Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium Tuberculosis During an Outbreak.

Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak.

PLoS One. 2013; 8(3): e58235
Kato-Maeda M, Ho C, Passarelli B, Banaei N, Grinsdale J, Flores L, Anderson J, Murray M, Rose G, Kawamura LM, Pourmand N, Tariq MA, Gagneux S, Hopewell PC

Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of over short time periods.To describe the microevolution of during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina’s sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case.Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission. HubMed – drug


Large-Scale Functional Purification of Recombinant HIV-1 Capsid.

PLoS One. 2013; 8(3): e58035
Hung M, Niedziela-Majka A, Jin D, Wong M, Leavitt S, Brendza KM, Liu X, Sakowicz R

During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. HubMed – drug


Posaconazole Exhibits In Vitro and In Vivo Synergistic Antifungal Activity with Caspofungin or FK506 against Candida albicans.

PLoS One. 2013; 8(3): e57672
Chen YL, Lehman VN, Averette AF, Perfect JR, Heitman J

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the ?-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen . Although current drug treatments for infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and infections. Our studies reported here demonstrate that posaconazole exhibits synergy with caspofungin or FK506 against drug susceptible or resistant strains. Furthermore, these combinations also show synergy against strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 ?-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future. HubMed – drug