Mimicking breast cancer-induced bone metastasis in vivo: Current transplantation models and advanced humanized strategies

Bone metastasis is a complication that occurs in 80 % of women with advanced breast cancer. Despite the prevalence of bone metastatic disease, the avenues for its clinical management are still restricted to palliative treatment options. In fact, the underlying mechanisms of breast cancer osteotropism have not yet been fully elucidated due to a lack of suitable in vivo models that are able to recapitulate the human disease. In this work, we review the current transplantation-based models to investigate breast cancer-induced bone metastasis and delineate the strengths and limitations of the use of different grafting techniques, tissue sources, and hosts. We further show that humanized xenograft models incorporating human cells or tissue grafts at the primary tumor site or the metastatic site mimic more closely the human disease. Tissue-engineered constructs are emerging as a reproducible alternative to recapitulate functional humanized tissues in these murine models. The development of advanced humanized animal models may provide better platforms to investigate the mutual interactions between human cancer cells and their microenvironment and ultimately improve the translation of preclinical drug trials to the clinic.

Altered formalin-induced pain and Fos induction in the periaqueductal grey of preadolescent rats following neonatal LPS exposure

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this process

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

High threshold of β1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2)

Background Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding ?1 integrins in collagen-stimulated MMP-2 activation. Methods Three ?1 integrin siRNAs were used to elucidate the involvement of ?1 integrins in the Col I-induced MMP-2 activation mechanism. ?1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of ?1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. Results All three ?1 integrin siRNAs were efficient at ?1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins ?2 and ?3, which are heterodimeric partners of ?1, but not ?V, which is not. All three ?1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of ?1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped ?1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the ?1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by ?1 integrin siRNA knockdown. Conclusion Together, the data reveals that strong abrogation of ?1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of ?1 integrin.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: Impact for allograft rejection

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell-matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

The prevalence, predictors and associations of hypertension in Sri Lanka: A cross-sectional population based national survey

We studied the community prevalence, patterns and predictors of hypertension in a large sub-population of South Asian adults with a view of identifying differential risk factors. Data were collected between years 2005-2006 and 5000 adults were invited for the study. The sample size was 4485, and about 39.5% were males. Mean systolic and diastolic blood pressures were 127.1 ± 19.8 mmHg and 75.4 ± 11.3 mmHg, respectively. Age-adjusted prevalence in all adults, males and females was 23.7%, 23.4% and 23.8%, respectively. Urban adults had a significantly higher prevalence of hypertension than rural adults. In the binary logistic-regression analysis, male gender (OR: 1.2), increasing age, Sri Lankan Moor ethnicity (OR: 1.6), physical inactivity (OR: 1.7), presence of diabetes (OR: 2.2) and central obesity (OR: 2.3) all were significantly associated with hypertension. In conclusion, nearly one-third of the Sri Lankan adult population is hypertensive. Hence, public health initiatives should encourage healthier lifestyles with emphasis on preventing obesity and increasing physical activity.

Moffatt vortices induced by the motion of a contact line

A recent hydrodynamic theory of liquid slippage on a solid substrate (Kirkinis & Davis, Phys. Rev. Lett., vol. 110, 2013, 234503) gives rise to a sequence of eddies (Moffatt vortices) that co-move with a moving contact line (CL) in a liquid wedge. The presence of these vortices is established through secular equations that depend on the dynamic contact angle α and capillary number Ca. The limiting case α→O is associated with the appearance of such vortices in a channel. The vortices are generated by the relative motion of the interfaces, which in turn is due to the motion of the CL. This effect has yet to be observed in experiment.

Patient reported adherence to hypertension treatment: A revalidation study

Background Adherence to hypertension management in patients with hypertension is known to influence their blood pressure control. It is important to measure patients’ adherence behaviours to assist with designing appropriate interventions to improve blood pressure control. Aims The purposes of this study were to use confirmatory factor analysis to revalidate the Therapeutic Adherence Scale for Hypertensive Patients (TASHP), and to calculate the cut-off score for classifying adherence behaviours into two groups: satisfactory and low adherence behaviours. Methods Systematic random sampling was used to recruit patients with hypertension in China. Demographic characteristics, the TASHP and blood pressure were collected. The psychometric tests of the TASHP included: construct validity, criteria-related validity, internal reliability, and split-half reliability. The area under the receiver operating characteristics curve and Youden index were used to identify the cut-off score of the TASHP for blood pressure control. Results This study involved 366 patients. Confirmatory factor analysis supported the four-component structure of the TASHP proposed in the original scale development study. The TASHP has a satisfactory internal reliability (Cronbach’s α > 0.7) and a satisfactory split-half reliability (Spearman–Brown coefficients > 0.7). The patients with overall scores of the TASHP ⩾ 109 points were considered to have satisfactory adherence behaviours. Conclusion The TASHP is a validated and reliable instrument to measure the adherence to hypertension management in Chinese patients with hypertension. The cut-off score of 109 points can be considered as an effective measure to classify the level of adherence into satisfactory and low adherence behaviours.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Nitrogen-doped graphene films from chemical vapor deposition of pyridine: Influence of process parameters on the electrical and optical properties

Graphene films were produced by chemical vapor deposition (CVD) of pyridine on copper substrates. Pyridine-CVD is expected to lead to doped graphene by the insertion of nitrogen atoms in the growing sp2 carbon lattice, possibly improving the properties of graphene as a transparent conductive film. We here report on the influence that the CVD parameters (i.e., temperature and gas flow) have on the morphology, transmittance, and electrical conductivity of the graphene films grown with pyridine. A temperature range between 930 and 1070 °C was explored and the results were compared to those of pristine graphene grown by ethanol-CVD under the same process conditions. The films were characterized by atomic force microscopy, Raman and X-ray photoemission spectroscopy. The optical transmittance and electrical conductivity of the films were measured to evaluate their performance as transparent conductive electrodes. Graphene films grown by pyridine reached an electrical conductivity of 14.3 × 105 S/m. Such a high conductivity seems to be associated with the electronic doping induced by substitutional nitrogen atoms. In particular, at 930 °C the nitrogen/carbon ratio of pyridine-grown graphene reaches 3%, and its electrical conductivity is 40% higher than that of pristine graphene grown from ethanol-CVD.

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Architectural changes of the biceps femoris after concentric or eccentric training

Purpose To determine i) the architectural adaptations of the biceps femoris long head (BFlf) following concentric or eccentric strength training interventions; ii) the time course of adaptation during training and detraining. Methods Participants in this randomized controlled trial (control [n=28], concentric training group [n=14], eccentric training group [n=14], males) completed a 4-week control period, followed by 6 weeks of either concentric- or eccentric-only knee flexor training on an isokinetic dynamometer and finished with 28 days of detraining. Architectural characteristics of BFlf were assessed at rest and during graded isometric contractions utilizing two-dimensional ultrasonography at 28 days pre-baseline, baseline, days 14, 21 and 42 of the intervention and then again following 28 days of detraining. Results BFlf fascicle length was significantly longer in the eccentric training group (p<0.05, d range: 2.65 to 2.98) and shorter in the concentric training group (p<0.05, d range: -1.62 to -0.96) after 42 days of training compared to baseline at all isometric contraction intensities. Following the 28-day detraining period, BFlf fascicle length was significantly reduced in the eccentric training group at all contraction intensities compared to the end of the intervention (p<0.05, d range: -1.73 to -1.55). There was no significant change in fascicle length of the concentric training group following the detraining period. Conclusions These results provide evidence that short term resistance training can lead to architectural alterations in the BFlf. In addition, the eccentric training-induced lengthening of BFlf fascicle length was reversed and returned to baseline values following 28 days of detraining. The contraction mode specific adaptations in this study may have implications for injury prevention and rehabilitation.

Solute induced liquid crystalline behaviour of a quaternary ammonium salt and its application to structure determination

A new liquid crystalline phase, induced by the addition of small amounts of a non-mesogenic solute (such as dimethyl sulphoxide or methyl iodide) to a quaternary ammonium salt, N-methyl-N,N,N-trioctadecylammonium iodide (MTAI), has been detected by NMR and optical microscopic studies. In some cases, there is a coexistence of nematic and smectic phases. Information on the ordering of the phases in the magnetic field of the spectrometer has been derived from NMR spectra of a dissolved molecule, C-13-enriched methyl iodide. The low order parameter of the pure thermotropic nematic phase of the salt provides first-order spectra of the dissolved oriented molecules. Analyses of spectra of cis,cis-mucononitrile exemplifies the utility of the MTAI nematic phase in the determination of structural parameters of the solute.

Gossypol-induced hypokalemia and role of exogenous potassium salt supplementation when used as an antispermatogenic agent in male langur monkey

In our earlier study, we have observed that hypokalemia in langur monkeys, following gossypol acetic acid (GAA) treatment (5 mg dose level) when used as an antispermatogenic agent, and potassium salt supplementation partially maintained body potassium level of the animals. The aims of the present investigation was to confirm further occurrence of hypokalemia in the monkey (comparatively at two higher dose levels) and the role of potassium salt in preventing occurrence of gossypol-induced hypokalemia. Highly purified gossypol acetic acid alone at two dose levels (7.5 and 10 mg/animal/day; oral) and in combination with potassium chloride (0.50 and 0.75 mg/animal/day; oral) was given for 180 days. Treatment with gossypol alone as well as with the supplementation of potassium salt resulted in severe oligospermia and azoospermia. Animals receiving gossypol alone showed significant potassium deficiency with signs of fatigue at both dose levels. Enhanced potassium loss through urine was found in potassium-deficient animals, whereas animals receiving gossypol acetic acid plus potassium salt showed normal serum potassium with a less significant increase in urine potassium level during treatment phases. Other parameters of the body remained within normal range except gradual and significant elevation in serum transaminases activity. The animals gradually returned to normalcy following 150 and 180 days of termination of the treatment.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.