphorylation events in the β cell (Jones and Persaud, 1998). PKA and protein phosphatase 2B (PP2B or calcineurin—a Ca²⁺/calmodulin-dependent enzyme) are examples of enzymes that can influence the release of insulin. The combined effects of these enzymes propagate GSIS, which is mediated intracellularly via an increase in ATP concentration, Ca²⁺ influx via the voltage-dependent Ca²⁺ channel (VDCC) and cyclic AMP (cAMP) signalling. At the same time, these enzymes can also regulate glucose usage (e.g., via glycogen synthase) in insulin-sensitive tissues such as the skeletal muscle.cAMP signalling serves to potentiate GSIS via either (1) PKA-dependent or (2) PKA-independent mechanisms (involving cAMP-binding protein Epac2A (exchange protein directly activated by cAMP 2)). A-kinase anchoring protein (AKAP) belongs to a group of regulatory proteins that interacts with cAMP-dependent PKA (Pidoux and Tasken, 2010; Welch et al, 2010). It can regulate the differential usage of kinase versus phosphatase, thereby controlling metabolic outcomes in specific tissues. Although it is known that PKA phosphorylation regulates β cell physiology, the role of such anchoring proteins is less clear (Faruque et al, 2009; Lester et al, 2001). For example, while disruption of the AKAP–PKA interaction has been reported to decrease insulin secretion (Lester et al, 1997), the specific regulatory protein that anchors PKA has yet to be identified.In this study, Hinke et al (2012) sought to identify the specific anchoring protein that tethers PKA, and to elucidate its function. Two AKAP proteins, namely, AKAP150 and AKAP220 were first shortlisted from an overlay assay used to detect RII (regulatory subunit of PKA) binding proteins. Subsequently, only AKAP150 was found to be important for nutrient-stimulated insulin secretion. Mice with a global knockout of AKAP150 (AKAP150KO) exhibited insulin secretory defects. AKAP150 binds to and regulates the phosphorylation-dependent VDCC. Thus, these AKAP150KO mice exhibited decreased basal Ca²⁺ current and glucose-stimulated Ca²⁺ influx in isolated β cells. One reason for the decrease in Ca²⁺ current could be attributed to a mislocation of its binding partner PP2B (discussed below). Glucose-stimulated cAMP fluctuation which is necessary for insulin secretion (Dyachok et al, 2008) was also abolished in AKAP150KO mice. Therefore, AKAP150KO mice exhibit an insulin secretory defect due to multiple impairments including (1) decreased Ca²⁺ influx and (2) defective cAMP production.Surprisingly, while the authors report that global AKAP150KO mice secrete less insulin, the skeletal muscle, an insulin-sensitive peripheral tissue, exhibited improved blood glucose clearance likely due to increased phosphorylation of IRS-1 and Akt/PKB, and activation of AMPK that resulted in improved insulin sensitivity. On the other hand, β cell-specific AKAP150KO mice secrete less insulin upon glucose stimulation despite increased insulin content in the β cell that occurs as an adaptation to the impaired glucose tolerance. These mice clearly exhibited an impaired glucose tolerance that is due to defective insulin secretion because they do not exhibit an increase in insulin sensitivity. Together, these data indicate that the skeletal muscle selectively adapts to the global absence of AKAP150 to compensate for the decrease in insulin in the body. Notably, AKAP150 is also expressed in the liver but does not exhibit compensatory effects while AKAP150 is not expressed in the adipose tissue.AKAP150 can anchor numerous enzymes with different metabolic activities. For instance, it binds PKA and PP2B, two enzymes with opposing functions, to the cell surface membrane. Hinke et al (2012) further investigated the impact of disrupting specific binding partners of AKAP150. Unexpectedly, AKAP150Δ36 mice that lack residues 705–724 and therefore cannot bind PKA exclusively are effectively metabolically normal. It is thus surprising that the anchoring of PKA to AKAP150 is not necessary for proper insulin release although this interaction is important in other cellular systems (Lu et al, 2008, 2011). AKAP150ΔPIX mice lacking residues 655–661 and thus unable to tether to PP2B at a seven-residue PIxIxIT motif demonstrate the same metabolic phenotype as global AKAP150KO mice. This suggests that AKAP150 is critical for tethering PP2B, and that PP2B is the key molecule necessary for insulin secretion in β cells. PP2B is also a determinant of the metabolic phenotypes such as improved insulin sensitivity and glucose handling upon loss of anchorage of PP2B.Overall, Hinke et al (2012) used complementary in vivo approaches including animal physiology, and in vitro islet culture and live-cell imaging to demonstrate the importance of the kinase/phosphatase anchoring protein AKAP150 in regulating nutrient-stimulated insulin secretion and modulating glucose homeostasis in mice (Figure 1). However, it is likely that there are AKAP150-independent mechanisms regulating insulin secretion since islets from AKAP150KO mice continued to respond to glucose stimulation and secrete insulin in both static and dynamic conditions, albeit at lower levels compared to wild-type mice. Importantly, the authors also identified AKAP150 tethering to PP2B as a key molecular event that regulates insulin secretion and glucose homeostasis (Figure 1). Thus, targeting the AKAP150–PP2B interface and the PIxIxIT motif could be therapeutically useful for increasing insulin sensitivity in patients with diabetes and metabolic syndromes. This could involve designing molecules or chemical compounds to bind the motif and block interaction between AKAP150 and PP2B. In parallel, the safety of systemic blockade of this interaction needs to be ascertained. Alternatively, skeletal muscle-specific AKAP150ΔPIX mice could be generated to determine if the metabolic phenotype is similar to global AKAP150ΔPIX mice. Should this be the case, then localized pharmacological blockade of AKAP150–PP2B interaction could be considered.Open in a separate windowFigure 1AKAP150 tethered to PP2B at a seven-residue PIxIxIT motif mediates nutrient-stimulated insulin secretion and glucose homeostasis. Both global AKAP150KO and AKAP150ΔPIX (AKAP150-PP2B binding abolished) mice exhibit insulin secretory defects, enhanced insulin sensitivity in skeletal muscle and overall improved glucose tolerance. This infers the importance of AKAP150-PP2B tethering for glucose homeostasis. ‘Tick'' indicates an increase or improvement. ‘Cross'' indicates a defect or impairment. ‘Equal sign'' indicates no change or no effect. Questions emerging from this study are highlighted in red.Several issues worth pursuing include (1) determining the differential adaptive response of the skeletal muscle versus the liver to alterations in insulin sensitivity in global AKAP150KO mice, (2) further investigating the functional relevance of AKAP150 tethering to PKA (by generating β cell-specific AKAP150Δ36 mice) as there is probably a biological rationale for their interaction, (3) exploring whether AKAP150-related or other proteins are expressed and act in different adipose tissue depots, (4) determining whether AKAP150 acts in a similar manner in ‘human'' skeletal muscle and β cells, and (5) examining if polymorphisms in human genes that encode AKAP150 tethering proteins are linked to disorders of glucose metabolism.