XRK3F2 inhibits p62 signaling and augments myeloma killing by proteasome inhibitors

Background: Despite advancements in therapy, multiple myeloma (MM) remains an incurable blood cancer. Our mission is to maximize the efficacy of a primary treatment for myeloma, proteasome inhibitors (PIs) which cause intracellular waste buildup, leading to ER stress and cell death. p62(sequestosome-1) provides an alternate pathway when the proteasome is inhibited, by breaking down cytotoxic material via autophagy. Upregulation of p62 is associated with PI resistance. We identified a small molecule, XRK3F2, that binds to the ZZ domain of p62 and inhibits its autophagic function. We hypothesized that XRK3F2 would improve MM killing when combined with PIs. Methods Used: We tested XRK3F2 and PI combinations in vitro, in ex vivo co-cultures, and in a human MM xenograft model. We tested XRK3F2 plus bortezomib in vitro and in ex vivo myeloma: bone cocultures and analyzed effects on tumor burden in a prior mouse xenograft experiment. Results: XRK3F2 induced cell death in various human MM cell lines, with a IC50s of 3-6 M. When combined with carfilzomib, the most potent approved PI, at physiologically relevant doses, there was strong synergy (Combinatorial index of 0.4 to 0.6, by Chou-Talalay analysis). The combination of the two agents significantly increased tumor killing in a tumor: bone co-culture model, where the microenvironment of the tumor provides MM survival signals and potential drug resistance. Enhanced tumor killing was further confirmed in a plasmacytoma model of the human MM cell line RPMI-8226 in NSG mice. We also identified soluble BCMA (B-cell maturation antigen, sBCMA) as a sensitive biomarker for tumor burden, which allowed for serial tumor measurements in all tested models. Conclusion and Potential Impact: Combining the p62-ZZ domain inhibitor XRK3F2 with PIs shows great promise in improving the killing of MM. Work is ongoing to validate the combination in xenograft models, where tumor cells colonize bones, and in immunocompetent models. Further mechanistic studies using primary MM cells from patients are also ongoing. sBCMA is a cheap, specific, and sensitive tool for serial tumor measurement and should be further validated for preclinical and clinical usage

Diagnostic Biomarkers for Alzheimer's Disease Using Non-Invasive Specimens

Studies in the field of Alzheimer's disease (AD) have shown the emergence of biomarkers in biologic fluids that hold great promise for the diagnosis of the disease. A diagnosis of AD at a presymptomatic or early stage may be the key for a successful treatment, with clinical trials currently investigating this. It is anticipated that preventative and therapeutic strategies may be stage-dependent, which means that they have a better chance of success at a very early stage-before critical neurons are lost. Several studies have been investigating the use of cerebrospinal fluid (CSF) and blood as clinical samples for the detection of AD with a number of established core markers, such as amyloid beta (Aβ), total tau (T-tau) and phosphorylated tau ( tau), being at the center of clinical research interest. The use of oral samples-including saliva and buccal mucosal cells-falls under one of the least-investigated areas in AD diagnosis. Such samples have great potential to provide a completely non-invasive alternative to current CSF and blood sampling procedures. The present work is a thorough review of the results and analytical approaches, including proteomics, metabolomics, spectroscopy and microbiome analyses that have been used for the study and detection of AD using salivary samples and buccal cells. With a few exceptions, most of the studies utilizing oral samples were performed in small cohorts, which in combination with the existence of contradictory results render it difficult to come to a definitive conclusion on the value of oral markers. Proteins such as Aβ, T-tau and tau, as well as small metabolites, were detected in saliva and have shown some potential as future AD diagnostics. Future large-cohort studies and standardization of sample preparation and (pre-)analytical factors are necessary to determine the use of these non-invasive samples as a diagnostic tool for AD

A dose-ranging, placebo-controlled, double-blind trial of nisoldipine in effort angina: Duration and extent of antianginal effects

Maximal treadmill exercise testing at 1, 3 and 8 hours was used to assess the onset, duration and antianginal efficacy of the d1hydropyridine slow channel calciumblocking agent, nisoldipine, in an oral dose range of 5, 10 and 20 mg. A double-blind, randomized, placebo-controlled design was used involving 12 patients with stable effort angina. Exercise tolerance was significantly increased 3 hours after each dose, when the maximal beneficial effect occurred. The improvement was observed as early as 1 hour after the 10 and 20 mg dose, and persisted for 8 hours after the 20 mg dose. At 3 hours, the onset of an exercise-induced ST segment depression of 0.1 mV or greater was increased by 62 (p < 0.05), 75 (p < 0.01) and 117 seconds (p < 0.01) with the 5,10 and 20 mg dose of nisoldipine, respectively, compared with placebo. Similarly, time to onset of angina was significantly increased. The sum of exerciseinduced ST segment depression at peak exercise was significantly decreased (p < 0.05) from 8.7 ± 2.3 to 6.7 ± 1.8 and 6.4 ± 2.0 mm, respectively, after the 10 and 20 mg dose of nisoldipine. The rate-pressure product was significantly greater with nisoldipine than with placebo at the onset of ischemia and at peak exercise (22.8 ± 1.1 versus 20 ± 1.4 × 103 U for the 20 mg dose; p ± 0.01).Thus, nisoldipine is an effective antianginal agent with a rapid onset of action that improves exercise tolerance, increases angina threshold and persists for at least 8 hours after oral dosing

Reliable in silico ranking of engineered therapeutic TCR binding affinities with MMPB/GBSA

Accurate and efficient in silico ranking of proteinprotein binding affinities is useful for protein design with applications in biological therapeutics. One popular approach to rank binding affinities is to apply the molecular mechanics Poisson-Boltzmann/generalized Born surface area (MMPB/ GBSA) method to molecular dynamics (MD) trajectories. Here, we identify protocols that enable the reliable evaluation of T-cell receptor (TCR) variants binding to their target, peptide-human leukocyte antigens (pHLAs). We suggest different protocols for variant sets with a few (&lt;= 4) or many mutations, with entropy corrections important for the latter. We demonstrate how potential outliers could be identified in advance and that just 5-10 replicas of short (4 ns) MD simulations may be sufficient for the reproducible and accurate ranking of TCR variants. The protocols developed here can be applied toward in silico screening during the optimization of therapeutic TCRs, potentially reducing both the cost and time taken for biologic development

Restenosis and its determinants in first and repeat coronary angioplasty

Restenosis is the main problem limiting long-term success of percutaneous transluminal coronary angioplasty (PTCA) and is most accurately evaluated by follow-up angiography. We compared the primary and long-term results of angioplasty in 268 consecutive patients (293 segments) with first PTCA (PTCA 1, angiographic follow-up 98%) and in 66 patients (76 segments) with repeat PTCA after restenosis (PTCA 2, angiographic follow-up 92%). Forty clinical, angiographic and procedural factors were assessed in relation to outcome. Primary success rate was higher in PTCA 2 (91% vs 67.5%) and major complications were fewer (4.5% vs 16%).Higher inflation pressure (7.9 ± 2.3 vs 6.8 ± 1.8 atm, P70%) after PTCA 1 and after PTC A 2 (27% vs 36%, P = NS) and the mean time to recurrence (4.7 vs 5.3 months, P = NS) were similar. Procedural factors were the main determinants of long-term success in primary PTCA. The restenosis risk was independently related to residual stenosis >45% (P<0.001), variant angina (P<0.05) and multivessel disease (P<0.05) after PTCA 1 and to male sex (P<0.001) and higher inflation pressure (P<0.05) after PTCA 2. Mild to moderate intimal tearing was associated with less restenosis after PTC A 1, but not after PTCA 2. Including 9 patients (10 segments) with a third PTCA, 70% of the 66 patients with repeat PTCA had a successful long-term outcome. Repeat angioplasty should therefore be considered as an integral part of PTCA therapy. Restenosis however remains a major concern. An optimal primary result with a minimal residual stenosis is decisive for first PTCA, whereas avoidance of a dissection by using lower inflation pressure on a restenosis might improve the long-term outcome of repeat PTC

TGFβ and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation

BACKGROUND: CCN2/CTGF is an established effector of TGFβ driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFβ leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function. RESULTS: From a panel of genes regulated by TGFβ and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFβ and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy. CONCLUSIONS: TGFβ and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response

Bridging the Gap Between Science, Economics and Policy to Develop and Implement a Pilot Market Based Instrument for Soil Carbon

Increasing soil organic carbon (SOC) has potential to offset greenhouse gas emissions, but the scope for on-farm carbon sequestration is poorly understood. A pilot scheme was developed in Central West NSW, Australia to trial the use of a market-based instrument to encourage farmers to increase soil organic carbon levels. The pilot considered the relationship between land use, management practices and soil carbon levels; offered alternative contract designs to attract landholders; and developed monitoring and reporting protocols. The pilot was rolled-out in 2011 and 2012 and had 11 successful tenders with an average price of $A37 per t CO2-e. The results of this conservation tender will assist the design of future programs aimed at encouraging mitigation effort from the agricultural sector

Peptide cargo tunes a network of correlated motions in human leukocyte antigens

Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug‐target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αβ T‐cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T‐cell compartment. We have applied high‐pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide–HLA complexes. We find that different peptide sequences affect peptide–HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide‐binding interface, in a manner that could influence T‐cell antigen discrimination