HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Binding of dantrolene-Na to the ciliated membrane ofParamecium aurelia

Summary Dantrolene-Na is a muscular relaxant which binds to sarcoplasmic reticulum (SR) with high affinity and decreases the availability of Ca²⁺ channels. The binding of fluorescent compounds, dantrolene-Na, nifedipine and chlortetracycline to the ciliary membrane ofParamecium aurelia has been studied. Dantrolene at the concentrations of 1.9 · 10^–5, 3.8 · 10^–5 and 7.9 · 10^–5 M manifested a punctuated binding pattern to the cell membrane. Isolated cilia also bound dantrolene at their basal portion, whereas deciliated cell bodies lost their dotted binding pattern. Chlortetracycline showed a similar but weaker fluorescent staining. Nifedipine treated cells revealed no sign of fluorescent binding to the membrane and was only taken up in food vacuoles.Based on these observations we propose that dantrolene binding regular arrays ofParamecium cell membrane could be identical to granular plaques observed by electron microscope. The possible functioning of these structures as Ca²⁺ reservoirs is also discussed.     

Computational Design and Analysis of a Poly-Epitope Fusion Protein: A New Vaccine Candidate for Hepatitis and Poliovirus

International Journal of Peptide Research and Therapeutics - Infections with HCV, HBV and poliovirus are still considered to be substantial global health burdens. Vaccination is one of the most...     

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells

Molecular Biology Reports - Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the...     

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

Exact results for fixation probability of bithermal evolutionary graphs

One of the most fundamental concepts of evolutionary dynamics is the “fixation” probability, i.e. the probability that a mutant spreads through the whole population. Most natural communities are geographically structured into habitats exchanging individuals among each other and can be modeled by an evolutionary graph (EG), where directed links weight the probability for the offspring of one individual to replace another individual in the community. EGs have recently spurred huge interest, as it has been shown that some topology can amplify or suppress the effect of beneficial mutations. Very few exact analytical results however are known for EGs. In this article we show that the use of a new technique, the fixed point of probability generating function, allows us to compute the exact fixation probability for a large subset of bithermal graphs. We also show by numerical simulations that the computed solution holds for all bithermal graphs. Moreover, the analytical solution allows us to clarify the opposing consequences of birth–death versus death–birth processes as amplifier or suppressor of beneficial mutations for the same bithermal topology.