The first clinical validation of whole-genome screening on standard trophectoderm biopsies of preimplantation embryos.

Objective: To validate the performance of our laboratory-developed whole-genome screening assay within clinical preimplantation genetic testing environments.

Design: Perform a laboratory-developed whole-genome assay on both cell lines and trophectoderm biopsies, subsequently employing the next-generation sequencing procedure to reach a sequencing depth of 30X. Adhere to the Genome Analysis Toolkit best practices for accuracy, sensitivity, specificity, and precision calculations by comparing samples with references.

The molecular basis of Rac-GTP action-promoting binding of p67 to Nox2 by disengaging the β hairpin from downstream residues.

p67 fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67 to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67 peptides (106-120) and (181-195), corresponding to the β hairpin and to a downstream region engaged in intramolecular bonds with the β hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67 to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67 , expressed in increased apparent molecular weights; (iv) p67 was bound to p67 peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67 failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67 -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67 deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers.

p67 -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism.

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67 . In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67 residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67 , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition.

p67 binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond-Canaan sighted?

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67 and there is evidence for a change in the configuration of p67 being required for binding to Nox2. To study this, we measured binding of p67 to a library of Nox2 peptides and binding of NusA-Nox2 fusion proteins to p67 .

Utility and First Clinical Application of Screening Embryos for Polygenic Disease Risk Reduction.

For over 2 decades preimplantation genetic testing (PGT) has been in clinical use to reduce the risk of miscarriage and genetic disease in patients with advanced maternal age and risk of transmitting disease. Recently developed methods of genome-wide genotyping and machine learning algorithms now offer the ability to genotype embryos for polygenic disease risk with accuracy equivalent to adults. In addition, contemporary studies on adults indicate the ability to predict polygenic disorders with risk equivalent to monogenic disorders.

Validation of concurrent preimplantation genetic testing for polygenic and monogenic disorders, structural rearrangements, and whole and segmental chromosome aneuploidy with a single universal platform.

Preimplantation genetic testing (PGT) has been successfully applied to reduce the risk of miscarriage, improve IVF success rates, and prevent inheritance of monogenic disease and unbalanced translocations. The present study provides the first method capable of simultaneous testing of aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic (PGT-M) disorders using a single platform. Using positive controls to establish performance characteristics, accuracies of 97 to >99% for each type of testing were observed.

Binding of p67 to Nox2 is stabilized by disulfide bonds between cysteines in the Cys-Gly-Cys triad in Nox2 and in p67.

A central event in the activation of the phagocyte NADPH oxidase involves binding of p67 to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67 to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357-383, as a potential binding site.

Turning off NADPH oxidase-2 by impeding p67 activation in infected mouse macrophages reduced viral entry and inflammation.

Background: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O and releasing HO. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages.

The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67 (phox.).

The superoxide (O(·-) 2)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b 558 (a heterodimer of Nox2 and p22 (phox) ), and four cytosolic components, p47 (phox) , p67 (phox) , p40 (phox) , and Rac. The catalytic component, responsible for O(·-) 2 generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67 (phox) .

Connexin43 in rat oocytes: developmental modulation of its phosphorylation.

It is well established that the 43-kDa connexin (Cx43) is predominantly expressed by ovarian somatic cells, whereas the identity of the connexins contributed by the oocyte to form gap junctions with its neighboring cells is not fully elucidated. Our study aimed to examine oocytes for the expression and regulation of Cx43 throughout oogenesis. Growing and fully grown rat oocytes that were meiotically incompetent and competent, respectively, were examined.

Temporal analysis of connexin43 protein and gene expression throughout the menstrual cycle in human endometrium.

Objective: To analyze the pattern of connexin43 gene and protein expression in human endometrium throughout the menstrual cycle.

Design: Controlled clinical study.

Setting: An academic research center.

Linking protein kinase C to the cell cycle: ectopic expression of PKC eta in NIH3T3 cells alters the expression of cyclins and Cdk inhibitors and induces adipogenesis.

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that ectopic expression of PKC eta in NIH3T3 fibroblasts blocks the normal phosphorylation of the Rb protein in quiescent cultures restimulated to enter the cell cycle; PKC eta activates a cellular program that includes increased expression of cyclins E (but not cyclin D), as well as the induced expression of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1.

The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus.

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus.