Antibody response in snakes with boid inclusion body disease.

Boid Inclusion Body Disease (BIBD) is a potentially fatal disease reported in captive boid snakes worldwide that is caused by reptarenavirus infection. Although the detection of intracytoplasmic inclusion bodies (IB) in blood cells serves as the gold standard for the ante mortem diagnosis of BIBD, the mechanisms underlying IB formation and the pathogenesis of BIBD are unknown. Knowledge on the reptile immune system is sparse compared to the mammalian counterpart, and in particular the response towards reptarenavirus infection is practically unknown. Herein, we investigated a breeding collection of 70 Boa constrictor snakes for BIBD, reptarenavirus viraemia, anti-reptarenavirus IgM and IgY antibodies, and population parameters. Using NGS and RT-PCR on pooled blood samples of snakes with and without BIBD, we could identify three different reptarenavirus S segments in the collection. The examination of individual samples by RT-PCR indicated that the presence of University of Giessen virus (UGV)-like S segment strongly correlates with IB formation. We could also demonstrate a negative correlation between BIBD and the presence of anti-UGV NP IgY antibodies. Further evidence of an association between antibody response and BIBD is the finding that the level of anti-reptarenavirus antibodies measured by ELISA was lower in snakes with BIBD. Furthermore, female snakes had a significantly lower body weight when they had BIBD. Taken together our findings suggest that the detection of the UGV-/S6-like S segment and the presence of anti-reptarenavirus IgY antibodies might serve as a prognostic tool for predicting the development of BIBD.

Randomized Controlled Trial of Physical Exercise in Diabetic Veterans With Length-Dependent Distal Symmetric Polyneuropathy.

Rationale: Physical exercise is an essential adjunct to the management of patients with type 2 diabetes mellitus. Therapeutic interventions that improve blood flow to peripheral nerves, such as exercise, may slow the progression of neuropathy in the diabetic patient. Aims: This randomized clinical trial was conducted to determine whether a structured program of aerobic, isokinetic strength, or the combination of aerobic-isokinetic strength exercise intervention alters peripheral nerve function in glycemic-controlled diabetic patients with advanced length-dependent distal symmetric polyneuropathy. Methods: Forty-five patients with type 2 diabetes mellitus exhibiting tight glycemic control (HbA1c intergroup range 7.2-8.0%) were randomized by block design across four experimental groups: sedentary controls (n = 12), aerobic exercise (n = 11), isokinetic strength (n = 11), or the combination of aerobic-isokinetic strength training (n = 11). Patients randomized to training groups exercised 3× per week for 12 weeks, whereas patients randomized to the sedentary control group received standard of care. To minimize attention and educational bias, all patients attended a 12-session health promotion educational series. At baseline, immediately following intervention, and again at 12-week post-intervention, detailed nerve conduction studies were conducted as a primary outcome measure. At these same intervals, all patients completed as secondary measures quantitative sensory testing, symptom-limited treadmill stress tests, and a Short-Form 36-Veterans Questionnaire (SF-36V). Results: Of the 45 patients randomized into this study, 37 (82%) had absent sural nerve responses, 19 (42%) had absent median sensory nerve responses, and 17 (38%) had absent ulnar sensory nerve responses. By comparison, responses from tibial nerves were absent in only three (7%) subjects while responses from peroneal nerves were absent in five (11%) subjects. Eleven (92%) of 12 patients that had volunteered to be biopsied exhibited abnormal levels of epidermal nerve fiber densities. Exercise, regardless of type, did not alter sensory or motor nerve electrodiagnostic findings among those patients exhibiting measurable responses (ANOVA). There was, however, a modest (p = 0.01) beneficial effect of exercise on sensory nerve function (Fisher's Exact Test). Importantly, the beneficial effect of exercise on sensory nerve function was enhanced (p = 0.03) during the post-intervention interval. In addition, three of six patients that had undergone exercise intervention exhibited a marked 1.9 ± 0.3-fold improvement in epidermal nerve fiber density. By comparison, none of three sedentary patients whom agreed to be biopsied a second time showed improvement in epidermal nerve fiber density. Compared to baseline values within groups, and compared with sedentary values across groups, neither aerobic, isokinetic strength, or the combination of aerobic-isokinetic strength exercise intervention altered peak oxygen uptake. Patients that underwent aerobic or the combined aerobic-isokinetic strength exercise intervention, however, demonstrated an increase in treadmill test duration that was sustained over the 12-week post-intervention period. Conclusion: A 12-week course of physical exercise, regardless of type, does not alter sensory or motor nerve electrodiagnostic findings. In a subset of patients, a short-term structured program of aerobic exercise may selectively improve sensory nerve fiber function. Large-scale exercise lifestyle intervention trials are warranted to further evaluate the impact of aerobic exercise on sensory nerve fiber function in diabetic neuropathic patients. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT00955201.

The Effect of Breathing Retraining Using Metronome-Based Acoustic Feedback on Exercise Endurance in COPD: A Randomized Trial.

BACKGROUND: During exercise-training patients with chronic obstructive pulmonary disease (COPD) can entrain their breathing pattern to visual-feedback cues as to achieve a slower respiratory rate and prolong exhalation. The result is an improvement in exercise tolerance and a reduction in dynamic hyperinflation. Acoustic stimuli, including metronome-generated acoustic stimuli, can entrain human movements. Accordingly, we hypothesized that exercise duration and dynamic hyperinflation would be less after exercise-training plus breathing-retraining using a metronome-based acoustic-feedback system than after exercise-training alone. METHODS: Of 205 patients with COPD [FEV1 = 44 ± 16% predicted (± SD)] recruited, 119 were randomly assigned to exercise-training plus breathing-retraining using acoustic feedback (n = 58) or exercise-training alone (n = 61). Patients exercised on a treadmill thrice-weekly for 12 weeks. Before and at completion of training, patients underwent constant-load treadmill testing with inspiratory capacity measures every 2 min. RESULTS: At completion of training, improvements in exercise duration in the breathing-retraining plus exercise-training and exercise-training alone groups were similar (p = 0.35). At isotime, inspiratory capacity increased (less exercise-induced dynamic hyperinflation) by 3% (p = 0.001) in the breathing-retraining plus exercise-training group and remained unchanged in the exercise-alone group. The between-group change in inspiratory capacity, however, was not significant (p = 0.08). CONCLUSIONS: In patients with COPD, breathing-retraining using a metronome-based acoustic feedback did not result in improved exercise endurance or decreased dynamic hyperinflation when compared to exercise-training alone. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT NCT01009099; URL: http://www.clinicaltrials.gov.

Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis).

In 2014 we observed a noticeable increase in the number of sudden deaths among green tree pythons (Morelia viridis). Pathological examination revealed the accumulation of mucoid material within the airways and lungs in association with enlargement of the entire lung. We performed a full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterize the pathogenesis of this mucinous pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung tissue samples from a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome whose sequence was <85% identical to that of nidoviruses previously identified in ball pythons and Indian pythons. We then employed reverse transcription-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cultures of Morelia viridis liver and brain cells, which we inoculated with homogenates of lung tissue from infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridization to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium, which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.IMPORTANCE Novel nidoviruses associated with severe respiratory disease were fairly recently identified in ball pythons and Indian pythons. Herein we report on the isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterized the pathological changes in the infected individuals and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death by inhibiting normal gas exchange in the lungs. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high rates of mortality in breeding collections, and most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

Structured Exercise in Obese Diabetic Patients with Chronic Kidney Disease: A Randomized Controlled Trial.

BACKGROUND: Patients with type 2 diabetes mellitus (DM), obesity, and chronic kidney disease (CKD) are generally physically inactive and may benefit from exercise. Our objective was to determine the effects of structured exercise on physical fitness, kidney function, endothelial function, inflammation, and body composition in such patients. METHODS: In this randomized, controlled trial, 36 male patients (age 49-81) were randomly assigned to exercise + diet management (n = 18) or diet alone (n = 18). Participants were eligible if they had type 2 DM, body mass index >30 kg/m2, CKD stages 2-4, and persistent proteinuria (>200 mg/g creatinine for >3 months). The exercise intervention was a 12-week (3 days per week) program of aerobic and resistance training followed by 40 weeks of home exercise. The primary outcome measure was change from baseline in urine protein to creatinine ratio (UPCR) at 12 and 52 weeks. RESULTS: Thirty-two participants completed the study (14 exercise + diet, 18 diet-alone group). The change from baseline in UPCR was slightly greater in the diet-alone group at 12 weeks but not at 52 weeks. Changes in both symptom-limited and constant-workrate treadmill times were significantly higher in the exercise + diet group at 12 weeks but not at 52 weeks. There were no significant differences in urine albumin to creatinine ratio, estimated glomerular filtration rate, endothelial function, inflammation, or body composition between the groups. CONCLUSIONS: In obese diabetic subjects with CKD, structured exercise improved exercise capacity but not body composition or renal function. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel.

An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine.

BACKGROUND: Many avenues have been proposed for a seamless transition between biomarker discovery data and selected reaction monitoring (SRM) assays for biomarker validation. Unfortunately, studies with the abundant urinary protein uromodulin have shown that these methods do not converge on a consistent set of surrogate peptides for targeted mass spectrometry. As an alternative, we present an empirical peptide selection work flow for robust protein quantification. METHODS: We compared the relative SRM signal intensity of 12 uromodulin-derived peptides between tryptic digests of 9 urine samples. Pairwise CVs between the 12 peptides were 0.19-0.99. We used a correlation matrix to identify peptides that reproducibly tracked the amount of uromodulin protein and selected 4 peptides with robust and highly correlated SRM signals. Absolute quantification was performed with stable isotope-labeled versions of these peptides as internal standards and a standard curve prepared from a tryptic digest of purified uromodulin. RESULTS: Absolute quantification of uromodulin in 40 clinical urine samples yielded interpeptide correlations of ≥0.984 and correlations of ≥0.912 with ELISA data. The SRM assays were linear over >3 orders of magnitude and had typical interdigest CVs of <10%, interinjection CVs of <7%, and intertransition CVs of <7%. CONCLUSIONS: Comparing the apparent abundance of a plurality of peptides derived from the same target protein makes it possible to select signature peptides that are unaffected by the unpredictable confounding factors inevitably present in biological samples.

Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.

Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC.

Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome.

The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis.

Contrasting breathing retraining and helium-oxygen during pulmonary rehabilitation in COPD: a randomized clinical trial.

BACKGROUND: Breathing-retraining and helium-oxygen (heliox) have been used to improve exercise tolerance in COPD. We hypothesized that, in patients with COPD, exercise duration after exercise-training plus breathing-retraining and oxygen would be longer than after exercise-training plus heliox or after exercise-training plus oxygen alone. We also explored the short-term maintenance of gains in exercise duration after using each technique. METHODS: Of 192 COPD patients recruited, 103 were randomly assigned to exercise-training plus heliox (n = 33), exercise-training plus breathing-retraining and oxygen (n = 35) and exercise-training and oxygen (n = 35). FiO2 was 0.30 during testing and training in all groups. Patients exercised on a treadmill thrice-weekly for eight weeks. Before, at completion of training, and six-weeks later, patients underwent constant-load treadmill testing. RESULTS: At completion of training, improvements in exercise duration in the heliox and breathing-retraining groups were not significantly different. Compared to the exercise-training plus oxygen group, exercise duration improved more in the breathing-retraining group (P = 0.008) but not in the heliox group (P = 0.142). Hyperinflation was reduced with breathing-retraining plus oxygen compared to the other two groups. Six-weeks later, improvements in exercise duration were still greater with breathing-retraining than with exercise-training (P = 0.015). In contrast, improvements in exercise duration with heliox did not differ from those in the other two groups. CONCLUSIONS: In moderate-to-severe COPD, exercise-training combined with either heliox or with breathing-retraining yielded not significantly different improvements in exercise duration - with only the latter being superior to exercise-training. Six-weeks after training, these improvements were still greater after exercise-training plus breathing-retraining than after exercise-training. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00123422.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

The Effects of Walking or Walking-with-Poles Training on Tissue Oxygenation in Patients with Peripheral Arterial Disease.

This randomized trial proposed to determine if there were differences in calf muscle StO(2) parameters in patients before and after 12 weeks of a traditional walking or walking-with-poles exercise program. Data were collected on 85 patients who were randomized to a traditional walking program (n = 40) or walking-with-poles program (n = 45) of exercise training. Patients walked for 3 times weekly for 12 weeks. Seventy-one patients completed both the baseline and the 12-week follow-up progressive treadmill tests (n = 36 traditional walking and n = 35 walking-with-poles). Using the near-infrared spectroscopy measures, StO(2) was measured prior to, during, and after exercise. At baseline, calf muscle oxygenation decreased from 56 ± 17% prior to the treadmill test to 16 ± 18% at peak exercise. The time elapsed prior to reaching nadir StO(2) values increased more in the traditional walking group when compared to the walking-with-poles group. Likewise, absolute walking time increased more in the traditional walking group than in the walking-with-poles group. Tissue oxygenation decline during treadmill testing was less for patients assigned to a 12-week traditional walking program when compared to those assigned to a 12-week walking-with-poles program. In conclusion, the 12-week traditional walking program was superior to walking-with-poles in improving tissue deoxygenation in patients with PAD.

Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS).

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. RESULTS: Using high performance liquid chromatography-coupled mass spectrometry (HPLC); we have established a highly specific and sensitive selected reaction monitoring (SRM) assay. Our multiplexed SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 26 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF were generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomol. We processed and analysed CSF samples from a total of 22 patients with SPMS, 7 patients with SPMS treated with lamotrigine, 12 patients with non-inflammatory neurological disorders (NIND) and 10 healthy controls (HC) for the levels of these 26 selected potential protein biomarkers. Our SRM data found one protein showing significant difference between SPMS and HC, three proteins differing between SPMS and NIND, two proteins between NIND and HC, and 11 protein biomarkers showing significant difference between a lamotrigine-treated and untreated SPMS group. Principal component analysis (PCA) revealed that these 26 proteins were correlated, and could be represented by four principal components. Overall, we established an efficient platform to develop and verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases. CONCLUSIONS: A highly specific and sensitive multiplex SRM-MS assay was established for development and verification of CSF protein biomarkers in SPMS. Five proteins were found to be expressed significantly differently between the three cohorts, SPMS, NIND and HC and 11 proteins associated with lamotrigine treatment, which we expect will further our current understanding of SPMS disease pathology and/or therapeutic intervention.

Comparison of walking with poles and traditional walking for peripheral arterial disease rehabilitation.

PURPOSE: The purpose of this study was to compare the effects of a 24-week walking with poles rehabilitation program with a traditional 24-week walking program on physical function in patients with peripheral arterial disease (PAD). METHODS: Patients with PAD (n = 103, age = 69.7 ± 8.9 years, ankle-brachial index < 0.90 or evidence of calcified vessels) were randomized into a rehabilitation program of traditional walking (n = 52) or walking with poles (n = 51). Patients exercised 3 times per week for 24 weeks. Exercise endurance was measured by time walked on a constant work rate treadmill test at 6, 12, and 24 weeks. Perceived physical function was measured by the Medical Outcomes Study Short Form-36 and Walking Impairment Questionnaire. Tissue oxygenation was measured using near-infrared spectroscopy. RESULTS: Patients assigned to the traditional walking group walked longer at 24 weeks than those assigned to the pole walking group (21.10 ± 17.07 minutes and 15.02 ± 12.32 minutes, respectively, P = .037). There were no differences between the groups in tissue oxygenation. However, there was a significant lengthening of time for which it took to reach minimum tissue oxygenation values (P < .001) within the groups on the constant work rate test. There were no differences between the groups in perceived physical function as measured by the Physical Function subscale on the Medical Outcomes Study Short Form-36 or perceived walking distance as measured by the Walking Distance subscale on the Walking Impairment Scale. CONCLUSIONS: Traditional walking was superior to walking with poles in increasing walking endurance on a constant work rate treadmill test for patients with PAD.

Analysis of Histone Modifications from Tryptic Peptides of Deuteroacetylated Isoforms.

The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.

Azalysine analogues as probes for protein lysine deacetylation and demethylation.

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.

Enzymatically active prostate-specific antigen promotes growth of human prostate cancers.

BACKGROUND: Prostate specific antigen (PSA) is the best-known member of the kallikrein-related peptidase family, with an established role as a prostatic disease biomarker. Although it is produced at high levels by all stages of prostate cancer, it is uncertain if PSA plays a role in prostate cancer initiation and progression. We decided to investigate the impact of PSA and its enzymatic activity on tumor cell growth rates. METHODS: A gene-specific shRNA lentiviral construct reduced endogenous PSA expression in the LNCaP human prostate cancer cell line. Resulting changes in growth rates in vitro and in vivo were determined. Using a mass spectroscopy-based approach, alterations to the LNCaP proteome due to reduced PSA were measured. Finally, to evaluate the importance of PSA's proteolytic activity, the PSA-null Du145 human prostate cancer cell line was engineered to express either enzymatically inactive pro-PSA (WT) or a furin-activated variant (FR) with high enzymatic activity. The resulting clones were evaluated for PSA-induced changes in growth rates in vivo and in vitro. RESULTS: Lowered PSA levels dramatically reduced LNCaP growth rates. Expressing active PSA (FR), but not the inactive WT variant, conferred a growth advantage on Du145 cells. Proteomics analysis revealed global changes to the LNCaP proteome as a result of reduced PSA expression. CONCLUSIONS: These studies demonstrate the importance of PSA to prostate cancer cell growth. We also show that the enzymatic activity of PSA confers an enhanced growth rate to human prostate cancer cells, suggesting a causal role in prostate cancer progression.

14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis.

BACKGROUND: During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. RESULTS: To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. CONCLUSIONS: 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics.

Predictors of weight loss in overweight veterans with knee osteoarthritis who participated in a clinical trial.

Individuals with osteoarthritis (OA) of the knee have a high prevalence of obesity. The objective of this study was to determine predictors of weight loss among participants with knee OA in a clinical trial. A secondary analysis of data from a randomized clinical trial was used. Multiple regression analysis was used to predict weight loss after identification of predictor variables from the data. Hypothesized predictors of weight loss included demographic, clinical, and behavioral characteristics. The only baseline variable that was significantly related to weight loss was the Center for Epidemiologic Studies Depression Scale (CES-D) score. The CES-D score was an independent predictor of weight loss at 16 (p < 0.01) and 32 weeks (p < 0.05). Receiving nutrition counseling was also predictive of weight loss at 16 weeks (p < 0.03). These two variables explained 24% and 22% of the variance in weight loss for weeks 16 and 32, respectively. Being less depressed and receiving nutritional counseling appeared predictive of weight loss in this group of veterans.

Aerobic exercise in obese diabetic patients with chronic kidney disease: a randomized and controlled pilot study.

BACKGROUND: Patients with obesity, diabetes, and chronic kidney disease (CKD) are generally physically inactive, have a high mortality rate, and may benefit from an exercise program. METHODS: We performed a 24-week randomized controlled feasibility study comparing aerobic exercise plus optimal medical management to medical management alone in patients with type 2 diabetes, obesity (body mass index [BMI] > 30 kg/m2), and stage 2-4 CKD (estimated glomerular filtration rate [eGFR] 15-90 mL/min/1.73 m2 with persistent proteinuria). Subjects randomized to exercise underwent thrice weekly aerobic training for 6 followed by 18 weeks of supervised home exercise. The primary outcome variable was change in proteinuria. RESULTS: Seven subjects randomized to exercise and 4 control subjects completed the study. Exercise training resulted in an increase in exercise duration during treadmill testing, which was accompanied by slight but insignificant decreases in resting systolic blood pressure and 24-hour proteinuria. Exercise did not alter GFR, hemoglobin, glycated hemoglobin, serum lipids, or C-reactive protein (CRP). Caloric intake and body weight and composition also did not change with exercise training. CONCLUSION: Exercise training in obese diabetic patients with CKD is feasible and may have clinical benefits. A large-scale randomized controlled trial to determine the effects of exercise on renal functions, cardiovascular fitness, inflammation, and oxidative stress in diabetic patients with CKD is planned.

Relationship between depression and functional measures in overweight and obese persons with osteoarthritis of the knee.

Depression is common in overweight and obese individuals with chronic illness. The purpose of this study was to determine the relationship between depression and functional status. Baseline data were used from 105 overweight/obese participants who enrolled in a clinical trial for overweight and obese adults with osteoarthritis of the knee. Forty-two percent of the sample was classified as depressed according to the Center for Epidemiologic Studies Depression Scale. A moderate relationship was seen between perceived physical function and physical performance in patients who were not depressed that did not exist in patients reporting depressive symptoms. In a stepwise regression analysis, poorer function (as measured by the Western Ontario and MacMaster Universities (Osteoarthritis Index) function subscale) and younger age accounted for 29 percent of the variance in depressive symptoms.