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Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, and PSMD12) as supporters of enzalutamide resistance in vitro. Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro.

Background/Aims Fibroblasts isolated by traditional explant culture from skin biopsies in systemic sclerosis (SSc) have a profibrotic and “migratory” phenotype. Single cell RNAseq (scRNAseq) studies suggest other fibroblast subpopulations may be important in SSc pathogenesis. We have isolated a second fibroblast population termed “resident” from whole skin in SSc. We used bulk and scRNAseq to further characterise these populations. Methods Forearm skin from dcSSc (n = 3) and HC (n = 3) was biopsied. “Migratory” fibroblasts were isolated by standard explant culture. The remaining biopsy fragments underwent collagenase digestion to yield a second “resident” fibroblast population. Functional characterization included 3-D collagen gel contraction, migratory scratch-wound assays and western blot comparing expression of pro-collagen I, CTGF and αSMA. Significant difference in gene expression was defined by fold change≥1.5, and adjusted p-value Results Both SSc fibroblast populations showed a fibrotic phenotype with overexpression of COL1, CTGF and αSMA compared to HC, but with reduced expression αSMA in resident compared to migratory SSc fibroblasts (p Conclusion We have isolated for the first time two distinct profibrotic fibroblast populations from SSc skin with altered expression of key genes associated with SSc pathogenesis. Future work will define their role in SSc pathogenesis, potentially facilitating a more targeted treatment approach. Disclosure K.E. Clark: None. S. Xu: None. V.H. Ong: None. C.D. Buckley: None. C. Denton: None.

Background/Aims Preclinical studies suggest that the S100A4, a Damage Associated Molecular Pattern (DAMP) protein upregulated upon stress or injury, may be a target for antifibrotic therapy. We have studied expression and function of S100A4 in systemic sclerosis (SSc) to further explore its relevance as a driver of fibroblast activation. Methods S100A4 protein concentration was measured by ELISA (Cusabio, USA) in serum of SSc (n = 94) and healthy controls (n = 15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n = 6) and healthy controls (NF, n = 6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate activation of profibrotic molecular and functional phenotype in SSc and control fibroblasts. Results Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p = 0.027). There was significant association with SSc-ILD (p = 0.025, n = 55), scleroderma renal crisis (p = 0.026, n = 4) and trend of increase in pulmonary hypertension (p = 0.075, n = 7). There was a trend for S100A4 to fall in longer disease duration (r=-0.18, p = 0.09) and a weak (r = 0.28, p = 0.07) correlation with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF controls (0.28 [0.02-3.29]; p 1.5) induced in NF by S100A4 were also constitutively overexpressed in SScF, and significantly downregulated by AX-202. Pathway mapping of these S100A4 dependent transcripts in SSc showed the most significant enriched Kegg® pathways (FDR Conclusion Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. Studies to elucidate the therapeutic potential of targeting S100A4 in SSc are warranted. Disclosure C.P. Denton: None. S. Xu: None. R.H. Maclean: None. K.E. Clark: None. S. Borchert: Corporate appointments; Employee of ARXX Therapeutics. R. Hussain: Corporate appointments; Employee of ARXX Therapeutics. J. Klingelhöfer: Corporate appointments; Employee of ARXX Therapeutics. J. Hallén: Corporate appointments; Employee of ARXX Therapeutics. V.H. Ong: None.

Working memory (WM) is the ability to maintain and manipulate internal representations. WM recruits varying brain regions based on task demands. Although the hippocampus has historically been associated with long-term memory (LTM), several studies provide evidence for its involvement during WM tasks. Slotnick (this issue) posits that this involvement is due to LTM processes. This argument rests on the assumption that processes are not shared among WM and LTM, and that WM processes are necessarily sustained. We argue that there are processes utilized by both WM and LTM, and that such processes need not be sustained to support WM.

Background Polysubstance use is a common, problematic behavior that increases risk of harm to self and others. Research suggests that rates may vary based on gender, sex and sexuality. Understanding the current state of this literature may inform prevention and treatment of polysubstance use, leading to reduced public health burden. Objectives This review aimed to synthesize research on gender, sex and sexuality differences in polysubstance use in adults and adolescents. Methods A scoping review was conducted using all EBSCO databases, PubMed and Google Scholar to identify articles examining the effects of gender, sex and sexuality on polysubstance use. Polysubstance use was defined broadly as the use of any combination of substances over any time period and included licit (alcohol, tobacco) and illicit substances, concurrent and simultaneous use, from lifetime to daily use and use at any frequency. Studies were considered if they were published in peer-reviewed journals between January 1990 and October 2020 and were written in English. Publicly available data sources were also utilized to fully capture prevalence data that has not been published elsewhere. Results Findings were mostly inconsistent and often conflicting. Only two findings were generally consistent: adult men were overall more likely to report polysubstance use than adult women, and sexual and gender minorities report more frequent polysubstance use than non-minorities. Conclusions Research has been unable to clearly elucidate differences in polysubstance use prevalence and patterns according to gender, sex and sexuality. Several recommendations are offered to advance future research and address limitations of current research.

To study the effect of surgeon-administered Transversus Abdominis Plane block (sTAP) on opioid usage and length of stay (LOS).Starting in April 2018, two surgeons at our institution gradually introduced sTAP for radical cystectomy (RC) patients. We performed a retrospective observational cohort analysis of RC patients catalogued in a prospectively maintained database using the Enhanced Recovery After Surgery Interactive Auditing System. Two surgeons adopted the sTAP block technique in April 2018. We included patients undergoing RC for bladder malignancy under Enhanced Recovery After Surgery protocol between January 2017 and August 2020. Primary outcomes included LOS, and postoperative day (POD) 0-3 total opioids consumption measured by morphine milligram equivalents (MME). Multivariable linear or logistic models evaluated the association of TAP with outcomes while controlling for potential confounders.Among 178 patients included in analysis, 84 patients underwent sTAP block and 94 did not. Multivariable analysis demonstrated significantly lower POD 0-3 total opioid usage (106.4 vs 192.2 MME, P = .004), and mean LOS (5.6 vs 7.7 days, P.001) among the sTAP group.sTAP appears to be an effective adjunct to RC care associated with improved LOS, and POD 0-3 opioid consumption. Further studies are needed to optimize TAP block technique and anesthetic composition.

Identifying collective variables (CVs) for chemical reactions is essential to reduce the 3

As sluggish cognitive tempo (SCT) shows similar inattention and neuropsychological dysfunction as youth with spina bifida (SB), it is important to examine whether neuropsychological functioning may affect the development of SCT in this population. Participants were 140 youth with SB and their parents who participated in five waves of a longitudinal study across eight years (ages 8-15 years at Time 1). At Time 1, teacher-, mother-, and father-report of SCT showed 9%, 8.3%, and 5.3% impairment in SCT respectively compared to other youth in the sample. Growth curves were used to examine changes over time in mother-, father-, and teacher-reported SCT. Four neurocognitive variables were included (attention, working memory, cognitive flexibility, plan/organize) as predictor variables of SCT growth and intercepts. Mother, father, and teacher-report of SCT were included in separate models. Age and shunt status were included as covariates in the growth models. Inattentive symptoms were also included as a covariate in working memory, shift, and plan/organize models. Worse working memory at Time 1 predicted an increase in mother-reported SCT symptoms over eight years (β = -.28) and poorer cognitive shifting skills predicted higher rates of teacher-reported SCT over time (β = -.61). All other neurocognitive variables predicted the intercept, but not the slope of SCT symptoms. Results indicate that worse neurocognitive functioning is associated with higher levels of SCT symptoms and that worse working memory and cognitive flexibility at baseline predicted an increase in SCT symptoms as youth with SB age. It may be important for clinicians to monitor and assess levels of SCT in youth with SB.

Background and Aims Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpp) for neutralization assays. Methods We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpp. Neutralization sensitivity of these HCVpp varied widely. HCVpp clustered into 15 distinct groups based on patterns of relative sensitivity to seven broadly neutralizing monoclonal antibodies (bNAbs). We used these data to select a final panel of 15 antigenically representative HCVpp. Results Both the 65 and 15 HCVpp panels span four tiers of neutralization sensitivity, and neutralizing breadth measurements for seven bNAbs were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpp were independent of genetic distances between E1E2 clones. Conclusions Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity, rather than panels selected solely for genetic diversity. We propose that this multi-tier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Background: \ud Outcomes for patients with blast-phase chronic myeloid leukaemia are poor. Long-term survival depends on reaching a second chronic phase, followed by allogeneic haematopoietic stem-cell transplantation (HSCT). We investigated whether the novel combination of the tyrosine-kinase inhibitor ponatinib with fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin (FLAG-IDA) could improve response and optimise allogeneic HSCT outcomes in patients with blast-phase chronic myeloid leukaemia. The aim was to identify a dose of ponatinib, which combined with FLAG-IDA, showed clinically meaningful activity and tolerability.\ud \ud Methods: \ud MATCHPOINT was a seamless, phase 1/2, multicentre trial done in eight UK Trials Acceleration Programme-funded centres. Eligible participants were adults (aged ≥16 years) with Philadelphia chromosome-positive or BCR-ABL1-positive blast-phase chronic myeloid leukaemia, suitable for intensive chemotherapy. Participants received up to two cycles of ponatinib with FLAG-IDA. Experimental doses of oral ponatinib (given from day 1 to day 28 of FLAG-IDA) were between 15 mg alternate days and 45 mg once daily and the starting dose was 30 mg once daily. Intravenous fludarabine (30 mg/m2 for 5 days), cytarabine (2 g/m2 for 5 days), and idarubicin (8 mg/m2 for 3 days), and subcutaneous granulocyte colony-stimulating factor (if used), were delivered according to local protocols. We used an innovative EffTox design to investigate the activity and tolerability of ponatinib–FLAG-IDA; the primary endpoints were the optimal ponatinib dose meeting prespecified thresholds of activity (inducement of second chronic phase defined as either haematological or minor cytogenetic response) and tolerability (dose-limiting toxicties). Analyses were planned on an intention-to-treat basis. MATCHPOINT was registered as an International Standard Randomised Controlled Trial, ISRCTN98986889, and has completed recruitment; the final results are presented.\ud \ud Findings: \ud Between March 19, 2015, and April 26, 2018, 17 patients (12 men, five women) were recruited, 16 of whom were evaluable for the coprimary outcomes. Median follow-up was 41 months (IQR 36–48). The EffTox model simultaneously considered clinical responses and dose-limiting toxicities, and determined the optimal ponatinib dose as 30 mg daily, combined with FLAG-IDA. 11 (69%) of 16 patients were in the second chronic phase after one cycle of treatment. Four (25%) patients had a dose-limiting toxicity (comprising cardiomyopathy and grade 4 increased alanine aminotransferase, cerebral venous sinus thrombosis, grade 3 increased amylase, and grade 4 increased alanine aminotransferase), fulfilling the criteria for clinically relevant activity and toxicity. 12 (71%) of 17 patients proceeded to allogeneic HSCT. The most common grade 3–4 non-haematological adverse events were lung infection (n=4 [24%]), fever (n=3 [18%]), and hypocalcaemia (n=3 [18%]). There were 12 serious adverse events in 11 (65%) patients. Three (18%) patients died due to treatment-related events (due to cardiomyopathy, pulmonary haemorrhage, and bone marrow aplasia).\ud \ud Interpretation: \ud Ponatinib–FLAG-IDA can induce second chronic phase in patients with blast-phase chronic myeloid leukaemia, representing an active salvage therapy to bridge to allogeneic HSCT. The number of treatment-related deaths is not in excess of what would be expected in this very high-risk group of patients receiving intensive chemotherapy. The efficient EffTox method is a model for investigating novel therapies in ultra-orphan cancers.\ud \ud Funding: \ud Blood Cancer UK and Incyte.

Theories of memory consolidation suggest the role of brain regions and connectivity between brain regions change as memories age. Human lesion studies indicate memories become hippocampus-independent over years, whereas animal studies suggest this process occurs across relatively short intervals, days to weeks. Human neuroimaging studies suggest that changes in hippocampal and cortical activity and connectivity can be detected over these short intervals, but many of these studies examined only two time periods. We examined memory and FMRI activity for photos of indoor and outdoor scenes across four time periods to examine these neural changes more carefully. Participants (N=21) studied scenes 1 hour, 1 day, 1 week, or 1 month before scanning. During scanning, participants viewed scenes, made old/new recognition memory judgments, and gave confidence ratings. Memory accuracy, confidence ratings, and response times changed with memory age. Brain activity in a widespread cortical network either increased or decreased with memory age, whereas hippocampal activity was not related to memory age. These findings were almost identical when effects of behavioral changes across time periods were minimized. Functional connectivity of the ventromedial prefrontal cortex with the posterior parietal cortex increased with memory age. By contrast, functional connectivity of the hippocampal with the parahippocampal cortex and fusiform gyrus decreased with memory age. In sum, we detected changes in cortical activity and changes in hippocampal and cortical connectivity with memory age across short intervals. These findings provide support for the predictions of systems consolidation and suggest that these changes begin soon after memories are formed.

The impact of homework completion on outcome from cognitive behavioral therapies (CBTs) for eating disorders (EDs) is unknown. We examined homework completion during two CBTs for bulimia-spectrum EDs and tested the associations among homework and treatment outcomes. After each session, therapists rated the quantity of self-monitoring completed (e.g. tracking food intake and ED symptoms), and degree of completion of the previous week's written (e.g. completing a worksheet) and behavioral (e.g. completing an at-home food exposure, regularly eating) homework on a Likert scale. On average, patients (

Caustic conditions are often employed for dissolution of a wide variety of minerals, where ion sorption, surface diffusion, and interfacial organization impact surface reactivity.

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. To investigate recurrently dysregulated genes in glioblastoma, we interrogated allele-specific expression (ASE), the difference in expression between two alleles of a gene, in glioblastoma stem cells (GSC) derived from 43 patients. A total of 118 genes were found with recurrent ASE preferentially in GSCs compared with normal tissues. These genes were enriched for apoptotic regulators, including schlafen family member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and PARP inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika. This discovery effort based upon ASE revealed novel points of vulnerability in GSCs, suggesting a potential alternative treatment strategy for chemotherapy-resistant glioblastoma.Significance:Assessing allele-specific expression reveals genes with recurrent cis-regulatory changes that are enriched in glioblastoma stem cells, including SLFN11, which modulates chemotherapy resistance and susceptibility to the oncolytic Zika virus.

PDF file - 16K, SLC1A5 protein is overexpressed in NSCLC tissues

Purpose: BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1–independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML.Experimental Design: We used primary cells from patients with CML and TKI-sensitive and -resistant BCR-ABL1+ CML cell lines and a novel ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small-molecule Axl inhibitor BGB324 in vitro and in vivo. In BCR-ABL1–unmutated cells, we also investigated BGB324 in combination with imatinib.Results: We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of patients with CML compared with healthy individuals and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34+ cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status.Conclusions: Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as -resistant CML and support the need for clinical trials. Clin Cancer Res; 23(9); 2289–300. ©2016 AACR.

Supplementary Data from Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma

PDF file - 28K, SLC1A5 mediates response to Gln-dependent growth

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Supplementary tables S1-S5 and supplementary figures S1-S5

Figure S1. Inhibition of Axl by shRNA. Figure S2. Assessment of half maximal inhibitory concentration (IC50) for BGB324 in human CD34+ cells from healthy donors. Figure S3. Treatment with BGB324 increased apoptosis and decreased proliferation of CML cell lines. Figure S4. Reduction of BCR-ABL and Crkl phosphorylation by imatinib. Figure S5. Treatment with imatinib and BGB324 does not induce an additive inhibitory effect on phosphorylation of Erk and Akt. Figure S6. BCR-ABL and Crkl phosphorylation are not affected by imatinib or BGB324 in KCL-22 T315I mutated cells. Figure S7. Colony forming assay using primary CD34+ cells isolated from healthy donors treated for 72 h with 1, 3 or 5 μM of BGB324 (n=2). Figure S8. BGB324 increases apoptosis of primary CML cells.

PDF file - 20K, Inhibition of SLC1A5 activity reduces Gln uptake and cell growth of NSCLC cells

PDF file - 15K, Silencing SLC1A5 in SCC cells by siRNA attenuates cell growth and reduces glutamine uptake