The timing of TCRα expression critically influences T cell development and selection

Sequential rearrangement of the T cell receptor for antigen (TCR) β and α chains is a hallmark of thymocyte development. This temporal control is lost in TCR transgenics because the α chain is expressed prematurely at the CD4−CD8− double negative (DN) stage. To test the importance of this, we expressed the HYα chain at the physiological CD4+CD8+ double positive (DP) stage. The reduced DP and increased DN cellularity typically seen in TCR transgenics was not observed when the α chain was expressed at the appropriate stage. Surprisingly, antigen-driven selection events were also altered. In male mice, thymocyte deletion now occurred at the single positive or medullary stage. In addition, no expansion of CD8αα intestinal intraepithelial lymphocytes (IELs) was observed, despite the fact that HY transgenics have been used to model IEL development. Collectively, these data establish the importance of proper timing of TCR expression in thymic development and selection and emphasize the need to use models that most accurately reflect the physiologic process

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

Mechanism of Assembly of the Dimanganese-Tyrosyl Radical Cofactor of Class Ib Ribonucleotide Reductase: Enzymatic Generation of Superoxide Is Required for Tyrosine Oxidation via a Mn(III)Mn(IV) Intermediate

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The Fe[superscript III][subscript 2]-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe[superscript II][subscript 2]-NrdB, O[subscript 2], and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn[superscript III][subscript 2]-Y• cofactor in their NrdF subunit. Mn[superscript II][subscript 2]-NrdF does not react with O[subscript 2], but it binds the reduced form of a conserved flavodoxin-like protein, NrdI[subscript hq], which, in the presence of O[subscript 2], reacts to form the Mn[superscript III][subscript 2]-Y• cofactor. Here we investigate the mechanism of assembly of the Mn[superscript III][subscript 2]-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn[superscript II][subscript 2]-NrdF, NrdI[subscript hq], and O[subscript 2] has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI[subscript hq] reduces O[subscript 2] to O[subscript 2]•– (40–48 s[superscript –1], 0.6 mM O[subscript 2]), the O[subscript 2]•– channels to and reacts with Mn[superscript II][subscript 2]-NrdF to form a Mn[superscript III]Mn[superscript IV] intermediate (2.2 ± 0.4 s[superscript –1]), and the Mn[superscript III]Mn[superscript IV] species oxidizes tyrosine to Y• (0.08–0.15 s[superscript –1]). Controlled production of O[subscript 2]•– by NrdI[subscript hq] during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn[superscript II][subscript 2] cluster with O[subscript 2] and satisfies the requirement for an “extra” reducing equivalent in Y• generation.National Institutes of Health (U.S.) (Grant GM81393)United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)