Pyrimidine Triones as Potential Activators of p53 Mutants.

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy.

Optimized protocol for CRISPR knockout of human iPSC-derived macrophages.

Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages.

A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk.

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers.

LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor.

The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains.

LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor.

The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains.

CHOPER filters enable rare mutation detection in complex mutagenesis populations by next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized genetics and enabled the accurate identification of many genetic variants across many genomes. However, detection of biologically important low-frequency variants within genetically heterogeneous populations remains challenging, because they are difficult to distinguish from intrinsic NGS sequencing error rates. Approaches to overcome these limitations are essential to detect rare mutations in large cohorts, virus or microbial populations, mitochondria heteroplasmy, and other heterogeneous mixtures such as tumors.

The systemic exposure to inhaled beclometasone/formoterol pMDI with valved holding chamber is independent of age and body size.

Background: Asthma guidelines recommend prescription of inhaled corticosteroids at a reduced dosage in children compared to older patients in order to minimize the systemic exposure and risk of unwanted side effects. In children, pressurized metered dose inhalers (pMDI) are recommended in combination with a valved holding chamber (VHC) to overcome the problem of coordinating inhalation with actuation. However, the influence of age and body size on the systemic exposure of drugs to be administered via a pMDI with VHC is still not fully elucidated.

Beclometasone-formoterol as maintenance and reliever treatment in patients with asthma: a double-blind, randomised controlled trial.

Background: According to international treatment guidelines, inhaled rapid-acting β2 agonists should be used for the control of symptoms in patients with asthma. We compared the efficacy and safety of an extrafine combination inhaler containing a corticosteroid (beclometasone) plus a rapid-onset, long-acting β2 agonist (formoterol) with a short-acting β2 agonist (salbutamol) as reliever strategies in patients taking beclometasone-formoterol combination as maintenance treatment.

Methods: In a double-blind trial undertaken in 183 centres in 14 European countries over 48 weeks, patients (aged ≥18 years) with asthma that was not fully controlled, with a forced expiratory volume in 1 s (FEV1) of at least 60% predicted, had a 2-week run in.

Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53.

The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive.

Relief of methacholine-induced bronchospasm with extrafine beclomethasone dipropionate/formoterol in comparison with salbutamol in asthma.

Background: Short-acting beta2-agonists like salbutamol and terbutaline are used as rescue medications for acute bronchoconstriction and relief of symptoms due to their rapid onset of action. The aim of this study was to assess whether inhaled beclomethasone dipropionate (BDP)/formoterol fumarate (FF) combination in extrafine formulation is non-inferior to salbutamol in the speed of reverting methacholine-induced bronchoconstriction and symptoms.

Methods: Fifty-six asthmatic patients were examined in a multicentre, randomised, double blind, double dummy, active treatment and placebo controlled three period cross-over study.

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants"). Activity can be restored by second-site suppressor mutations ("rescue mutants"). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details.

Src kinases are required for a balanced production of IL-12/IL-23 in human dendritic cells activated by Toll-like receptor agonists.

Background: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation.

All-codon scanning identifies p53 cancer rescue mutations.

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes.

Neoadjuvant chemotherapy with trastuzumab followed by adjuvant trastuzumab versus neoadjuvant chemotherapy alone, in patients with HER2-positive locally advanced breast cancer (the NOAH trial): a randomised controlled superiority trial with a parallel HER2-negative cohort.

Background: The monoclonal antibody trastuzumab has survival benefit when given with chemotherapy to patients with early, operable, and metastatic breast cancer that has HER2 (also known as ERBB2) overexpression or amplification. We aimed to assess event-free survival in patients with HER2-positive locally advanced or inflammatory breast cancer receiving neoadjuvant chemotherapy with or without 1 year of trastuzumab.

Methods: We compared 1 year of treatment with trastuzumab (given as neoadjuvant and adjuvant treatment; n=117) with no trastuzumab (118), in women with HER2-positive locally advanced or inflammatory breast cancer treated with a neoadjuvant chemotherapy regimen consisting of doxorubicin, paclitaxel, cyclophosphamide, methotrexate, and fluorouracil.

Predicting positive p53 cancer rescue regions using Most Informative Positive (MIP) active learning.

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results.

Introduction of zwitterionic motifs into bacterial polysaccharides generates TLR2 agonists able to activate APCs.

It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS.

Dynamics of chromophore binding to Lhc proteins in vivo and in vitro during operation of the xanthophyll cycle.

Three plant xanthophylls are components of the xanthophyll cycle in which, upon exposure of leaves to high light, the enzyme violaxanthin de-epoxidase (VDE) transforms violaxanthin into zeaxanthin via the intermediate antheraxanthin. Previous work () showed that xanthophylls are bound to Lhc proteins and that substitution of violaxanthin with zeaxanthin induces conformational changes and fluorescence quenching by thermal dissipation. We have analyzed the efficiency of different Lhc proteins to exchange violaxanthin with zeaxanthin both in vivo and in vitro.