Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Multi-centre evaluation of the speed-oligo Mycobacteria assay for differentiation of Mycobacterium spp. in clinical isolates

<p>Abstract</p> <p>Background</p> <p>A new DNA line probe assay (Speed-oligo Mycobacteria, Vircell) has been launched for rapid differentiation of <it>Mycobacterium </it>spp. from cultures. Compared to other line-probe assays, Speed-oligo Mycobacteria covers a relatively limited spectrum of species but uses a simpler and faster dip-stick technique. The present multi-centre, multi-country study aimed at evaluating the utility and usability of Speed-oligo Mycobacteria in routine mycobacteriology diagnostics. Results from Speed-oligo Myobacteria were compared to those from Genotype CM (HAIN lifescience, Nehren, Germany), another line-probe assay.</p> <p>Methods</p> <p>Speed-oligo Mycobacteria assay was performed in three main steps: 1) DNA extraction from cultured material 2) PCR amplification of the target gene and an internal control and 3) hybridization of the PCR products to specific probes by means of a dip-stick.</p> <p>Results</p> <p>Two hundred forty-two clinical isolates were recovered from consecutive positive mycobacterial cultures at two German (IML Gauting, Bioscientia Ingelheim), one Czech (KLINLAB Prague), and at a Sudanese (Khartoum) laboratory. All <it>Mycobacterium </it>species covered by the assay were reliably recognized. The rate of false positive results was 1.2% and concerned only the species <it>M. marinum </it>and <it>M. peregrinum</it>. The identification rate, i.e. the proportion of isolates which was correctly differentiated to the level of species or complex by the assay, differed significantly among laboratories being 94.9%, 90.7%, and 75.0% at the study sites IML Gauting, KLINLAB Prague and Bioscientia Ingelheim, respectively. This difference was caused by different spectra of NTM species encountered by the laboratory centres in daily routine diagnostics.</p> <p>Conclusions</p> <p>Speed-oligo Mycobacteria assay was proved a rapid and easy-to-perform alternative to conventional line-probe assays. The assay showed excellent sensitivity with regard to identification of genus <it>Mycobacterium </it>and species/complexes covered by the test. However, due to its relatively limited spectrum of taxa, a varying proportion of NTM may not be identified by the assay in daily diagnostics demanding further analyses. The only significant shortcoming in terms of specificity was the misidentification of the clinically relevant species <it>M. marinum</it>.</p

Monte Carlo Simulations of HIV Capsid Protein Homodimer

Capsid protein (CA) is the building block of virus coats. To help understand how the HIV CA proteins self-organize into large assemblies of various shapes, we aim to computationally evaluate the binding affinity and interfaces in a CA homodimer. We model the N- and C-terminal domains (NTD and CTD) of the CA as rigid bodies and treat the five-residue loop between the two domains as a flexible linker. We adopt a transferrable residue-level coarse-grained energy function to describe the interactions between the protein domains. In seven extensive Monte Carlo simulations with different volumes, a large number of binding/unbinding transitions between the two CA proteins are observed, thus allowing a reliable estimation of the equilibrium probabilities for the dimeric vs monomeric forms. The obtained dissociation constant for the CA homodimer from our simulations, 20–25 μM, is in reasonable agreement with experimental measurement. A wide range of binding interfaces, primarily between the NTDs, are identified in the simulations. Although some observed bound structures here closely resemble the major binding interfaces in the capsid assembly, they are statistically insignificant in our simulation trajectories. Our results suggest that although the general purpose energy functions adopted here could reasonably reproduce the overall binding affinity for the CA homodimer, further adjustment would be needed to accurately represent the relative strength of individual binding interfaces

The Future Landscape of High-Redshift Galaxy Cluster Science

Large scale structure and cosmolog

Isolation of Mycobacterium malmoense in the island of Crete, Greece

Mycobacterium malmoense was isolated from a broncho-alveolar lavage sample of a 73-year-old cancer (small cell lung carcinoma) patient in Crete, representing the first reported case of this pathogen in Greece. The isolate was considered to be a colonizer and the patient did not receive any antimycobacterial treatment while he received chemotherapy to which he responded favourably. No signs of pulmonary infection were noted during the course of his disease. This case provides evidence of the ubiquitous nature of this mycobacterial species, believed until recently to favour cooler climates. We, therefore, propose that the index of suspicion for this pathogen should be raised particularly in patients with underlying immunodeficiency, cancer and chronic lung disease, irrespective of the geographic location

Molecular diagnostic tools in mycobacteriology

Although the diagnosis of mycobacteriosis and susceptibility testing are still primarily based on conventional methods (staining, culture, biochemical analysis, proportional method), a series of molecular assays are increasingly introduced and incorporated in the workflow of clinical mycobacteriology laboratories worldwide. These assays are rapid and offer high sensitivities and specificities. In the present review, we describe the molecular assays concerning the early detection of Mycobacteria in clinical specimens, the identification of mycobacterial species, the detection of drug resistance and the typing for epidemiological investigations. (C) 2008 Elsevier B.V. All rights reserved

Co-limitation of potato growth by Potato Cyst Nematode (Globodera rostochiensis) and Rhizoctonia solani

Globodera rostochiensis and Rhizoctonia solani are the most important growth limiting factors influencing potato production in Iran. The effects of inoculation with Potato Cyst Nematodes (PCN) (0, 50, 75 and 100 cysts/3.5 kg soil) and R. solani (with or without inoculation) on potato growth and development were investigated in cultivars Santé and Marfona. Inoculation with R. solani induced severe damage, especially when inoculation was accompanied with high density of PCN. The damage caused by R. solani tended to increase with an increase in PCN density, especially in Marfona. In Santé, number of stems or branches per plant significantly increased by inoculation with R. solani, while in Marfona it was significantly affected either by R. solani inoculation or PCN density. In Santé, number of stolons per plant was significantly increased by PCN, but not by R. solani. In Marfona, however, the number of stolons per plant was significantly affected either by R. solani inoculation or by presence of PCN, but not affected by PCN density. The general effect of R. solani or PCN inoculation treatments on shoot, below-ground and total dry weight of potato was significant, but strongly affected by cultivar. In general, our study supports the synergistic interaction between R. solani and PCN and its moderation by the use of a resistant cultivar such as Santé

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly froth the clinical sample. (C) 2009 Elsevier Inc. All rights reserved