Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

Are mice good models for human neuromuscular disease? Comparing muscle excursions in walking between mice and humans

The mouse is one of the most widely used animal models to study neuromuscular diseases and test new therapeutic strategies. However, findings from successful pre-clinical studies using mouse models frequently fail to translate to humans due to various factors. Differences in muscle function between the two species could be crucial but often have been overlooked. The purpose of this study was to evaluate and compare muscle excursions in walking between mice and humans

The study protocol of a cluster-randomised controlled trial of family-mediated personalised activities for nursing home residents with dementia

<p>Abstract</p> <p>Background</p> <p>Following admission to a nursing home, the feelings of depression and burden that family carers may experience do not necessarily diminish. Additionally, they may experience feelings of guilt and grief for the loss of a previously close relationship. At the same time, individuals with dementia may develop symptoms of depression and agitation (BPSD) that may be related to changes in family relationships, social interaction and stimulation. Until now, interventions to alleviate carer stress and BPSD have treated carers and relatives separately rather than focusing on maintaining or enhancing their relationships. One-to-one structured activities have been shown to reduce BPSD and also improve the caring experience, but barriers such as a lack of resources impede the implementation of activities in aged care facilities. The current study will investigate the effect of individualised activities based on the Montessori methodology administered by family carers in residential care.</p> <p>Methods/Design</p> <p>We will conduct a cluster-randomised trial to train family carers in conducting personalised one-to-one activities based on the Montessori methodology with their relatives. Montessori activities derive from the principles espoused by Maria Montessori and subsequent educational theorists to promote engagement in learning, namely task breakdown, guided repetition, progression in difficulty from simple to complex, and the careful matching of demands to levels of competence. Persons with dementia living in aged care facilities and frequently visiting family carers will be included in the study. Consented, willing participants will be randomly assigned by facility to a treatment condition using the Montessori approach or a control waiting list condition. We hypothesise that family carers conducting Montessori-based activities will experience improvements in quality of visits and overall relationship with the resident as well as higher self-rated mastery, fewer depressive symptoms, and a better quality of life than carers in the waiting list condition.</p> <p>Discussion</p> <p>We hypothesise that training family carers to deliver personalised activities to their relatives in a residential setting will make visits more satisfying and may consequently improve the quality of life for carers and their relatives. These beneficial effects might also reduce nursing staff burden and thus impact positively on residential facilities.</p> <p>Trial Registration</p> <p>Australian New Zealand Clinical Trials Registry - <a href="http://www.anzctr.org.au/ACTRN12611000998943.aspx">ACTRN12611000998943</a></p

Association of Blood Lead (Pb) and Plasma Homocysteine: A Cross Sectional Survey in Karachi, Pakistan

Background: High blood lead (Pb) and hyperhomocysteinemia have been found to be associated with cardiovascular disease (CVD). Mean blood Pb and mean plasma homocysteine levels have been reported to be high in Pakistani population. The objective of the present study was to assess the relationship of blood Pb to the risk of hyperhomocysteinemia in a low income urban population of Karachi, Pakistan. Methodology/Principal Findings: In a cross sectional survey, 872 healthy adults (355 males, 517 females, age 18-60 years) were recruited from a low income urban population of Karachi. Fasting venous blood was obtained and assessed for blood Pb and plasma/serum homocysteine, folate, pyridoxal phosphate (PLP, a coenzymic form of vitamin B6) and vitamin B12. The study population had median (IQR) blood Pb of 10.82 microg/dL (8.29-13.60). Prevalence of high blood Pb (levels\u3e10 microg/dL) was higher in males compared to females (62.5% males vs 56% females, p value=0.05). Mean+/-SD/median (IQR) value of plasma homocysteine was significantly higher in the highest quartile of blood Pb compared to the lowest quartile 16.13+/-11.2 micromol/L vs 13.28+/-9.7micromol/L/13.15 (10.33-17.81) micromol/L vs 11.09 (8.65 14.31) micromol/L (p valu

Characterization of the linkage disequilibrium structure and identification of tagging-SNPs in five DNA repair genes

BACKGROUND: Characterization of the linkage disequilibrium (LD) structure of candidate genes is the basis for an effective association study of complex diseases such as cancer. In this study, we report the LD and haplotype architecture and tagging-single nucleotide polymorphisms (tSNPs) for five DNA repair genes: ATM, MRE11A, XRCC4, NBS1 and RAD50. METHODS: The genes ATM, MRE11A, and XRCC4 were characterized using a panel of 94 unrelated female subjects (47 breast cancer cases, 47 controls) obtained from high-risk breast cancer families. A similar LD structure and tSNP analysis was performed for NBS1 and RAD50, using publicly available genotyping data. We studied a total of 61 SNPs at an average marker density of 10 kb. Using a matrix decomposition algorithm, based on principal component analysis, we captured >90% of the intragenetic variation for each gene. RESULTS: Our results revealed that three of the five genes did not conform to a haplotype block structure (MRE11A, RAD50 and XRCC4). Instead, the data fit a more flexible LD group paradigm, where SNPs in high LD are not required to be contiguous. Traditional haplotype blocks assume recombination is the only dynamic at work. For ATM, MRE11A and XRCC4 we repeated the analysis in cases and controls separately to determine whether LD structure was consistent across breast cancer cases and controls. No substantial difference in LD structures was found. CONCLUSION: This study suggests that appropriate SNP selection for an association study involving candidate genes should allow for both mutation and recombination, which shape the population-level genomic structure. Furthermore, LD structure characterization in either breast cancer cases or controls appears to be sufficient for future cancer studies utilizing these genes

Alterations in ethanol-induced behaviors and consumption in knock-in mice expressing ethanol-resistant NMDA receptors

Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75-2.0 g/kg; IP) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol. © 2013 den Hartog et al

Specific Thiazolidinediones Inhibit Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARγ Independent Manner

Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs--Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)--on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity.Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G(0)/G(1) stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent.CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle

The “Flexi-Chamber”: A Novel Cost-Effective In Situ Respirometry Chamber for Coral Physiological Measurements

Coral reefs are threatened worldwide, with environmental stressors increasingly affecting the ability of reef-building corals to sustain growth from calcification (G), photosynthesis (P) and respiration (R). These processes support the foundation of coral reefs by directly influencing biogeochemical nutrient cycles and complex ecological interactions and therefore represent key knowledge required for effective reef management. However, metabolic rates are not trivial to quantify and typically rely on the use of cumbersome in situ respirometry chambers and/or the need to remove material and examine ex situ, thereby fundamentally limiting the scale, resolution and possibly the accuracy of the rate data. Here we describe a novel low-cost in situ respirometry bag that mitigates many constraints of traditional glass and plexi-glass incubation chambers. We subsequently demonstrate the effectiveness of our novel "Flexi-Chamber" approach via two case studies: 1) the Flexi-Chamber provides values of P, R and G for the reef-building coral Siderastrea cf. stellata collected from reefs close to Salvador, Brazil, which were statistically similar to values collected from a traditional glass respirometry vessel; and 2) wide-scale application of obtaining P, R and G rates for different species across different habitats to obtain inter- and intra-species differences. Our novel cost-effective design allows us to increase sampling scale of metabolic rate measurements in situ without the need for destructive sampling and thus significantly expands on existing research potential, not only for corals as we have demonstrated here, but also other important benthic groups

A Nomenclature for Vertebral Fossae in Sauropods and Other Saurischian Dinosaurs

The axial skeleton of extinct saurischian dinosaurs (i.e., theropods, sauropodomorphs), like living birds, was pneumatized by epithelial outpocketings of the respiratory system. Pneumatic signatures in the vertebral column of fossil saurischians include complex branching chambers within the bone (internal pneumaticity) and large chambers visible externally that are bounded by neural arch laminae (external pneumaticity). Although general aspects of internal pneumaticity are synapomorphic for saurischian subgroups, the individual internal pneumatic spaces cannot be homologized across species or even along the vertebral column, due to their variability and absence of topographical landmarks. External pneumatic structures, in contrast, are defined by ready topological landmarks (vertebral laminae), but no consistent nomenclatural system exists. This deficiency has fostered confusion and limited their use as character data in phylogenetic analysis.We present a simple system for naming external neural arch fossae that parallels the one developed for the vertebral laminae that bound them. The nomenclatural system identifies fossae by pointing to reference landmarks (e.g., neural spine, centrum, costal articulations, zygapophyses). We standardize the naming process by creating tripartite names from “primary landmarks,” which form the zygodiapophyseal table, “secondary landmarks,” which orient with respect to that table, and “tertiary landmarks,” which further delineate a given fossa.The proposed nomenclatural system for lamina-bounded fossae adds clarity to descriptions of complex vertebrae and allows these structures to be sourced as character data for phylogenetic analyses. These anatomical terms denote potentially homologous pneumatic structures within Saurischia, but they could be applied to any vertebrate with vertebral laminae that enclose spaces, regardless of their developmental origin or phylogenetic distribution

Right-to-left shunt with hypoxemia in pulmonary hypertension

<p>Abstract</p> <p>Background</p> <p>Hypoxemia is common in pulmonary hypertension (PH) and may be partly related to ventilation/perfusion mismatch, low diffusion capacity, low cardiac output, and/or right-to-left (RL) shunting.</p> <p>Methods</p> <p>To determine whether true RL shunting causing hypoxemia is caused by intracardiac shunting, as classically considered, a retrospective single center study was conducted in consecutive patients with precapillary PH, with hypoxemia at rest (PaO₂< 10 kPa), shunt fraction (Qs/Qt) greater than 5%, elevated alveolar-arterial difference of PO₂(AaPO₂), and with transthoracic contrast echocardiography performed within 3 months.</p> <p>Results</p> <p>Among 263 patients with precapillary PH, 34 patients were included: pulmonary arterial hypertension, 21%; PH associated with lung disease, 47% (chronic obstructive pulmonary disease, 23%; interstitial lung disease, 9%; other, 15%); chronic thromboembolic PH, 26%; miscellaneous causes, 6%. Mean pulmonary artery pressure, cardiac index, and pulmonary vascular resistance were 45.8 ± 10.8 mmHg, 2.2 ± 0.6 L/min/m², and 469 ± 275 dyn.s.cm^-5, respectively. PaO₂in room air was 6.8 ± 1.3 kPa. Qs/Qt was 10.2 ± 4.2%. AaPO₂under 100% oxygen was 32.5 ± 12.4 kPa. Positive contrast was present at transthoracic contrast echocardiography in 6/34 (18%) of patients, including only 4/34 (12%) with intracardiac RL shunting. Qs/Qt did not correlate with hemodynamic parameters. Patients' characteristics did not differ according to the result of contrast echocardiography.</p> <p>Conclusion</p> <p>When present in patients with precapillary PH, RL shunting is usually not related to reopening of patent <it>foramen ovale</it>, whatever the etiology of PH.</p