Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Role of MARCKS in regulated secretion from mast cells and airway goblet cells.

MARCKS (myristoylated alanine-rich C kinase substrate) is postulated to regulate the passage of secretory granules through cortical actin in the early phase of exocytosis. There are, however, three proposed mechanisms of action, all of which were derived from studies using synthetic peptides representing either the central phosphorylation site domain or the upstream, NH2-terminal domain: it tethers actin to the plasma membrane and/or to secretory granules, and/or it sequesters PIP2. Using MARCKS-null mice, we probed for a loss of function secretory phenotype in mast cells harvested from embryonic livers and maturated in vivo [embryonic hepatic-derived mast cells (eHMCs)]. Both wild-type (WT) and MARCKS-null eHMCs exhibited full exocytic responses upon FcϵRI receptor activation with DNP-BSA (2,4-dinitrophenyl-BSA), whether they were in suspension or adherent. The secretory responses of MARCKS-null eHMCs were consistently higher than those of WT cells, but the differences had sporadic statistical significance. The MARCKS-null cells exhibited faster secretory kinetics, however, achieving the plateau phase of the response with a t½ ∼2.5-fold faster. Hence, MARCKS appears to be a nonessential regulatory protein in mast cell exocytosis but exerts a negative modulation. Surprisingly, the MARCKS NH2-terminal peptide, MANS, which has been reported to inhibit mucin secretion from airway goblet cells (Li Y, Martin LD, Spizz G, Adler KB. J Biol Chem 276: 40982-40990, 2001), inhibited hexosaminidase secretion from WT and MARCKS-null eHMCs, leading us to reexamine its effects on mucin secretion. Results from studies using peptide inhibitors with human bronchial epithelial cells and with binding assays using purified mucins suggested that MANS inhibited the mucin binding assay, rather than the secretory response.

Thiocyanate and nitrite analysis using miniaturised isotachophoresis on a planar polymer chip.

A new method has been developed to improve the determination of thiocyanate using isotachophoresis. This method uses complexation with copper(II) as a mechanism for improving the separation of thiocyanate from chlorate and perchlorate. By using a pH of 3.25 the method can also be used to analyse nitrite. Separations were carried out using a miniaturised poly(methyl methacrylate) (PMMA) separation device. Linearity was observed from 1.25 to 75 mg dm(-3) with a correlation coefficient of 0.998 for both thiocyanate and nitrite. Limits of detection for these two species were calculated to be 0.8 mg dm(-3) and 0.9 mg dm(-3) respectively. The method was successfully applied to the analysis of these anions in a range of samples including explosive residues.

Secondary Interpretations of Diagnostic Imaging Examinations: Patient Liabilities and Out-of-Pocket Costs.

BACKGROUND: Secondary interpretations of diagnostic imaging examinations are increasingly performed to improve care for complex patients. We sought to determine associated patient-billed liabilities and out-of-pocket payments and to identify patient and imaging study characteristics that correlate with higher patient bills and out-of-pocket payments. METHODS: Data extracted for 7,740 secondary imaging interpretations performed across our large metropolitan health system over 25 months included total professional charges, insurance payments, patient-billed liabilities, and patient out-of-pocket payments. Multivariable linear regression analyses were performed to identify patient and imaging factors associated with higher patient bills and out-of-pocket payments. RESULTS: Mean secondary interpretation professional charges, insurance payments, patient-billed liabilities, and patient out-of-pocket payments were $306.50, $108.02, $27.80, and $14.55, respectively. Patients received bills for 47.5% of services and made out-of-pocket payments for 17.1%. Patient-billed liabilities and out-of-pocket payments were higher for patients who were younger and uninsured and for secondary interpretations requested for patients seen in outpatient (versus inpatient) settings. Patient-billed liabilities and out-of-pocket payments were lower for patients who were Black (versus White) and had government-sponsored (versus commercial) insurance and for secondary interpretations performed during the second, third, or fourth (versus first) quarter of each calendar year. CONCLUSION: Observed differences between patient-billed liabilities and out-of-pocket payments suggest that secondary interpretations of diagnostic imaging examinations can result in small but real patient financial burdens. Improved price transparency and enhanced patient communication about the value of secondary interpretations could reduce potential surprises when patients receive these bills.

Determination of the potassium content of explosive residues using miniaturised isotachophoresis.

A new method has been developed to allow the determination of potassium in post-explosion residues to be made using miniaturised isotachophoresis. The method is based on the use of a caesium leading ion with 4.5 mM 18-crown-6 ether added to retard the potassium to allow reliable determinations to be made. With the conditions selected no interference was noted from other small inorganic cations, such as ammonium, barium, calcium, magnesium, sodium or strontium. The method was successfully applied to the analysis of seven samples containing explosive residues obtained from the unconfined burning of several flash powders. The procedure was found to offer good linearity for potassium determinations over the concentration range of 1.25-150 µg/mL with a coefficient of determination of 0.999 achieved.

Determination of chlorine containing species in explosive residues using chip-based isotachophoresis.

A new method has been developed to allow the determination of the chlorate, chloride and perchlorate anions in inorganic explosive residues to be made using isotachophoresis (ITP). To enable a good separation of these species to be achieved the method involves the use of two complexing agents. Indium(III) is used to allow the determination of chloride whilst using nitrate as the leading ion and alpha-cyclodextrin is used to allow the separation of chlorate and perchlorate. Separations were carried out using a miniaturised poly(methyl methacrylate) (PMMA) separation device. The method was applied to analysing both model samples and actual inorganic explosive containing residue samples. Successful determinations of these samples were achieved with no interference from other anions typically found in inorganic explosive residues. Limits of detection (LOD) for the species of interest were calculated to be 0.80 mg l(-1) for chloride, 1.75 mg l(-1) for chlorate and 1.40 mg l(-1) for perchlorate.

The role of erlotinib and the Optune device in a patient with an epidermal growth factor receptor viii amplified glioblastoma.

The standard treatment for patients diagnosed with glioblastoma is surgical resection of tumor followed by high dose radiation and chemotherapy with temozolomide. For patients who experience allergic reactions to temozolomide despite desensitization protocols, alternative therapies must be considered. In this report, we present such a patient who then received treatment with an epidermal growth factor receptor inhibitor, erlotinib, concurrent with a tumor-treating field device, Optune. Through this combination of a targeted molecular therapy and the Optune device, the patient has been able to achieve stable disease 9 months after completing radiation.

Quality assurance testing of an explosives trace analysis laboratory--further improvements.

The Forensic Explosives Laboratory (FEL) operates within the Defence Science and Technology Laboratory (DSTL) which is part of the UK Government Ministry of Defence (MOD). The FEL provides support and advice to the Home Office and UK police forces on matters relating to the criminal misuse of explosives. During 1989 the FEL established a weekly quality assurance testing regime in its explosives trace analysis laboratory. The purpose of the regime is to prevent the accumulation of explosives traces within the laboratory at levels that could, if other precautions failed, result in the contamination of samples and controls. Designated areas within the laboratory are swabbed using cotton wool swabs moistened with ethanol water mixture, in equal amounts. The swabs are then extracted, cleaned up and analyzed using Gas Chromatographs with Thermal Energy Analyzer detectors. This paper follows on from a previous published paper describing the regime and summarizing subsequent results from approximately 6 years of tests. Lessons learned and improvements made over the period are also discussed. Monitoring samples taken from surfaces within the trace laboratories and trace vehicle examination bay have, with few exceptions, revealed only low levels of contamination, predominantly of RDX. Analysis of the control swabs, processed alongside the monitoring swabs, has demonstrated that in this environment the risk of forensic sample contamination, assuming all the relevant anti-contamination procedures have been followed, is so small that it is considered to be negligible. The monitoring regime has also been valuable in assessing the process of continuous improvement, allowing sources of contamination transfer into the trace areas to be identified and eliminated.

Effectiveness of contamination prevention procedures in a trace explosives laboratory.

The effectiveness of a number of the explosives contamination prevention controls that are adopted within the Forensic Explosives Laboratory (FEL) principal trace laboratory has been scrutinised. Within the trace laboratory, rigorous procedures for processing forensic swab samples for traces of organic explosives are routinely adopted by forensic scientists. In order to demonstrate the effectiveness of these procedures, and the principle of separating the sample from the laboratory and the forensic scientist, explosives-free swab samples and appropriate controls have been processed, in accordance with trace laboratory procedures, in several explosives contaminated environments. In all cases, no explosives were detected in the post-processing samples, demonstrating that the contamination prevention procedures are effective, robust and fit-for-purpose.

Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores.

Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5-2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.