Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.     

Considerations in the grouping of plant and fungal taxa for an epidemiologic study

Although exposure to airborne pollen grains and fungal spores has been implicated as a causative factor for acute exacerbation of asthma, the few epidemiologic studies that have attempted to evaluate the relationship between these bioaerosols and asthma have used only total counts (ignoring the relative importance of different taxa) or a few predominant pollen or spore types (ignoring less abundant but potentially relevant groups). This paper reports the development of hypothesis‐driven exposure metrics (based on known aeroallergen associations with allergic asthma and other hypersensitivity diseases, pollen allergen cross‐reactivity, and the presence of local sources in the city of Fresno, California, USA) for a 3.5 year epidemiologic study of childhood asthma. Outdoor regional and neighborhood concentrations of pollen and spores were measured using Hirst‐type, 7‐day samplers. Indoor and outdoor residential concentrations were measured at 84 selected homes with similar 24‐hour slit impactors. All pollen and spore concentrations were recorded in 2‐hour intervals to assist in understanding diurnal fluctuations in aeroallergen concentrations, identify exposures during the time periods that children are outdoors, and study interaction between aeroallergens and other air contaminants, which were the primary focus of the study. The 124 pollen taxa that were observed were reduced to 15 categories and the 66 fungal and algal taxa were reduced to five categories that will be used in microenvironmental models to generate individual daily exposure estimates for each of the 315 children. These new exposure metrics will allow examination of health effects for taxa traditionally associated with allergy and those with locally elevated concentrations in combination with exposures to other indoor and outdoor air contaminants.     

Callus from Pyrostegia venusta (Ker Gawl.) Miers: a source of phenylethanoid glycosides with vasorelaxant activities

Plant Cell, Tissue and Organ Culture (PCTOC) - Pyrostegia venusta (Ker Gawl.) Miers is a plant used for the treatment of respiratory diseases, diarrhea, vitiligo, jaundice and to attenuate...     

Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion. METHODS: We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment. RESULTS: The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms. CONCLUSIONS: The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.