Expanding the DNA editing toolbox: Novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness.

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches.

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility.

Directed co-evolution of interacting protein-peptide pairs by compartmentalized two-hybrid replication (C2HR).

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic β-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects.

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets.

Structure-activity studies of Mdm2/Mdm4-binding stapled peptides comprising non-natural amino acids.

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan.

Functional characterization of p53 pathway components in the ancient metazoan Trichoplax adhaerens.

The identification of genes encoding a p53 family member and an Mdm2 ortholog in the ancient placozoan Trichoplax adhaerens advocates for the evolutionary conservation of a pivotal stress-response pathway observed in all higher eukaryotes. Here, we recapitulate several key functionalities ascribed to this known interacting protein pair by analysis of the placozoan proteins (Tap53 and TaMdm2) using both in vitro and cellular assays. In addition to interacting with each other, the Tap53 and TaMdm2 proteins are also able to respectively bind human Mdm2 and p53, providing strong evidence for functional conservation.

The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution.

Conservative site-specific and single-copy transgenesis in human LINE-1 elements.

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility.

Directed evolution of λ integrase activity and specificity by genetic derepression.

Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using β-lactamase and the β-lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin.

Rapid screening of protein-protein interaction inhibitors using the protease exclusion assay.

We have previously developed a sensitive and modular homogenous biosensor system using peptides to detect target ligands. By transposing the basic mechanistic principle of the nuclease protection assay into this biosensor framework, we have developed the protease exclusion (PE) assay which can discern antagonists of protein-protein interactions in a rapid, single-step format. We demonstrate the concept with multiple protein-peptide pairs and validate the method by successfully screening a small molecule library for compounds capable of inhibiting the therapeutically relevant p53-Mdm2 interaction.

Analysis of p53 binding to DNA by fluorescence imaging microscopy.

Transcription factors play a central role in cell biology through binding to target DNA elements and regulating gene expression. In this study, we used the p53 tumour suppressor as a model transcription factor to develop an imaging based assay to measure DNA binding. The assay utilizes fluorescence imaging microscopy to detect labelled p53 bound to DNA coated on microbeads.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways.

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown.

Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide.

The gaseous mediators hydrogen sulphide (H2S) and nitric oxide (*NO) are synthesised in the body from L-cysteine and L-arginine, respectively. In the cardiovascular system, *NO is an important regulator of vascular tone and its over- or under-production has been linked to a variety of diseases. The physiological significance of H2S is not yet clear but, like *NO, it exhibits vasodilator activity and may play a part in septic and haemorrhagic shock, hypertension, regulation of cardiac contractility, and in inflammation.

Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat.

The biosynthesis of both nitric oxide (NO) and hydrogen sulfide (H2S) is increased in lipopolysaccharide (LPS)-injected mice and rats but their interaction in these models is not known. In this study we examined the effect of the NO donor, nitroflurbiprofen (and the parent molecule flurbiprofen) on NO and H2S metabolism in tissues from LPS-pretreated rats. Administration of LPS (10 mg kg(-1), i.

Hypochlorous acid-mediated mitochondrial dysfunction and apoptosis in human hepatoma HepG2 and human fetal liver cells: role of mitochondrial permeability transition.

Liver cirrhosis is often preceded by overt signs of hepatitis, including parenchymal cell inflammation and infiltration of polymorphonuclear (PMN) leukocytes. Activated PMNs release both reactive oxygen species and reactive halogen species, including hypochlorous acid (HOCl), which are known to be significantly cytotoxic due to their oxidizing potential. Because the role of mitochondria in the hepatotoxicity attributed to HOCl has not been elucidated, we investigated the effects of HOCl on mitochondrial function in the human hepatoma HepG2 cell line, human fetal liver cells, and isolated rat liver mitochondria.

Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain?

Hydrogen sulphide (H(2)S) is a cytotoxic gas that has recently been proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM, and considerably lower H(2)S levels are reported in the brains of Alzheimer's disease (AD) patients. Levels of myeloperoxidase (MPO), an enzyme that catalyses the formation of the oxidant hypochlorous acid (HOCl), are elevated in the prefrontal cortex, hippocampal microglia, and neurons of AD patients where MPO co-localised with beta-amyloid plaques.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death.

Nitrite-mediated protection against hypochlorous acid-induced chondrocyte toxicity: a novel cytoprotective role of nitric oxide in the inflamed joint?

Objective: To examine the potential consequences of overproduction of nitric oxide (NO) and nitrite (NO(2) (-)) in the inflamed rheumatoid joint.

Methods: Human articular chondrocytes in culture were exposed to HOCl (hypochlorous acid, a physiologic oxidant formed in increased amounts at sites of chronic inflammation), and assays of cell viability, intracellular ATP and glutathione (GSH), and lactate dehydrogenase (LDH) were performed. HOCl-induced lipid peroxidation and activation of the MAP kinases ERK-1/2, JNK-1/2, and p38 were also measured.

Lack of tyrosine nitration by hypochlorous acid in the presence of physiological concentrations of nitrite. Implications for the role of nitryl chloride in tyrosine nitration in vivo.

Elevated levels of reactive nitrogen species (RNS) such as peroxynitrite have been implicated in over 50 diverse human diseases as measured by the formation of the RNS biomarker 3-nitrotyrosine. Recently, an additional RNS was postulated to contribute to 3-nitrotyrosine formation in vivo; nitryl chloride formed from the reaction of nitrite and neutrophil myeloperoxidase-derived hypochlorous acid (HOCl). Whether nitryl chloride nitrates intracellular protein is unknown.

Megestrol acetate in cancer anorexia and weight loss.

High-dose megestrol acetate has been associated with increased appetite and weight. To examine the effects of high-dose megestrol acetate in the treatment of anorexia and weight loss in patients with advanced hormone-insensitive malignant lesions, a randomized double-blind placebo-controlled trial was conducted. Patients receiving megestrol acetate for 1 month reported a significant improvement in appetite and adequacy of food intake compared with those receiving placebo.

Psychological functioning of daughters of breast cancer patients. Part II: Characterizing the distressed daughter of the breast cancer patient.

Sixty daughters whose mothers had breast cancer were cross-sectionally studied. Daughters age 11-20 reported feeling significantly more uncomfortable about involvement in their mothers' illness than daughters age 20+. Daughters whose mothers died were more likely to report long-term life plan changes and role changes with their mothers during the mothers' illness.

Quality of life assessment. An independent prognostic variable for survival in lung cancer.

Improved quality of life has long been the goal of cancer treatment, but only recently have investigators begun to include a systematic assessment of quality of life in clinical trials. The major interest for its inclusion in clinical trials has been to assess treatment outcome. An evaluation of the relationship between patient-rated quality of life and survival is reported in a homogeneous sample of patients with advanced metastatic lung cancer participating in a clinical trial.

Psychological functioning of daughters of breast cancer patients. Part I: Daughters and comparison subjects.

Sixty daughters of mothers with breast cancer were matched for age, educational level, and race with 60 comparison subjects without a maternal history of breast cancer to assess the impact on psychological adjustment, coping, body image, sexual functioning, and health knowledge and practices of having had a mother with breast cancer. Daughters of breast cancer patients showed significantly less frequent sexual intercourse, lower sexual satisfaction, and greater feelings of vulnerability to breast cancer, and they could identify a greater number of symptoms of breast cancer. No differences between groups were found in psychological symptoms, coping styles, breast self-examination practices, mammography practices, health knowledge, or body-image ratings.