XBP1 variant 1 promotes mitosis of cancer cells involving upregulation of the polyglutamylase TTLL6.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients' survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1ß. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

Critical role of water conditions in the responses of autumn phenology of marsh wetlands to climate change on the Tibetan Plateau.

The Tibetan Plateau, housing 20% of China's wetlands, plays a vital role in the regional carbon cycle. Examining the phenological dynamics of wetland vegetation in response to climate change is crucial for understanding its impact on the ecosystem. Despite this importance, the specific effects of climate change on wetland vegetation phenology in this region remain uncertain. In this study, we investigated the influence of climate change on the end of the growing season (EOS) of marsh wetland vegetation across the Tibetan Plateau, utilizing satellite-derived Normalized Difference Vegetation Index (NDVI) data and observational climate data. We observed that the regionally averaged EOS of marsh vegetation across the Tibetan Plateau was significantly (p < .05) delayed by 4.10 days/decade from 2001 to 2020. Warming preseason temperatures were found to be the primary driver behind the delay in the EOS of marsh vegetation, whereas preseason cumulative precipitation showed no significant impact. Interestingly, the responses of EOS to climate change varied spatially across the plateau, indicating a regulatory role for hydrological conditions in marsh phenology. In the humid and cold central regions, preseason daytime warming significantly delayed the EOS. However, areas with lower soil moisture exhibited a weaker or reversed delay effect, suggesting complex interplays between temperature, soil moisture, and EOS. Notably, in the arid southwestern regions of the plateau, increased preseason rainfall directly delayed the EOS, while higher daytime temperatures advanced it. Our results emphasize the critical role of hydrological conditions, specifically soil moisture, in shaping marsh EOS responses in different regions. Our findings underscore the need to incorporate hydrological factors into terrestrial ecosystem models, particularly in cold and dry regions, for accurate predictions of marsh vegetation phenological responses to climate change. This understanding is vital for informed conservation and management strategies in the face of current and future climate challenges.

A quantitative high-throughput screen identifies compounds that lower expression of the SCA2-and ALS-associated gene ATXN2.

CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS.

Novel inflammatory mediator profile observed during pediatric heart surgery with cardiopulmonary bypass and continuous ultrafiltration.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with systemic inflammation, featuring increased levels of circulating pro-inflammatory cytokines. Intra-operative ultrafiltration extracts fluid and inflammatory factors potentially dampening inflammation-related organ dysfunction and enhancing post-operative recovery. This study aimed to define the impact of continuous subzero-balance ultrafiltration (SBUF) on circulating levels of major inflammatory mediators. METHODS: Twenty pediatric patients undergoing cardiac surgery, CPB and SBUF were prospectively enrolled. Blood samples were collected prior to CPB initiation (Pre-CPB Plasma) and immediately before weaning off CPB (End-CPB Plasma). Ultrafiltrate effluent samples were also collected at the End-CPB time-point (End-CPB Effluent). The concentrations of thirty-nine inflammatory factors were assessed and sieving coefficients were calculated. RESULTS: A profound increase in inflammatory cytokines and activated complement products were noted in plasma following CBP. Twenty-two inflammatory mediators were detected in the ultrafiltrate effluent. Novel mediators removed by ultrafiltration included cytokines IL1-Ra, IL-2, IL-12, IL-17A, IL-33, TRAIL, GM-CSF, ET-1, and the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCL10. Mediator extraction by SBUF was significantly associated with molecular mass < 66 kDa (Chi2 statistic = 18.8, Chi2 with Yates' correction = 16.0, p < 0.0001). There was a moderate negative linear correlation between molecular mass and sieving coefficient (Spearman R = - 0.45 and p = 0.02). Notably, the anti-inflammatory cytokine IL-10 was not efficiently extracted by SBUF. CONCLUSIONS: CPB is associated with a burden of circulating inflammatory mediators, and SBUF selectively extracts twenty of these pro-inflammatory factors while preserving the key anti-inflammatory regulator IL-10. Ultrafiltration could potentially function as an immunomodulatory therapy during pediatric cardiac surgery. Trial registration ClinicalTrials.gov, NCT05154864. Registered retrospectively on December 13, 2021. https://clinicaltrials.gov/ct2/show/record/NCT05154864 .

A transgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis.

Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)-based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.

Revolutionizing Health Professions Admissions to Achieve an Inclusive Workforce.

This article describes the "The Admissions Revolution: Bold Strategies for Diversifying the Healthcare Workforce" conference, which preceded the 2022 Beyond Flexner Alliance Conference and called for health professions institutions to boldly reimagine the admission process to diversify the health care workforce. Proposed strategies encompassed 4 key themes: admission metrics, aligning admission practices with institutional mission, community partnerships to fulfill social mission, and student support and retention. Transformation of the health professions admission process requires broad institutional and individual effort. Careful consideration and implementation of these practices will help institutions achieve greater workforce diversity and catalyze progress toward health equity.

Frontiers in the Application of RF Vacuum Electronics.

The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy.

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson's disease pathology.

SAXS study of the formation and structure of polynuclear thorium(IV) colloids and thorium dioxide nanoparticles.

Stable actinide colloids and nanoparticles are of interest because of their potential to affect the transportation of radionuclides in the near-field of a nuclear waste repository. At high concentrations, thorium(IV) can precipitate to form intrinsic colloids. In the present study, polynuclear thorium colloids and thorium dioxide crystallites, formed by the condensation of hydrolyzed Th4+ solutions (3 mM; initial pH 5.5) aged for up to 18 months, were studied using small-angle X-ray scattering. Scattering profiles were fitted using a unified Guinier/power-law model (Beaucage model) to extract the radii of gyration and Porod exponents. Analysis of the scattering profiles from a dispersion aged for 5 months indicated that both polymer coils and more compacted structures (radius of gyration Rg ≃ 10 nm) were present, which translated in the Kratky plots as a plateau and a peak maximum, respectively. After 18 months, the SAXS data were consistent with the presence of agglomerates of ThO2 particles suspended in aqueous solution (pH 3.2; [Th] = 1.45 mM). The measured radius of gyration (Rg) of the agglomerates was 5.8 nm, whereas the radius of the ThO2 particles was 2.5 nm.

Discovery of Small-Molecule VapC1 Nuclease Inhibitors by Virtual Screening and Scaffold Hopping from an Atomic Structure Revealing Protein-Protein Interactions with a Native VapB1 Inhibitor.

Nontypeable Haemophilus influenzae (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics. The vapBC-1 locus of NTHi encodes a type II TA system, comprising the ribonuclease toxin VapC1 and its cognate antitoxin VapB1. The activity of VapC1 has been linked to the survival of NTHi during antibiotic treatment both in vivo and ex vivo. Therefore, inhibitors of VapC1 might serve as adjuvants to antibiotics, preventing NTHi from entering growth arrest and surviving; however, none have been reported to date. A truncated VapB1 peptide from a crystal structure of the VapBC-1 complex was used to generate pharmacophore queries to facilitate a scaffold hopping approach for the identification of small-molecule VapC1 inhibitors. The National Center for Advancing Translational Sciences small-molecule library was virtually screened using the shape-based method rapid overlay of chemical structures (ROCS), and the top-ranking hits were docked into the VapB1 binding pocket of VapC1. Two hundred virtual screening hits with the best docking scores were selected and tested in a biochemical VapC1 activity assay, which confirmed eight compounds as VapC1 inhibitors. An additional 60 compounds were selected with structural similarities to the confirmed VapC1 inhibitors, of which 20 inhibited VapC1 activity. Intracellular target engagement of five inhibitors was indicated by the destabilization of VapC1 within bacterial cells from a cellular thermal shift assay; however, no impact on bacterial growth was observed. Thus, this virtual screening and scaffold hopping approach enabled the discovery of VapC1 ribonuclease inhibitors that might serve as starting points for preclinical development.

Use and outcomes of dual antiplatelet therapy for acute coronary syndrome in patients with chronic kidney disease: insights from the Canadian Observational Antiplatelet Study (COAPT).

Chronic kidney disease (CKD) increases the risk of adverse outcomes in acute coronary syndrome (ACS). The optimal regimen of dual antiplatelet therapy (DAPT) post-percutaneous coronary intervention (PCI) in CKD poses a challenge due to the increased bleeding and clotting tendencies, particularly since patients with CKD were underrepresented in randomized controlled trials. We examined the practice patterns of DAPT prescription stratified by the presence of CKD. The multicentre prospective Canadian Observational Antiplatelet Study (COAPT) enrolled patients with ACS between December 2011 and May 2013. The present study is a subgroup analysis comparing type and duration of DAPT and associated outcomes among patients with and without CKD (eGFR < 60 ml/min/1.73 m2, calculated by CKD-EPI). Patients with CKD (275/1921, 14.3%) were prescribed prasugrel/ticagrelor less (18.5% vs 25.8%, p = 0.01) and had a shorter duration of DAPT therapy versus patients without CKD (median 382 vs 402 days, p = 0.003). CKD was associated with major adverse cardiovascular events (MACE) at 12 months (p < 0.001) but not bleeding when compared to patients without CKD. CKD was associated with MACE in both patients on prasugrel/ticagrelor (p = 0.017) and those on clopidogrel (p < 0.001) (p for heterogeneity = 0.70). CKD was associated with increased bleeding only among patients receiving prasugrel/ticagrelor (p = 0.007), but not among those receiving clopidogrel (p = 0.64) (p for heterogeneity = 0.036). Patients with CKD had a shorter DAPT duration and were less frequently prescribed potent P2Y12 inhibitors than patients without CKD. Overall, compared with patients without CKD, patients with CKD had higher rates of MACE and similar bleeding rates. However, among those prescribed more potent P2Y12 inhibitors, CKD was associated with more bleeding than those without CKD. Further studies are needed to better define the benefit/risk evaluation, and establish a more tailored and evidence-based DAPT regimen for this high-risk patient group.

Discovery of endoplasmic reticulum calcium stabilizers to rescue ER-stressed podocytes in nephrotic syndrome.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Subzero balance - simple modified ultrafiltration (SBUF-SMUF) technique for pediatric cardiopulmonary bypass.

The use of cardiopulmonary bypass (CPB) can be associated with significant hemodilution, coagulopathy and a systemic inflammatory response for infants and children undergoing cardiac surgery. Intra-operative ultrafiltration has been used for decades to ameliorate these harmful effects. The novel combination of a continuous and non-continuous form of ultrafiltration, Subzero Balance Simple Modified Ultrafiltration (SBUF-SMUF) here described, seeks to enhance recovery from pediatric cardiac surgery and CPB.

Compound screening in cell-based models of tau inclusion formation: Comparison of primary neuron and HEK293 cell assays.

The hallmark pathological features of Alzheimer's disease (AD) brains are senile plaques, comprising ß-amyloid (Aß) peptides, and neuronal inclusions formed from tau protein. These plaques form 10-20 years before AD symptom onset, whereas robust tau pathology is more closely associated with symptoms and correlates with cognitive status. This temporal sequence of AD pathology development, coupled with repeated clinical failures of Aß-directed drugs, suggests that molecules that reduce tau inclusions have therapeutic potential. Few tau-directed drugs are presently in clinical testing, in part because of the difficulty in identifying molecules that reduce tau inclusions. We describe here two cell-based assays of tau inclusion formation that we employed to screen for compounds that inhibit tau pathology: a HEK293 cell-based tau overexpression assay, and a primary rat cortical neuron assay with physiological tau expression. Screening a collection of ∼3500 pharmaceutical compounds with the HEK293 cell tau aggregation assay, we obtained only a low number of hit compounds. Moreover, these compounds generally failed to inhibit tau inclusion formation in the cortical neuron assay. We then screened the Prestwick library of mostly approved drugs in the cortical neuron assay, leading to the identification of a greater number of tau inclusion inhibitors. These included four dopamine D2 receptor antagonists, with D2 receptors having previously been suggested to regulate tau inclusions in a Caenorhabditis elegans model. These results suggest that neurons, the cells most affected by tau pathology in AD, are very suitable for screening for tau inclusion inhibitors.

KDM5 histone demethylases repress immune response via suppression of STING.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy.

The Gap in Prescribing Stroke Prevention Therapies in Postoperative Atrial Fibrillation After Isolated Coronary Artery Bypass Grafting Surgery.

BACKGROUND: Postoperative atrial fibrillation (POAF) commonly occurs after coronary artery bypass graft (CABG) surgery. Patients with POAF are at higher risk of stroke and mortality. The use of oral anticoagulation (OAC) in POAF remains inconsistent, and the care gap is unknown. OBJECTIVE: We sought to evaluate rate of adherence and factors associated with OAC use in POAF patients after isolated CABG. METHODS: In this study, all patients who had an isolated CABG between April 1, 2019 and March 30, 2020, at Health Sciences North, Sudbury retrospectively were evaluated. We identified all patients who developed POAF and captured the use of OAC. Multivariable logistic regression models were used to identify predictors of OAC prescription. RESULTS: In total, 339 CABG patients between April 1, 2019 and March 30, 2020, were identified; 86 patients developed POAF. No patients had major perioperative bleeding. All POAF patients had an indication for OAC therapies based on CHADS 65 score. However, only 17 (19.8%) patients with POAF had OAC prescription at the time of hospital discharge.  Baseline characteristics were similar between those with or without OAC. In multivariable analysis, the use of dual antiplatelet therapy was associated with a decreased use of OAC therapy in POAF (OR 0.037 (95% CI 0.005, 0.29), P = .002). Moreover, in comparison to surgeon A, surgeon B was associated with a decreased use of OAC therapy, whereas surgeon C was associated with an increased use of OAC therapy in POAF (OR 0.15 (95% CI 0.03, 0.83), P = .03 and OR 6.49 (95% CI 1.21, 34.82), P = .03, respectively). None of the CHADS 65 elements, including age, hypertension, stroke, diabetes mellitus or congestive heart failure, correlated with the use of OAC. KEY FINDINGS: Eighty percent of patients who developed POAF after isolated CABG failed to receive stroke prevention therapies. The use of dual antiplatelet therapy and individual surgeons' preference were associated with the use of OAC therapy among POAF. CONCLUSIONS: This data set suggests that approximately only 1 in 5 patients with POAF after isolated CABG got prescribed OAC.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Structural Study of Polystyrene-b-poly(acrylic acid) Micelles Complexed with Uranyl: A SAXS Core-Shell Model Analysis.

The interactions between natural colloidal organic matter and actinides in solutions are complex and not fully understood. In this work, a crew-cut polystyrene-b-poly(acry1ic acid) (PS-b-PAA) micelle is proposed as a model particle for humic acid (HA) colloid with the aim to better understand the sequestration, aggregation, and mobility of HA colloids in the presence of uranyl ions. The effects of uranyl ions on the structure of PS29k-b-PAA5k micelles in aqueous solution were mainly investigated by synchrotron small-angle X-ray scattering. A core-shell model, accounting for the thickness and contrast changes of the PAA corona induced by the adsorption of uranyl, was employed to analyze the scattering data. A combination of transmission electron microscopy, dynamic light scattering, and zetametry showed a strong affinity of uranyl ions to PAA segments in water at pH 4-5 that resulted in the shrinkage and improved contrast of the PAA corona, as well as colloidal destabilization at a high uranyl concentration.

Bioethics: cancelling patient operations.

PURPOSE OF REVIEW: This review aims to surmise a bioethical approach to the phenomenon of cancelling patient operations. There is increasing public and political interest in the matter with a rise in the frequency of cancellations. Cancellations are emotional for patients and are difficult clinical decisions. RECENT FINDINGS: Reasons for cancellation involve patient factors and resource allocation applying to elective and emergency surgery. The four pillars of bioethics are easily applied, (autonomy, beneficence, nonmaleficence and justice), although their failings are becoming more prominent with the rise of more encompassing virtue ethics. These include dignity, solidarity, phronesis and trust. Importantly patient dignity should be preserved, this complimenting solidarity and trust in specialist knowledge more than autonomy does. Beauchamp and Childress have provided a descriptive framework describing futility, which may aid communication and mental clarity when deliberating if it is the right choice to cancel. With regards to resource factors, ideally managerial staff should be involved in these decisions leaving the physician to be the patient's clinical advocate. SUMMARY: Although cancellations are undesirable, they are inevitable and form part of the duties of a doctor. When they do occur, care must remain patient-centred, asking how we can improve this situation.

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2.

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.