ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient.

Identification of human remains using Rapid DNA analysis.

Rapid identification of human remains following mass casualty events is essential to bring closure to family members and friends of the victims. Unfortunately, disaster victim identification, missing persons identification, and forensic casework analysis are often complicated by sample degradation due to exposure to harsh environmental conditions. Following a mass disaster, forensic laboratories may be overwhelmed by the number of dissociated portions that require identification and reassociation or compromised by the event itself.

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay.

Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types.

Sirt2 deacetylase is a novel AKT binding partner critical for AKT activation by insulin.

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr(308) and Ser(473), mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis.

Variants in the Signaling Protein TSAd are Associated with Susceptibility to Ovarian Cancer in BRCA1/2 Negative High Risk Families.

A substantial fraction of familial ovarian cancer cases cannot be attributed to specific genetic factors. The discovery of additional susceptibility genes will permit a more accurate assessment of hereditary cancer risk and allow for monitoring of predisposed women in order to intervene at the earliest possible stage. We focused on a population with elevated familial breast and ovarian cancer risk.

Bimodal regulation of FoxO3 by AKT and 14-3-3.

FoxO3 is a member of FoxO family transcription factors that mediate cellular functions downstream of AKT. FoxO3 phosphorylation by AKT generates binding sites for 14-3-3, which in-turn regulates FoxO3 transcriptional activity and localization. We examine here the functional significance of AKT-FoxO3 interaction and further detail the mechanistic aspects of FoxO3 regulation by AKT and 14-3-3.

Life span extension and neuronal cell protection by Drosophila nicotinamidase.

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases.

Construction of an active acetohydroxyacid synthase I with a flexible linker connecting the catalytic and the regulatory subunits.

Acetohydroxyacid synthase I (AHAS I), one of three isozymes in Escherichia coli catalyzing the first common step in the biosynthesis of branched amino acids, is composed of two kinds of subunits. The large catalytic (B) and small regulatory (N) subunits of the holoenzyme dissociate and associate freely and rapidly and are quite different in size, charge and hydrophobicity, so that high resolution purification methods lead to partial separation of subunits and to heterogeneity. We have prepared several linked AHAS I proteins, in which the large subunit B with a hexahistidine-tag at the N-terminus, was covalently joined by a flexible linker, containing several (X) amino acids, to the small subunit N to form His6-BuXN polypeptides.

The F-box protein, Ufo1, maintains genome stability by recruiting the yeast mating switch endonuclease, Ho, for rapid proteasome degradation.

We describe a unique E3, the F-box protein, Ufo1, of yeast. Ufo1 recruits the mating switch endonuclease, Ho, to the SCF complex for ubiquitylation. In addition to the F-box and WD40 protein-protein interaction domains found in all F-box proteins, Ufo1 has a unique domain comprising multiple copies of the ubiquitin-interacting motif.

Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes.

SCF complexes are E3 ubiquitin-protein ligases that mediate degradation of regulatory and signaling proteins and control G1/S cell cycle progression by degradation of G1 cyclins and the cyclin-dependent kinase inhibitor, Sic1. Interchangeable F-box proteins bind the core SCF components; each recruits a specific subset of substrates for ubiquitylation. The F-box proteins themselves are rapidly turned over by autoubiquitylation, allowing rapid recycling of SCF complexes.

The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease.

Ho endonuclease initiates a mating type switch by making a double-strand break at the mating type locus, MAT. Ho is marked by phosphorylation for rapid destruction by functions of the DNA damage response, MEC1, RAD9, and CHK1. Phosphorylated Ho is recruited for ubiquitylation via the SCF ubiquitin ligase complex by the F-box protein, Ufo1.

DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export.

Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1. A PEST sequence marks Ho for degradation. Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation.