Monomeric APOBEC3G is catalytically active and has antiviral activity

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.     

Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein-disulfide isomerase with superoxide dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.     

Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development

During metamorphic development, bullfrogs (Rana catesbeiana) undergo substantial morphological, anatomical, and physiological changes as the animals prepare for the transition from a fully-aquatic to a semi-terrestrial existence. Using BrdU incorporation and immunohistochemistry, we quantify changes in cell proliferation in two key auditory brainstem nuclei, the dorsolateral nucleus and the superior olivary nucleus, over the course of larval and early postmetamorphic development. From hatchling through early larval stages, numbers of proliferating cells increase in both nuclei, paralleling the overall increase in total numbers of cells available for labeling. Numbers of proliferating cells in the superior olivary nucleus decrease during the late larval and deaf periods, and significantly increase during metamorphic climax. Proliferating cells in the dorsolateral nucleus increase in number from hatchling to late larval stages, decrease during the deaf period, and increase during climax. In both nuclei, numbers of proliferating cells decrease during the postmetamorphic froglet stage, despite increases in the number of cells available for label. Newly generated cells express either glial- or neural-specific phenotypes beginning between 1 week and 1 month post-BrdU injection, respectively, while some new cells express gamma-aminobutyric acid within 2 days of mitosis. Our data show that these auditory nuclei dramatically up-regulate mitosis immediately prior to establishment of a transduction system based on atmospheric hearing.     

The Effect of Leflunomide on Cycling and Activation of T-Cells in HIV-1-Infected Participants

Background

The pathogenesis of immunodeficiency due to human immunodeficiency virus (HIV)-1 is incompletely understood, but immune activation is believed to play a central role. Immunomodulatory agents that decrease immune activation may be useful in the treatment of HIV-1 infection.

Methodology

A randomized, double blind, placebo-controlled pilot study of leflunomide for 28 days was performed in participants with HIV-1 infection who were not receiving antiretroviral therapy. Participants randomized to leflunomide were subsequently treated with cholestyramine until leflunomide levels were below detection limit.

Findings

Treatment with leflunomide was well tolerated with mostly low-grade adverse events. Leflunomide administration reduced cycling of CD4 T cells (by ex vivo bromodeoxyuridine uptake and Ki67 expression) and decreased expression of activation markers (HLA-DR/CD38 co-expression) on CD8 T cells in peripheral blood. In addition, decreased expression of HIV-1 co-receptors was observed in both CD4 and CD8 T cells in the leflunomide group. There were no significant changes in naïve and memory T cell subsets, apoptosis of T cells or markers of microbial translocation.

Conclusions

Leflunomide was effective in reducing immune activation in the setting of chronic HIV-1 infection suggesting that targeting immune activation with immunomodulatory agents may be a feasible strategy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00101374","term_id":"NCT00101374"}}NCT00101374     

60 kD Ro and nRNP A Frequently Initiate Human Lupus Autoimmunity

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns.     

Fibulin-1 Is Increased in Asthma – A Novel Mediator of Airway Remodeling?

Background

The extracellular matrix is a dynamic and complex network of macromolecules responsible for maintaining and influencing cellular functions of the airway. The role of fibronectin, an extracellular matrix protein, is well documented in asthma. However, the expression and function of fibulin-1, a secreted glycoprotein which interacts with fibronectin, has not been reported. Fibulin-1 is widely expressed in basement membranes in many organs including the lung. There are four isoforms in humans (A–D) of which fibulin-1C and 1D predominate. The objective of this study was to study the expression of fibulin-1 in volunteers with and without asthma, and to examine its function in vitro.

Methodology/Principal Findings

We used immunohistochemistry and dot-blots to examine fibulin-1 levels in bronchial biopsies, bronchoalveolar lavage fluid and serum. Real-time PCR for fibulin-1C and 1D, and ELISA and western blotting for fibulin-1 were used to study the levels in airway smooth muscle cells. The function of fibulin-1C was determined by assessing its role, using an antisense oligonucleotide, in cell proliferation, migration and wound healing. A murine model of airway hyperresponsiveness (AHR) was used to explore the biological significance of fibulin-1. Levels of fibulin-1 were significantly increased in the serum and bronchoalveolar lavage fluid of 21 asthmatics compared with 11 healthy volunteers. In addition fibulin-1 was increased in asthma derived airway smooth muscle cells and fibulin-1C contributed to the enhanced proliferation and wound repair in these cells. These features were reversed when fibulin-1C was suppressed using an antisense oligomer. In a mouse model of AHR, treatment with an AO inhibited the development of AHR to methacholine.

Conclusions

Our data collectively suggest fibulin-1C may be worthy of further investigation as a target for airway remodeling in asthma.     

Nanoparticulate transport of oximes over an in vitro blood-brain barrier model

Background

Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB.

Methodology/Principal Findings

In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes.

Conclusions/Significance

With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.     

Bottlenecks for High Coverage of Intermittent Preventive Treatment in Pregnancy: The Case of Adolescent Pregnancies in Rural Burkina Faso

Background

While IPTp-SP is currently being scaled up in sub-Saharan Africa (SSA), the coverage with the required ≥2 doses of SP remains considerably short of the Roll Back Malaria (RBM) goal of 80%, not to mention of the recently advocated universal coverage.

Methods

The study triangulates quantitative data from a health center randomized community-based trial on IPTp-SP effectiveness and the additional benefit of a promotional campaign with qualitative data from focused ethnography.

Findings

In rural Burkina Faso, despite the significantly higher risk of malaria infection among adolescent primigravidae (PG) (OR 2.44 95%CI 1.81–3.28, p<0.001), making them primary target beneficiaries of IPTp-SP, adolescents adhered to the required three or more ANC visits significantly less (PG: 46.6%; SG 43.7%) than adults (PG: 61.9%; SG 54.9%) and had lower SP uptake during the malaria transmission season, further showing the difficulty of reaching this age group. Adolescents'' structural constraints (such as their social position and household labor requirements) and needs (such as anonymity in the health encounter) leave them highly vulnerable during their pregnancies and, especially, during the high malaria transmission season.

Conclusion

Our study shows that adolescents need to be targeted specifically, prior to their first pregnancy and with measures adapted to their social context, addressing their structural constraints and needs and going beyond standard health promotion campaigns. Unless such specific measures are taken, adolescents'' social vulnerability will present a serious bottleneck for the effectiveness of IPTi-SP.     

Behavior and Welfare of Domestic Cats Housed in Cages Larger than U.S. Norm

The effect of providing additional floor space on cat behavior and welfare is not well documented. This study involved replication of an investigation of cats’ responses to enhanced cage and room environments using cages of 0.56 m² with the same methodology but an increased space allowance of 1.1 m². Singly housed adult cats (n = 59) were randomly assigned to a treatment group that was a combination of a managed or unmanaged room and an enriched or unenriched cage environment. Cats were observed for 2 days for maintenance, affiliative, and avoidant behaviors using scan sampling and 5-min, continuous focal sampling. At the end of Day 2, cats’ reactions to the approach of an unfamiliar person were assessed. Cats housed in enriched/managed environments exhibited more maintenance and affiliative behaviors and fewer avoidant behaviors than cats in unmanaged/unenriched environments, suggesting that macro and micro environments may be equally relevant to the cat. Increased space did not enhance the cats’ welfare outcomes, suggesting that the provision of additional cage space may not be as important to the cat as a managed housing environment.