NEST 2014: views from the trainees-talking about what matters in efforts to diversify the STEM workforce.

This paper summarizes the outcomes of a retreat designed to cultivate interactions between trainees at various training levels and provide them opportunities to share their training perspectives and expectations. Retreat outcomes are used to support the development of better science, technology, engineering, and mathematics training practices by informing the trainers’ perspective.

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain.

The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication.

A method for imputing the impact of health problems on at-work performance and productivity from available health data.

Objective: To develop a method for imputing the work performance and productivity impact of illness and treatment from available data.

Methods: Using data from four studies of musculoskeletal disorders (eg, osteoarthritis) and pain, we modeled the relationships between scores from the Work Limitations Questionnaire (WLQ), a validated measure of health-related limitations in work performance and productivity, and a series of validated health measures (eg, a pain scale).

Results: The 15 health and 5 WLQ variables were significantly associated in 115 of 116 study-specific models (P < 0.

Proton transfer in the mechanism of polyadenylate polymerase.

PAP (polyadenylate polymerase) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, the pH dependence of the steady-state kinetic parameters of yeast PAP were determined for the forward (adenyl transfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the forward reaction.

The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA.

DNA replication is initiated upon binding of "initiators" to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA.

Repertoire diversification in mice with an IgH-locus-targeted transgene for the rearranged VH domain of a physiologically selected anti-ssDNA antibody.

To test the fate of developing B cells with autoreactive receptor components, we studied mice homozygous for a knock-in transgene coding the VH domain of an IgM ssDNA-binding antibody. The transgene has unmutated C57 BL/6 V gene segments. Homozygous knock-in mice developed normal numbers of spleen and bone marrow B cells and normal serum Ig concentrations, and had the same low level of serum anti-ssDNA antibody as non-transgenic mice.

Altered memory B-cell homeostasis in human aging.

Previous studies of age-associated immune system changes revealed alterations in expressed immunoglobulin heavy chain variable domain repertoires, and variability in the fraction of expressed heavy chain variable domain genes with mutations. To test whether the latter finding reflected a variation in memory B-cell numbers, we measured circulating memory B cells of 11 healthy elderly subjects, 173 nursing-home residents, and 34 healthy young adults. A large fraction of old adults have low values for memory cells both as a percentage of all B cells and as an absolute memory B-cell concentration.

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain.

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments.

Human immunoglobulin variable region gene analysis by single cell RT-PCR.

This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19.

Immunoglobulin VH gene expression in human aging.

Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly.