Neuropilin-2 Mediated β-Catenin Signaling and Survival in Human Gastro-Intestinal Cancer Cell Lines

NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI) cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p<0.01). Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of β-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p<0.05). Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01). Collectively, our results demonstrate that tumor cell–derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Low lung function and incident lung cancer in the United States: data From the First National Health and Nutrition Examination Survey follow-up

Background Obstructive lung disease and lung cancer are tobacco-related diseases that can remain clinically silent until late in the disease process. We sought to define the risk for incident lung cancer among a national cohort of US adults with and without obstructive lung disease. Methods We studied participants in the First National Health and Nutrition Examination Survey, who had up to 22 years of follow-up. We classified subjects as having moderate or severe obstructive lung disease at baseline if the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) was less than 70% and the FEV1 was less than 80% of the predicted value. We also determined incident cases of lung cancer during the follow-up period. Results A total of 113 lung cancers occurred in the 5402 adults in the cohort. In the proportional hazards model adjusted for covariates of age, sex, race, education, smoking status, and duration and intensity of smoking, the presence of moderate or severe obstructive lung disease was associated with a higher risk for incident lung cancer (hazard ratio, 2.8; 95% confidence interval, 1.8-4.4). Conclusions The presence of moderate or severe obstructive lung disease is a significant predictor of incident lung cancer in long-term follow-up. This finding may be useful clinically and in studies evaluating the utility of new tools for the early detection of lung cancer

Algorithmic extraction of smartphone accelerometer-derived mechano-biological descriptors of resistance exercise is robust to changes in intensity and velocity

Background It was shown that single repetition, contraction-phase specific and total time-under-tension (TUT) can be extracted reliably and validly from smartphone accelerometer-derived data of resistance exercise machines using user-determined resistance exercise velocities at 60% one repetition maximum (1-RM). However, it remained unclear how robust the extraction of these mechano-biological descriptors is over a wide range of movement velocities (slowversus fast-movement velocity) and intensities (30% 1-RM versus 80% 1-RM) that reflect the interindividual variability during resistance exercise. Objective In this work, we examined whether the manipulation of velocity or intensity would disrupt an algorithmic extraction of single repetitions, contraction-phase specific and total TUT. Methods Twenty-seven participants performed four sets of three repetitions of their 30% and 80% 1- RM with velocities of 1 s, 2 s, 6 s and 8 s per repetition, respectively. An algorithm extracted the number of repetitions, single repetition, contraction-phase specific and total TUT. All exercises were video-recorded. The video recordings served as the gold standard to which algorithmically-derived TUT was compared. The agreement between the methods was examined using Limits of Agreement (LoA). The Pearson correlation coefficients were used to calculate the association, and the intraclass correlation coefficient (ICC 2.1) examined the interrater reliability. Results The calculated error rate for the algorithmic detection of the number of single repetitions derived from two smartphones accelerometers was 1.9%. The comparison between algorithmically- derived, contraction-phase specific TUT against video, revealed a high degree of correlation (r > 0.94) for both exercise machines. The agreement between the two methods was high on both exercise machines, intensities and velocities and was as follows: LoA ranged from -0.21 to 0.22 seconds for single repetition TUT (2.57% of mean TUT), from -0.24 to 0.22 seconds for concentric contraction TUT (6.25% of mean TUT), from -0.22 to 0.24 seconds for eccentric contraction TUT (5.52% of mean TUT) and from -1.97 to 1.00 seconds for total TUT (5.13% of mean TUT). Interrater reliability for single repetition, contraction- phase specific TUT was high (ICC > 0.99). Conclusion Neither intensity nor velocity disrupts the proposed algorithmic data extraction approach. Therefore, smartphone accelerometers can be used to extract scientific mechano-biological descriptors of dynamic resistance exercise with intensities ranging from 30% to 80% of the 1-RM with velocities ranging from 1 s to 8 s per repetition, respectively, thus making this simple method a reliable tool for resistance exercise mechano-biological descriptors extraction.ISSN:1932-620

Assessment of NRP-2 expression in human gastric cancer tissues and cell lines.

<p>(A) Immunohistochemical staining for NRP-2 expression in representative tissue sections (20X) of normal human gastric mucosa and gastric cancer specimens (B) Immunoblot analysis of NRP-2 expression in six human GI cancer cell lines. Vinculin served as an internal loading control. (C) Generation of stable CNDT 2.5 cell lines with NRP-2 knockdown. Immunoblot analysis of NRP-1 and -2 expression in CNDT 2.5 cells transfected with shcntr or shNRP-2 plasmids (shNRP-2 clones; C6 and C10). Vinculin served as a loading control. (D) MTT assay results. Growth rates were no different between the control cells and NRP-2 knockdown clones. Bars indicate SEM. (E) Top: Mean number of cells that migrated in a Boyden chamber assay. Bottom: Representative images (10X) of migration assays. (F) Top: Mean number of cells that invaded in BioCoat Matrigel invasion chamber assay. Bottom: Representative images (10X) of invasion assays.</p

Effect of NRP-2 knockdown on expression of known metastatic genes.

<p>(<i>A</i>) Autoradiographic image of the array membrane showing differential expression of metastatic genes in shcntr (<i>left</i>) and shNRP-2 (<i>right</i>) cells. Circled spots indicate the position of the S100A4 gene. (B) Validation of S100A4 protein expression level in cells by immunoblot analysis. Actin served as a loading control. (C) Reduction of the steady-state level of β-catenin by NRP-2 knockdown. β-catenin expression in shcntr and shNRP-2 cells was determined by immunoblotting. Vinculin served as a loading control. (D) Verification of reduced β-catenin expression in shNRP-2 cells by immunofluorescence staining. shcntr and shNRP-2 CNDT 2.5 cells growing in chamber slides were fixed and immunostained with anti-β-catenin antibody (red). Nuclei were counterstained with DAPI (blue). (E) Validation of β-catenin reduction in a second NRP-2 knockdown gastric cancer cell line. NCI-N87 gastric cancer cells were infected with lentivirus containing shcntr or shNRP-2 constructs. Immunoblot analysis showed reduction in β-catenin expression in NCI-N87 NRP-2 knockdown cells. Actin served as a loading control. (F) β-catenin level in the cytoplasmic (Cyto) and nuclear (Nuc) fractions of shcntr and shNRP-2 cells. HSP90 (cytoplasmic) and LaminB (nuclear) served as fractionation controls.</p

Hypothetical schema for NRP-2-mediated activation of β-catenin signaling in gastrointestinal cancer cells.

<p>Left: In a GI cancer cell, increased inactive GSK3β inhibits destruction complex function, leading to stabilization of β-catenin that is free to translocate to the nucleus and activate transcription of target genes. Right: In a GI cancer cell with NRP-2 knockdown, the active destruction complex phosphorylates β-catenin and targets it for proteasome-mediated degradation, resulting in decreased activation of downstream target genes.</p