Parallel analysis of genetic selections using whole genome oligonucleotide arrays

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein–protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.     

The Incidence of Primary vs Secondary Focal Segmental Glomerulosclerosis: A Clinicopathologic Study

Objectives

To describe the change in the incidence rates of primary and secondary focal segmental glomerulosclerosis (FSGS) from 1994 through 2013 in Olmsted County, Minnesota, and to identify the clinical and biopsy characteristics that distinguish primary from secondary FSGS.

Risk of bias reporting in Cochrane systematic reviews

Risk of bias is an inherent quality of primary research and therefore of systematic reviews. This column addresses the Cochrane Collaboration's approach to assessing, risks of bias, the meaning of each, indicators of low, high and uncertain, and ways that risk of bias can be represented in a Cochrane systematic review report. The sources of risk of bias that reviewers evaluate include selection, performance, detection, attrition and reporting bias. Each poses threat to the internal validity of the primary studies and requires the reviewer to judge the level of risk as high, low or unclear. Reviewers need to address how studies of higher risk of bias might impact the pooled effect.     

Focused antibody response to influenza linked to antigenic drift

The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.     

Determinants of Diuretic Responsiveness and Associated Outcomes During Acute Heart Failure Hospitalization: An Analysis From the NHLBI Heart Failure Network Clinical Trials

Background

Poor response to loop diuretic therapy is a marker of risk during heart failure hospitalization. We sought to describe baseline determinants of diuretic response and to further explore the relationship between this response and clinical outcomes.