Visually Impaired OLder people's Exercise programme for falls prevenTion (VIOLET): a feasibility study protocol.
In the UK, 1 in 5 people aged 75 and over live with sight loss. Visually impaired older people (VIOP) have an above average incidence of falls and 1.3-1.9 times more likely to experience hip fractures, than the general population. Older people with eye diseases are ∼3 times more likely than those with good vision, to limit activities due to fear of falling. This feasibility study aims to adapt the group-based Falls Management Exercise (FaME) programme to the needs of VIOP and carry out an external pilot trial to inform the design of a future definitive randomised controlled trial. A UK based 2-centre mixed methods, randomised, feasibility study will be conducted over 28 months. Stakeholder panels, including VIOP, will make recommendations for adaptations to an existing exercise programme (FaME), to meet the needs of VIOP, promoting uptake and adherence, while retaining required effective components of the exercise programme. 80 VIOP aged 60 and over, living at home, ambulant with or without a walking aid, will be recruited in Newcastle (n=40) and Glasgow (n=40) through National Health Service (NHS) Trusts and third sector partners. Participants randomised into the intervention arm will receive the adapted FaME programme. Participants randomised into the control arm will continue with usual activity. Outcomes are, recruitment rate, adherence and validated measures including fear of falling and quality of life. Postintervention in-depth qualitative interviews will be conducted with a purposive sample of VIOP (N=10). Postural stability instructors will be interviewed, before trial-specific training and following the intervention. Ethics approval was secured through the National Research Ethics Service (NRES) Committee North East, Newcastle and North Tyneside 2. Glasgow Caledonian University was approved as a non-NHS site with local ethics approval. Findings will be disseminated through peer-reviewed journals, national and international conferences. ISRCTN16949845.


Metabolomics reveals amino acids contribute to variation in response to simvastatin treatment.
Statins are widely prescribed for reducing LDL-cholesterol (C) and risk for cardiovascular disease (CVD), but there is considerable variation in therapeutic response. We used a gas chromatography-time-of-flight mass-spectrometry-based metabolomics platform to evaluate global effects of simvastatin on intermediary metabolism. Analyses were conducted in 148 participants in the Cholesterol and Pharmacogenetics study who were profiled pre and six weeks post treatment with 40 mg/day simvastatin: 100 randomly selected from the full range of the LDL-C response distribution and 24 each from the top and bottom 10% of this distribution ("good" and "poor" responders, respectively). The metabolic signature of drug exposure in the full range of responders included essential amino acids, lauric acid (p<0.0055, q<0.055), and alpha-tocopherol (p<0.0003, q<0.017). Using the HumanCyc database and pathway enrichment analysis, we observed that the metabolites of drug exposure were enriched for the pathway class amino acid degradation (p<0.0032). Metabolites whose change correlated with LDL-C lowering response to simvastatin in the full range responders included cystine, urea cycle intermediates, and the dibasic amino acids ornithine, citrulline and lysine. These dibasic amino acids share plasma membrane transporters with arginine, the rate-limiting substrate for nitric oxide synthase (NOS), a critical mediator of cardiovascular health. Baseline metabolic profiles of the good and poor responders were analyzed by orthogonal partial least square discriminant analysis so as to determine the metabolites that best separated the two response groups and could be predictive of LDL-C response. Among these were xanthine, 2-hydroxyvaleric acid, succinic acid, stearic acid, and fructose. Together, the findings from this study indicate that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism may play a role in modulating the response to simvastatin treatment. ClinicalTrials.gov NCT00451828.


Pressure and volume limited ventilation for the ventilatory management of patients with acute lung injury: a systematic review and meta-analysis.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life threatening clinical conditions seen in critically ill patients with diverse underlying illnesses. Lung injury may be perpetuated by ventilation strategies that do not limit lung volumes and airway pressures. We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) comparing pressure and volume-limited (PVL) ventilation strategies with more traditional mechanical ventilation in adults with ALI and ARDS. We searched Medline, EMBASE, HEALTHSTAR and CENTRAL, related articles on PubMed™, conference proceedings and bibliographies of identified articles for randomized trials comparing PVL ventilation with traditional approaches to ventilation in critically ill adults with ALI and ARDS. Two reviewers independently selected trials, assessed trial quality, and abstracted data. We identified ten trials (n = 1,749) meeting study inclusion criteria. Tidal volumes achieved in control groups were at the lower end of the traditional range of 10-15 mL/kg. We found a clinically important but borderline statistically significant reduction in hospital mortality with PVL [relative risk (RR) 0.84; 95% CI 0.70, 1.00; p = 0.05]. This reduction in risk was attenuated (RR 0.90; 95% CI 0.74, 1.09, p = 0.27) in a sensitivity analysis which excluded 2 trials that combined PVL with open-lung strategies and stopped early for benefit. We found no effect of PVL on barotrauma; however, use of paralytic agents increased significantly with PVL (RR 1.37; 95% CI, 1.04, 1.82; p = 0.03). This systematic review suggests that PVL strategies for mechanical ventilation in ALI and ARDS reduce mortality and are associated with increased use of paralytic agents.


Determinants of VH1-46 Cross-Reactivity to Pemphigus Vulgaris Autoantigen Desmoglein 3 and Rotavirus Antigen VP6.
Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease Ag. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign Ag prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from two PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and three PV patients identified specific cross-reactive Abs in one PV patient, but notably all six cross-reactive clonotypes used VH1-46. Site-directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.


Conjugation of multiple copies of polyethylene glycol to hemoglobin facilitated through thiolation: influence on hemoglobin structure and function.
PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)(6)-Hb, that carries approximately six PEG5K chains/Hb - HexaPEGylated Hb. PEGylation increased the O(2) affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)(6)-Hb, its molecular volume, O(2) affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)(2)-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)(6)-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)(2)-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.


Evaluation of insulin sensitivity with a new lipid-based index in non-diabetic postmenopausal overweight and obese women before and after a weight loss intervention.
To evaluate the validity of a new lipid-based index (Disse index) in assessing insulin sensitivity (IS) compared with the hyperinsulinemic-euglycemic (HIEG) clamp in overweight and obese, non-diabetic, postmenopausal women, before and after a weight loss intervention. Association between Disse index and the HIEG clamp was evaluated in 86 non-diabetic postmenopausal overweight and obese women before and after weight loss. Percentage changes (%Delta) were calculated for several fasting indices and compared with %Delta of HIEG clamp. We observed a strong correlation between Disse index and HIEG clamp (r=0.69, P<0.001). This association was higher than those of homeostasis model assessment (HOMA), quantitative insulin sensitivity check index (QUICKI), and McAuley indices while no significant difference was observed with Revised-QUICKI. Percent change of Disse index (pre- versus post-weight loss program) was significantly correlated with %Delta of HIEG clamp (r=0.34, P<0.01). This correlation was similar to those observed for the other indices tested. We validated the reliability of Disse index in assessing IS in non-diabetic post-menopausal overweight and obese women, before and after weight loss intervention. Disse index may be useful not only for insulin resistant diagnostics in this type of population, but also for the IS follow-up after a weight-loss program and weight stabilization. The presence of lipid elements in this fasting index improves the estimation of IS in overweight and obese non-diabetic post-menopausal women and could add more information about peripheral IS.


Ybp1 and Gpx3 signaling in Candida albicans govern hydrogen peroxide-induced oxidation of the Cap1 transcription factor and macrophage escape.
As Candida albicans is the major fungal pathogen of humans, there is an urgent need to understand how this pathogen evades toxic reactive oxygen species (ROS) generated by the host immune system. A key regulator of antioxidant gene expression, and thus ROS resistance, in C. albicans is the AP-1-like transcription factor Cap1. Despite this, little is known regarding the intracellular signaling mechanisms that underlie the oxidation and activation of Cap1. Therefore, the aims of this study were; (i) to identify the regulatory proteins that govern Cap1 oxidation, and (ii) to investigate the importance of Cap1 oxidation in C. albicans pathogenesis. In response to hydrogen peroxide (H2O2), but not glutathione-depleting/modifying oxidants, Cap1 oxidation, nuclear accumulation, phosphorylation, and Cap1-dependent gene expression, is mediated by a glutathione peroxidase-like enzyme, which we name Gpx3, and an orthologue of the Saccharomyces cerevisiae Yap1 binding protein, Ybp1. In addition, Ybp1 also functions to stabilise Cap1 and this novel function is conserved in S. cerevisiae. C. albicans cells lacking Cap1, Ybp1, or Gpx3, are unable to filament and thus, escape from murine macrophages after phagocytosis, and also display defective virulence in the Galleria mellonella infection model. Ybp1 is required to promote the stability of fungal AP-1-like transcription factors, and Ybp1 and Gpx3 mediated Cap1-dependent oxidative stress responses are essential for the effective killing of macrophages by C. albicans. Activation of Cap1, specifically by H2O2, is a prerequisite for the subsequent filamentation and escape of this fungal pathogen from the macrophage.


Epcam, CD44, and CD49f distinguish sphere-forming human prostate basal cells from a subpopulation with predominant tubule initiation capability.
Human prostate basal cells expressing alpha-6 integrin (CD49f(Hi)) and/or CD44 form prostaspheres in vitro. This functional trait is often correlated with stem/progenitor (S/P) activity, including the ability to self-renew and induce differentiated tubules in vivo. Antigenic profiles that distinguish tubule-initiating prostate stem cells (SCs) from progenitor cells (PCs) and mature luminal cells (LCs) with less regenerative potential are unknown. Prostasphere assays and RT-PCR analysis was performed following FACS separation of total benign prostate cells based upon combinations of Epcam, CD44, and/or CD49f expression. Epithelial cell fractions were isolated, including Epcam(+)CD44(+) and Epcam+CD44+CD49f(Hi) basal cells that formed abundant spheres. When non-sphere-forming Epcam(+)CD44(-) cells were fractionated based upon CD49f expression, a distinct subpopulation (Epcam(+)CD44(-)CD49f(Hi)) was identified that possessed a basal profile similar to Epcam(+)CD44(+)CD49f(Hi) sphere-forming cells (p63(+)AR(Lo)PSA(-)). Evaluation of tubule induction capability of fractionated cells was performed, in vivo, via a fully humanized prostate tissue regeneration assay. Non-sphere-forming Epcam(+)CD44(-) cells induced significantly more prostate tubular structures than Epcam(+)CD44(+) sphere-forming cells. Further fractionation based upon CD49f co-expression identified Epcam(+)CD44(-)CD49f(Hi) (non-sphere-forming) basal cells with significantly increased tubule induction activity compared to Epcam(+)CD44(-)CD49f(Lo) (true) luminal cells. Our data delineates antigenic profiles that functionally distinguish human prostate epithelial subpopulations, including putative SCs that display superior tubule initiation capability and induce differentiated ductal/acini structures, sphere-forming PCs with relatively decreased tubule initiation activity, and terminally differentiated LCs that lack both sphere-forming and tubule-initiation activity. The results clearly demonstrate that sphere-forming ability is not predictive of tubule-initiation activity. The subpopulations identified are of interest because they may play distinct roles as cells of origin in the development of prostatic diseases, including cancer.


Factors influencing duration of exposure with symptoms and costs of occupational asthma.
The most important factor for the prognosis of occupational asthma is the length of exposure with symptoms prior to removal from exposure. We wanted to identify factors, including socioeconomic status, that can influence the delay in submitting a claim to a medicolegal agency after the onset of asthmatic symptoms, and to confirm that this delay is associated with worse respiratory prognosis and higher direct costs. This is a cross-sectional study of subjects who claimed compensation for occupational asthma at the Workers' Compensation Board of Quebec, Canada. Data were collected at re-evaluation ∼2.5 yrs after diagnosis. Information on the number of years with symptoms and removal from exposure was obtained from the medicolegal file. 60 subjects were included in the study. Being older, having a revenue of >30,000 Canadian dollars and having occupational asthma due to high molecular weight agents were all positively associated with the number of years of exposure with symptoms before removal from exposure. Subjects with persistent airway hyperresponsiveness at follow-up had a higher number of years with symptoms. Experiencing symptoms in the workplace for <1 yr generated lower direct costs. These findings might help in surveillance programmes that could be preferentially targeted for these subgroups of workers.


Investigation of dopamine content, synthesis, and release in the rabbit retina in vitro: II. Effects of high potassium, adenylate cyclase activators, and N-n-propyl-3-(3-hydroxyphenyl) piperidine.
The modulation of 3,4-dihydroxyphenylethylamine (dopamine, DA) synthesis and release in rabbit retina in vitro by high K+; adenylate cyclase activators such as forskolin, 2-chloroadenosine, vasoactive intestinal polypeptide (VIP); and the putative DA autoreceptor agonist N-n-propyl-3-(3-hydroxyphenyl) piperidine (3-PPP) has been investigated. Incubation of retinas in 50 mM K+ resulted in the activation of tyrosine hydroxylase (TH). Activation did not require the presence of extracellular Ca2+. K+ 50 mM also induced a Ca2+-dependent release of DA. Forskolin 50 microM stimulated TH but 100 microM 2-chloroadenosine and 650 nM VIP did not. Individually, (+)-3-PPP, (-)-3-PPP, and (+/-)-3-PPP reduced DA synthesis and increased its release. The effects of (+/-)-3-PPP were dose-dependent and did not require the presence of extracellular Ca2+. The activation of TH induced by 50 mM K+, but not that induced by 50 microM forskolin, was abolished by 100 microM (+/-)-3-PPP.


Identification of an Unconventional Subpeptidome Bound to the Behçet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1).
Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position Ω). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ∼40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.


Methanol loading dependent methoxylation in zeolite H-ZSM-5.
We evaluate the effect of the number of methanol molecules per acidic site of H-ZSM-5 on the methoxylation reaction at room temperature by applying operando diffuse reflectance infrared Fourier transformed spectroscopy (DRIFTS) and mass spectrometry (MS), which capture the methoxylation reaction by simultaneously probing surface adsorbed species and reaction products, respectively. To this end, the methanol loading in H-ZSM-5 (Si/Al ≈ 25) pores is systematically varied between 32, 16, 8 and 4 molecules per unit cell, which corresponds to 8, 4, 2 and 1 molecules per Brønsted acidic site, respectively. The operando DRIFTS/MS data show that the room temperature methoxylation depends on the methanol loading: the higher the methanol loading, the faster the methoxylation. Accordingly, the reaction is more than an order of magnitude faster with 8 methanol molecules per Brønsted acidic site than that with 2 molecules, as evident from the evolution of the methyl rock band of the methoxy species and of water as a function of time. Significantly, no methoxylation is observed with ≤1 molecule per Brønsted acidic site. However, hydrogen bonded methanol occurs across all loadings studied, but the structure of hydrogen bonded methanol also depends on the loading. Methanol loading of ≤1 molecule per acidic site leads to the formation of hydrogen bonded methanol with no proton transfer (i.e. neutral geometry), while loading ≥2 molecules per acidic site results in a hydrogen bonded methanol with a net positive charge on the adduct (protonated geometry). The infrared vibrational frequencies of methoxy and hydrogen bonded methanol are corroborated by Density Functional Theory (DFT) calculations. Both the experiments and calculations reflect the methoxy bands at around 940, 1180, 2868-2876 and 2980-2973 cm-1 which correspond to ν(C-O), ρ(CH3), ν s(C-H) and ν as(C-H), respectively.


Environmental stresses inhibit and stimulate different protein import pathways in plant mitochondria.
The impact of various environmental stresses (drought, chilling or herbicide treatment) on the capacity of plant mitochondria to import precursor proteins was investigated. Drought treatment stimulated import and processing of various precursor proteins via the general import pathway. The stimulatory effect of drought on the general import pathway was due to an increased rate of import, was accompanied by an increased rate of processing, and could be attributed to the presequence of the precursor protein. Interestingly, drought decreased the import of the F(A)d subunit of ATP synthase suggesting a bypass of the point of stimulation during import of this precursor. Both chilling and herbicide treatment of plants, on the other hand, caused inhibition of import with all precursors tested. No decrease in processing of imported proteins was observed by these stress treatments. Western analysis of several mitochondrial proteins indicated that the steady-state level of several mitochondrial components, including the TOM20 receptor and the core subunits of the cytochrome bc(1) complex responsible for processing, remained largely unchanged. Thus environmental stresses differentially affect import of precursor proteins in a complicated manner dependent on the import pathway utilised.


An amyotrophic lateral sclerosis hot spot in the French Alps associated with genotoxic fungi.
Between 1990 and 2018, 14 cases of amyotrophic lateral sclerosis (ALS) were diagnosed in residents of, and in visitors with second homes to, a mountainous hamlet in the French Alps. Systematic investigation revealed a socio-professional network that connected ALS cases. Genetic risk factors for ALS were excluded. Several known environmental factors were scrutinized and eliminated, notably lead and other chemical contaminants in soil, water or home-grown vegetation used for food, radon and electromagnetic fields. Some lifestyle-related behavioral risk factors were identified: Prior to clinical onset of motor neuron disease, some patients had a high degree of athleticism and smoked tobacco. Recent investigations on site, based on a new hypothesis, showed that all patients had ingested wild mushrooms, notably poisonous False Morels. Half of the ALS cohort reported acute illness following Gyromitra gigas mushroom consumption. This finding supports the hypothesis that genotoxins of fungal origin may induce motor neuron degeneration.


Non-invasive detection of a palifermin-mediated adaptive response following chemotherapy-induced damage to the distal small intestine of rats.
Pre-clinical studies have indicated that palifermin may be an effective treatment modality for intestinal mucositis, a debilitating complication of cancer chemotherapy. We determined whether palifermin was protective in rats with experimentally induced intestinal mucositis and the applicability of the sucrose breath test (SBT) to monitor palifermin for its efficacy as an anti-mucositis agent. SBT values and sucrase activity were reduced in all 5-FU-treated groups compared with untreated controls (p < 0.05). At 72 h post 5-FU, sucrase activity was higher in rats treated with palifermin compared with 5-FU controls (p < 0.05). Jejunal and ileal villus heights were lower in all 5-FU groups compared with saline controls. Dark agouti rats (n = 10) were subcutaneously injected with palifermin or vehicle for 3 d after which they were injected with 5-fluorouracil (5-FU) and sacrificed after 72 h. The in vivo SBT and in vitro sucrase assay were used to evaluate small intestinal function and damage. Intestinal disease severity was determined by histological assessment of villus height and crypt depth. The SBT can monitor the ability of palifermin to modify the functional capacity of the small intestine in rats with intestinal mucositis. Further studies are indicated to investigate the prophylactic potential of palifermin against intestinal mucositis.


TNF-α, a good or bad factor in hematological diseases?
Tumor necrosis factor-alpha (TNF-α) is a highly pleiotropic cytokine involved in a spectrum of physiological processes that control inflammation, anti-tumor responses and homeostasis through two receptors, TNF-R1 and TNF-R2. In general, TNF-R1 mediates cytotoxicity, resistance to infection and stimulation of NF-κB. By contrast, TNF-R2 has been implicated in proliferation of T-cell line, thymocytes and human mononuclear cells. Hematological malignancies are the types of cancer that affect normal hematopoiesis, have a speedy development, high lethal rate and until now still have no effective treatment. Several studies have shown that inflammatory cytokines play an important role in the onset and progress of these diseases. In this review, we summarize the recent studies and evaluate the positive or negative role of TNF-α in some hematological malignancies or diseases with a malignant tendency.


Sperm morphologic features as a prognostic factor in in vitro fertilization.
To determine whether there is a prognostic value in the percentage normal sperm morphologic features in a human in vitro fertilization (IVF) program, the authors conducted a prospective study in women with bilateral tubal damage. Based on the percentage of morphologically normal spermatozoa, the patients were divided into four groups: group I, normal morphologic features between 0% and 14%; group II, 15% to 30%; group III, 31% to 45%; and group IV, 46% to 60%. One hundred ninety successful laparoscopic cycles were evaluated. In group I, 104 oocytes were obtained, of which 37% fertilized, but no pregnancy resulted; in group II, 81% of 324 oocytes were fertilized, with a pregnancy rate per embryo transfer (ET) of 22%; in group III, 82% of 309 oocytes were fertilized, with a 31% pregnancy rate; and in group IV, 91% of 69 oocytes were fertilized, with a pregnancy rate of 12%. Probability models indicated that there was a clear threshold in normal sperm morphologic features at 14%, with high fertilization and pregnancy rate in the groups with normal sperm morphologic features greater than 14%.


Suppression of GRASP65 phosphorylation by tetrahydrocurcumin protects against cerebral ischemia/reperfusion injury via ERK signaling.
The aim of the present study was to assess the neuroprotective effects of tetrahydrocurcumin (THC) in a mouse model of cerebral ischemia/reperfusion (I/R) injury, and to investigate the involvement of Golgi reassembly and stacking protein 65 (GRASP65) and the extracellular signal‑regulated kinase (ERK) signaling pathway. Cerebral I/R injury was induced using the Pulsinelli four‑vessel occlusion method. After 5 min of reperfusion, mice received THC (5, 10 or 25 mg/kg) or saline by intraperitoneal injection. After 24 h of reperfusion, mice underwent neurological evaluation. Infarct volumes were determined by triphenyltetrazolium chloride staining, and levels of superoxide dismutase and malondialdehyde were measured in brain tissue homogenates. Expression of GRASP65, phosphorylated‑GRASP65, ERK and phosphorylated‑ERK was determined by western blotting. THC induced a dose‑dependent decrease in the phosphorylation of ERK and GRASP65. Thus, THC attenuated I/R injury‑induced activation of the ERK signaling pathway and reduced the phosphorylation of GRASP65. THC exhibited a dose‑dependent protective effect against cerebral I/R injury, mediated by suppression of the ERK signaling pathway and a subsequent reduction in GRASP65 phosphorylation. The current study provided new information in the research of the cerebral ischemia‑reperfusion injury mechanism.


Effects of antemortem ingestion of ethanol on insect successional patterns and development of Phormia regina (Diptera: Calliphoridae).
The effects of antemortem ingestion of ethanol by domestic pigs, Sus scrofa L., on postmortem insect successional patterns and the development of Phormia regina (Meigen) were studied during summer 2003 in Blacksburg, VA. Insect samples were collected from the carcasses of ethanol-treated and untreated pigs for 10 d postmortem during two successional studies. In total, 32 insect taxa were collected during the two studies, with 29 and 27 taxa observed on the carcasses of ethanol-treated and untreated pigs, respectively. The earliest arrivers to both carcass types were dipterans. This group was represented by six families, with P. regina and Phaenicia coeruleiviridis (Macquart) being the most common calliphorids. Beetles in six families were collected on the carcasses of ethanol-treated pigs, but only three of the families were collected on carcasses of the untreated pigs. Permutation analyses to test the null hypothesis of no similarity between successional patterns of insect taxa from carcasses of ethanol-treated and untreated pigs showed that the successional patterns were similar between carcass types in the first (P = 0.003) and the second (P = 0.01) studies. The results of the development study of P. regina maggots in the field show that there was a significant difference between the distributions of length for maggots reared on loin tissue from ethanol-treated and untreated pigs. Maggots that fed on tissue from ethanol-treated pigs took approximately 11.9 h longer to reach the pupal stage than maggots that fed on tissue from untreated pigs. The longer developmental time for maggots on tissue from ethanol-treated pigs was due mainly to the longer postfeeding period of the third instar.


Prevention and management of osteoporosis: consensus statements from the Scientific Advisory Board of the Osteoporosis Society of Canada. 4. Calcium nutrition and osteoporosis.
To recommend appropriate levels of calcium intake in light of the most recent studies. Dietary calcium intake, calcium supplementation, calcium and vitamin D supplementation; ovarian hormone therapy in postmenopausal women. Fracture and loss of bone mineral density in osteoporosis; increased bone mass, prevention of fractures and improved quality of life associated with osteoporosis prevention. Relevant clinical studies and reports were examined, in particular those published since the 1988 Osteoporosis Society of Canada position paper on calcium nutrition. Only studies in humans were considered, including controlled, randomized trials and prospective studies, using bone mass and fractures as end-points. Studies in early and later phases of skeletal growth were noted. The analysis was designed to eliminate menopause as a confounding variable. Preventing osteoporosis and maximizing quality of life were given a high value. Adequate calcium nutrition increases bone mineral density during skeletal growth and prevents bone loss and osteoporotic fractures in the elderly. Risks associated with high dietary calcium intake are low, and a recent study extends this conclusion to the risk of kidney stones. Lactase-deficient patients may substitute yogurt and lactase-treated milk for cow's milk. True milk allergy is probably rare; its promotion of diabetes mellitus in susceptible people is being studied. Current recommended intakes of calcium are too low. Revised intake guidelines designed to reduce bone loss and protect against osteoporotic fractures are suggested. Canadians should attempt to meet their calcium requirements principally through food sources. Pharmaceutical calcium supplements and a dietician's advice should be considered where dietary preferences or lactase deficiency restrict consumption of dairy foods. Further research is necessary before recommending the general use of calcium supplements by adolescents. Calcium supplementation cannot substitute for hormone therapy in the prevention of postmenopausal bone loss and fractures. Adequate amounts of vitamin D are necessary for optimal calcium absorption and bone health. Elderly people and those who use heavy sun screens should have a dietary intake of 400 to 800 IU of vitamin D per day.


miRvestigator: web application to identify miRNAs responsible for co-regulated gene expression patterns discovered through transcriptome profiling.
Transcriptome profiling studies have produced staggering numbers of gene co-expression signatures for a variety of biological systems. A significant fraction of these signatures will be partially or fully explained by miRNA-mediated targeted transcript degradation. miRvestigator takes as input lists of co-expressed genes from Caenorhabditis elegans, Drosophila melanogaster, G. gallus, Homo sapiens, Mus musculus or Rattus norvegicus and identifies the specific miRNAs that are likely to bind to 3' un-translated region (UTR) sequences to mediate the observed co-regulation. The novelty of our approach is the miRvestigator hidden Markov model (HMM) algorithm which systematically computes a similarity P-value for each unique miRNA seed sequence from the miRNA database miRBase to an overrepresented sequence motif identified within the 3'-UTR of the query genes. We have made this miRNA discovery tool accessible to the community by integrating our HMM algorithm with a proven algorithm for de novo discovery of miRNA seed sequences and wrapping these algorithms into a user-friendly interface. Additionally, the miRvestigator web server also produces a list of putative miRNA binding sites within 3'-UTRs of the query transcripts to facilitate the design of validation experiments. The miRvestigator is freely available at http://mirvestigator.systemsbiology.net.


S100A4 May Be a Good Prognostic Marker and a Therapeutic Target for Colon Cancer.
Globally, the colorectal cancers rank the third in terms of cancer incidence and rank the fourth in cancer-associated deaths. S100A4, an important member of the S100 protein family, serves to promote tumor progression and metastasis. By conducting this study, we aim to examine the role of S100A4 in the prognosis of colon cancer and to demonstrate its prognostic significance. Tissue samples of colon cancer from 148 patients who underwent colon resection due to colon cancer were analyzed by immunohistochemical staining to determine the protein expression levels of S100A4. The protein expression levels of S100A4 in tumor tissue were matched with the clinicopathologic factors including patient survival. Cytoplasmic expression of S100A4 protein was demonstrated in the tumor tissue of 132 patients (89.2%) out of a total of 148 study patients. Statistically, the expression levels of the cytoplasmic S100A4 protein correlated significantly with the TNM stages and patient survival. The distribution of the S100A4 protein staining in the tumor tissue was associated with the age groups, tumor localization, TNM staging, and patient survival with statistical significance. The levels of S100A4 protein expression were found to be an independent prognostic factor for TNM staging and poor survival. Expression of the S100A4 protein in colon cancers may be an indicator of tumor progression and lymph node metastasis and may be useful for predicting the overall survival of the patients with colon cancer. In patients with colon cancer, it may be used as an indicator of poor prognosis.


RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients.
Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.


Lifestyle, cardiovascular risk knowledge and patient counselling among selected sub-Saharan African family physicians and trainees.
Cardiovascular disease (CVD)-related deaths in sub-Saharan Africa (SSA) are on the rise, and primary care physicians could facilitate the reversal of this trend through treatment and prevention strategies. The aim of this study was to determine the relationship between physician lifestyle practices, CVD prevention knowledge and patient CVD counselling practices among family physicians (FPs) and family medicine (FM) trainees affiliated to FM colleges and organisations in SSA. FPs and FM trainees affiliated to FM colleges and organisations in Anglophone SSA. A web-based cross-sectional analytical study was conducted using validated, self-administered questionnaires. Following collation of responses, the relationship between the participants' CVD prevention knowledge, lifestyle practices and CVD counselling rates was assessed. Of the 174 participants (53% response rate), 83% were married, 51% were females and the mean age was 39.2 (standard deviation [SD] 7.6) years. Most of the participants responded accurately to the CVD prevention knowledge items, but few had accurate responses on prioritising care by 10-year risk. Most participants had less than optimal lifestyle practices except for smoking, vegetable or fruit ingestion and sleep habits. Most participants (65%) usually counselled patients on nutrition, but less frequently on weight management, exercise, smoking and alcohol. The region of practice and physicians with poor lifestyle were predictive of patient counselling rates. Training on patient counselling and self-awareness for CVD prevention may influence patient counselling practice. Promoting quality training on patient counselling among FPs as well as a healthy self-awareness for CVD prevention is thus needed. The complex relationship between physician lifestyle and patient counselling warrants further study.


Role of electrode placement as a contributor to variability in cochlear implant outcomes.
Suboptimal cochlear implant (CI) electrode array placement may reduce presentation of coded information to the central nervous system and, consequently, limit speech recognition. Generally, mean speech reception scores for CI recipients are similar across different CI systems, yet large outcome variation is observed among recipients implanted with the same device. These observations suggest significant recipient-dependent factors influence speech reception performance. This study examines electrode array insertion depth and scalar placement as recipient-dependent factors affecting outcome. Scalar location and depth of insertion of intracochlear electrodes were measured in 14 patients implanted with Advanced Bionics electrode arrays and whose word recognition scores varied broadly. Electrode position was measured using computed tomographic images of the cochlea and correlated with stable monosyllabic word recognition scores. Electrode placement, primarily in terms of depth of insertion and scala tympani versus scala vestibuli location, varies widely across subjects. Lower outcome scores are associated with greater insertion depth and greater number of contacts being located in scala vestibuli. Three patterns of scalar placement are observed suggesting variability in insertion dynamics arising from surgical technique. A significant portion of variability in word recognition scores across a broad range of performance levels of CI subjects is explained by variability in scalar location and insertion depth of the electrode array. We suggest that this variability in electrode placement can be reduced and average speech reception improved by better selection of cochleostomy sites, revised insertion approaches, and control of insertion depth during surgical placement of the array.


Development of a robust classifier for quality control of reverse-phase protein arrays.
High-throughput reverse-phase protein array (RPPA) technology allows for the parallel measurement of protein expression levels in approximately 1000 samples. However, the many steps required in the complex protocol (sample lysate preparation, slide printing, hybridization, washing and amplified detection) may create substantial variability in data quality. We are not aware of any other quality control algorithm that is tuned to the special characteristics of RPPAs. We have developed a novel classifier for quality control of RPPA experiments using a generalized linear model and logistic function. The outcome of the classifier, ranging from 0 to 1, is defined as the probability that a slide is of good quality. After training, we tested the classifier using two independent validation datasets. We conclude that the classifier can distinguish RPPA slides of good quality from those of poor quality sufficiently well such that normalization schemes, protein expression patterns and advanced biological analyses will not be drastically impacted by erroneous measurements or systematic variations. The classifier, implemented in the "SuperCurve" R package, can be freely downloaded at http://bioinformatics.mdanderson.org/main/OOMPA:Overview or http://r-forge.r-project.org/projects/supercurve/. The data used to develop and validate the classifier are available at http://bioinformatics.mdanderson.org/MOAR.


An educational leaflet improves response to invitation for screening for arthritis in patients with psoriasis in primary care, but only in practices in the most deprived areas.
This study hypothesises that an educational leaflet about psoriatic arthritis (PsA) will improve psoriasis patients' attendance for screening for PsA. A random sample of patients ≥18 years old with a coded diagnosis of psoriasis and no diagnosis of PsA, rheumatoid arthritis or ankylosing spondylitis were identified from five GP surgeries in Yorkshire, UK. Patients were randomised 1:1 to receive study information alone or with the educational leaflet, with an invitation to attend for a screening examination by a dermatologist and rheumatologist. Nine hundred thirty-two invitation packs were sent to recruit 191 (20.5%) participants. One hundred sixty-nine (88.5%) had current or previous psoriasis and 17 (10.1%) had previously undiagnosed PsA. The estimated prevalence of PsA was 18.1% (95% CI: 16.2, 20.1%).The response rate was lower than expected and was not significantly higher when patients received the educational leaflet (22.8 vs 18.3%, p = 0.08). Response rates varied by practice (14.7 to 30.6%). However, deprivation scores for each practice revealed a significant increase in response with the leaflet for deprivation decile of 3 (p < 0.001) but no significant differences in the other practices. An educational leaflet about PsA improves attendance for screening in primary care, but only in those practices with higher levels of socioeconomic deprivation.


Phthalate-Induced Fetal Leydig Cell Dysfunction Mediates Male Reproductive Tract Anomalies.
Male fetal Leydig cells in the testis secrete androgen and insulin-like 3, determining the sexual differentiation. The abnormal development of fetal Leydig cells could lead to the reduction of androgen and insulin-like 3, thus causing the male reproductive tract anomalies in male neonates, including cryptorchidism and hypospadias. Environmental pollutants, such as phthalic acid esters (phthalates), can perturb the development and differentiated function of Leydig cells, thereby contributing to the reproductive toxicity in the male. Here, we review the epidemiological studies in humans and experimental investigations in rodents of various phthalates. Most of phthalates disturb the expression of various genes encoded for steroidogenesis-related proteins and insulin-like 3 in fetal Leydig cells and the dose-additive effects are exerted after exposure in a mixture.


Electric-field-induced quantum coherence of the intersubband transition in semiconductor quantum wells.
An intense coherent laser field couples two electron subbands in quantum wells. Using generalized self-consistent density functional theory, we study the nature of the Coulomb and light-induced electronic renormalization. We show electric-field-induced transparency and light amplification without population inversion of a weak probe beam in such a system. The effects of the pump field strength, electron density, and delta-doping position are investigated.


Cystic Echinococcosis in Three Locations in the Middle Atlas, Morocco: Estimation of the Infection Rate in the Dog Reservoir.
A longitudinal study was carried out in Middle atlas, Morocco (locality of Had Oued Ifrane) in a population of 255 dogs from three localities, including two categories of dogs (owned and stray dogs). The dogs were investigated three times over a period ranging from 4 to 8 months between December and August. At each investigation, dogs were treated with arecoline, inducing defecation and allowing feces collection. Dogs were further treated with praziquantel to clear them from Echinococcus granulosus. Microscopic examination of feces was performed to assess the infection status of dogs at each investigation, and positive samples underwent copro-PCR to determine the circulating strain of E. granulosus. A high prevalence of infestation ranging from 23.5% to 38.8% and from 51.3% to 68.5% was, respectively, found in owned and in stray dogs. The PCR results revealed the presence of G1 strain in all positive samples. A logistic regression model was used to determine the incidence of infestation and showed that stray dogs underwent a significantly higher risk of infection (odds ratio = 14; 95% confidence interval: 6-30; p < 0.001) compared with owned dogs. Only anthelmintic treatment intervals of 2 months efficiently prevented egg shedding in owned and stray dogs. The seasonal effect was also significant, with the highest risk of reinfestation in winter and the lowest risk in summer. This study confirms that stray dogs undergo an increased risk of infestation by E. granulosus and indicate that infective pressure is influenced by season.


Risks to normal tissues from radionuclide therapy.
The development of radionuclide therapies during the past few decades provides a growing body of data on radiobiologic effects, including normal tissue toxicities and antitumor efficacy. Information on normal tissue toxicity from radionuclides is more limited than that from external beam radiation and appears to be more variable. Much of the increased variability is attributed to heterogeneous distribution, which complicates the potential for whole-organ toxicity, and the differences in dosimetry methodology. Although new tools are becoming available, quantitation of heterogeneous dose for radionuclides is usually less precise than dosimetry that is used in external beam radiation practice. The correlation between reported dose estimates and toxicity has improved during the past 2 decades, partly as the result of increased accuracy and standardization of dosimetry techniques and to adjustment for biologic effects. This review provides an updated compendium of dose-response relationships and consideration of dosimetry as well as radiobiologic factors that influence the reported results. Data presented are mainly derived from studies involving deliver of radiation to adults with malignancies, with most experience from radionuclides that predominantly emit beta radiation.


High rate of unintended pregnancies after knowing of HIV infection among HIV positive women under antiretroviral treatment in Kigali, Rwanda.
More than 90% of pediatric AIDS results from the transmission of the virus through HIV positive pregnant mothers to their children. However, little has been known about factors associated with unintended pregnancies after knowing their HIV seropositive status, or contraceptive use among HIV positive women under antiretroviral treatment (ART). We investigated thus factors associated with unintended pregnancies after knowing seropositive status, and also factors associated with the non-use of contraceptives among HIV positive women under ART. We carried out a cross-sectional study in Kigali, Rwanda in 2007. A total of 565 HIV positive women under ART were interviewed. We examined the associated factors of unintended pregnancies or non-use of contraceptives using logistic regression analysis. Among all the respondents (n = 565), 132 women became pregnant after knowing their HIV seropositive status. Among them, 82 (62.7%) got pregnant unintentionally. Those who had two or more children (adjusted OR, 3.83) were more likely to get pregnant unintentionally. Meanwhile, among all, 263 had sexual intercourse during the last three months. Of them, 85 women did not use any contraceptives. Those who did not agree that 'HIV positive children can survive as long as HIV negative children' (adjusted OR, 2.28), and those who 'can always ask partner to use a condom' (adjusted OR, 9.83), were more likely to use contraceptives. This study suggests that HIV positive women under ART need special support to avoid unintended pregnancies especially those who have two or more children. Moreover, interventions are also needed to improve women's understanding of the prognosis of pediatric AIDS, and condom-use negotiation skills.


Intrinsic positive end-expiratory pressure in ambulatory patients with airways obstruction.
In patients with severe expiratory airflow limitation, dynamic hyperinflation often occurs when inspiratory efforts are initiated at a thoracic volume above the relaxation point of the respiratory system. The result is intrinsic positive end-expiratory alveolar pressure (PEEPi). To determine whether PEEPi occurs in ambulatory patients, we measured alveolar pressure (Palv) noninvasively during tidal breathing in 8 normal subjects, 15 asthmatic subjects, and 19 patients with COPD, using a body plethysmographic technique that includes computerized corrections for nonlinear pneumotachometer output and for plethysmograph leakage. In all 8 normal subjects, 9 asthmatic subjects, and 3 COPD patients, Palv descended smoothly to zero at end expiration. In contrast, among each of the remaining 22 patients, there was an abrupt change in slope of the Palv tracing near end expiration, identifying the onset of the next inspiratory effort and indicating the presence of PEEPi, ranging from 0.2 to 9.5 cm H2O. PEEPi was significantly correlated with FRC (% of predicted); PEEPi = (0.040 x %FRC) - 3.65, r = 0.73, p < 0.001, and with the reciprocal of FEV1 (% of predicted), PEEPi = (138/%FEV1) - 1.34, r = 0.69, p < 0.001. PEEPi could be elicited in normal subjects by severe expiratory resistive loading but not by the increased expiratory muscle activity occurring during an MVV maneuver. We conclude that PEEPi is common in patients with airways obstruction, even without overt ventilatory failure, and that its severity is generally in proportion to the severity of the hyperinflation and the airways obstruction.


Classic Kaposi's sarcoma in Morocco: clinico-epidemiological study at the National Institute of Oncology.
Classic Kaposi's sarcoma (CKS) is a rare disease likely associated with human herpes virus 8 (HHV-8) infection, and occurs predominantly in Jewish, Mediterranean and middle eastern men. There is a dearth of data in Moroccan patients with CKS regarding epidemiology, clinical characteristics and outcomes. This report examines a cohort of patients with CKS evaluated at the national institute of oncology over 11-year period. A retrospective analysis of patients referred to the national institute of oncology with classical Kaposi sarcoma, between January 1998 and February 2008, was performed. Reviewed information included demographics, clinical and pathological staging, death or last follow-up. During the study period, 56 patients with a diagnosis of CKS have been referred to our hospital. There were 11 (19.7%) females and 45 (80.3%) males (male-to-female ratio: 4:1). Mean age at diagnosis was 61.7 ± 15 (range: 15-86 years). Nodules and/or plaques were the most frequent type of lesion. The most common location was the lower limbs, particularly the distal lower extremity (90%). In addition to skin involvement, visceral spread was evident in 9 cases. The most common visceral involvement sites were lymph nodes (44%), lung (22%), and gastrointestinal tract (22%). Associated lymphoedema was seen in 24 (42%) of the patients. There were 18 stage I patients (32.14%), 8: stage II (14.28%), 21 stage III (37.5%) and 9 stage IV (16.07%). A second primary malignancy was diagnosed in 6 cases (10.7%), none of the reticuloendothelial system. With a median follow-up of 45 months, 38 (67.8) patients are alive, of whom 25 (65.78%) patients with stable disease, five with progressive disease currently under systemic chemotherapy and 8 (21.05%) are alive and free of disease, over a mean interval of 5 years. This is the largest reported series in our context. In Morocco, CKS exhibits some special characteristics including a disseminated skin disease at diagnosis especially in men, a more common visceral or lymph node involvement and a less frequent association with second malignancies.


Protease activity as a prognostic factor for wound healing in venous leg ulcers.
Venous leg ulcers (VLUs) are a common type of complex wound that have a negative impact on people's lives and incur high costs for health services and society. It has been suggested that prolonged high levels of protease activity in the later stages of the healing of chronic wounds may be associated with delayed healing. Protease modulating treatments have been developed which seek to modulate protease activity and thereby promote healing in chronic wounds. To determine whether protease activity is an independent prognostic factor for the healing of venous leg ulcers. In February 2018, we searched the following databases: Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, Ovid Embase and CINAHL. We included prospective and retrospective longitudinal studies with any follow-up period that recruited people with VLUs and investigated whether protease activity in wound fluid was associated with future healing of VLUs. We included randomised controlled trials (RCTs) analysed as cohort studies, provided interventions were taken into account in the analysis, and case-control studies if there were no available cohort studies. We also included prediction model studies provided they reported separately associations of individual prognostic factors (protease activity) with healing. Studies of any type of protease or combination of proteases were eligible, including proteases from bacteria, and the prognostic factor could be examined as a continuous or categorical variable; any cut-off point was permitted. The primary outcomes were time to healing (survival analysis) and the proportion of people with ulcers completely healed; the secondary outcome was change in ulcer size/rate of wound closure. We extracted unadjusted (simple) and adjusted (multivariable) associations between the prognostic factor and healing. Two review authors independently assessed studies for inclusion at each stage, and undertook data extraction, assessment of risk of bias and GRADE assessment. We collected association statistics where available. No study reported adjusted analyses: instead we collected unadjusted results or calculated association measures from raw data. We calculated risk ratios when both outcome and prognostic factor were dichotomous variables. When the prognostic factor was reported as continuous data and healing outcomes were dichotomous, we either performed regression analysis or analysed the impact of healing on protease levels, analysing as the standardised mean difference. When both prognostic factor and outcome were continuous data, we reported correlation coefficients or calculated them from individual participant data.We displayed all results on forest plots to give an overall visual representation. We planned to conduct meta-analyses where this was appropriate, otherwise we summarised narratively. We included 19 studies comprising 21 cohorts involving 646 participants. Only 11 studies (13 cohorts, 522 participants) had data available for analysis. Of these, five were prospective cohort studies, four were RCTs and two had a type of case-control design. Follow-up time ranged from four to 36 weeks. Studies covered 10 different matrix metalloproteases (MMPs) and two serine proteases (human neutrophil elastase and urokinase-type plasminogen activators). Two studies recorded complete healing as an outcome; other studies recorded partial healing measures. There was clinical and methodological heterogeneity across studies; for example, in the definition of healing, the type of protease and its measurement, the distribution of active and bound protease species, the types of treatment and the reporting of results. Therefore, meta-analysis was not performed. No study had conducted multivariable analyses and all included evidence was of very low certainty because of the lack of adjustment for confounders, the high risk of bias for all studies except one, imprecision around the measures of association and inconsistency in the direction of association. Collectively the research indicated complete uncertainty as to the association between protease activity and VLU healing. This review identified very low validity evidence regarding any association between protease activity and VLU healing and there is complete uncertainty regarding the relationship. The review offers information for both future research and systematic review methodology.


Cognitive performance in healthy older adults relates to spontaneous switching between states of functional connectivity during rest.
Growing evidence has shown that brain activity at rest slowly wanders through a repertoire of different states, where whole-brain functional connectivity (FC) temporarily settles into distinct FC patterns. Nevertheless, the functional role of resting-state activity remains unclear. Here, we investigate how the switching behavior of resting-state FC relates with cognitive performance in healthy older adults. We analyse resting-state fMRI data from 98 healthy adults previously categorized as being among the best or among the worst performers in a cohort study of >1000 subjects aged 50+ who underwent neuropsychological assessment. We use a novel approach focusing on the dominant FC pattern captured by the leading eigenvector of dynamic FC matrices. Recurrent FC patterns - or states - are detected and characterized in terms of lifetime, probability of occurrence and switching profiles. We find that poorer cognitive performance is associated with weaker FC temporal similarity together with altered switching between FC states. These results provide new evidence linking the switching dynamics of FC during rest with cognitive performance in later life, reinforcing the functional role of resting-state activity for effective cognitive processing.


Leukocyte transcriptome from chickens infected with avian pathogenic Escherichia coli identifies pathways associated with resistance.
Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which is responsible for morbidity and mortality in chickens. Gene expression patterns have previously been demonstrated to differ between chicken populations that are resistant vs. susceptible to bacterial infection, but little is currently known about gene expression response to APEC. Increased understanding of gene expression patterns associated with resistance will facilitate genetic selection to increase resistance to APEC. Male broiler chicks were vaccinated at 2 weeks of age and challenged with APEC at 4 weeks of age. Peripheral blood leukocytes were collected at 1 and 5 day post-infection. Lesions on the liver, pericardium, and air sacs were used to assign a mild or severe pathology status to non-vaccinated, challenged chicks. Ten treatment groups were therefore generated with a priori factors of vaccination, challenge, day post-infection, and the a posteriori factor of pathology status. Global transcriptomic response was evaluated using the Agilent 44K chicken microarray. APEC infection resulted in more up-regulation than down-regulation of differentially expressed genes. Immune response and metabolic processes were enriched with differentially expressed genes. Although vaccination significantly reduced lesions in challenged bird, there was no detectable effect of vaccination on gene expression. This study investigated the transcriptomic differences in host responses associated with mild vs. severe pathology, in addition to the effects of vaccination and challenge, thus revealing genes and networks associated with response to APEC and providing a foundation for future studies on, and genetic selection for, genetic resistance to APEC.


Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase.
Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)](i) mobilization, thromboxane A(2) (TxA(2)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH(●)) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA(2) cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.


Autism in a recently arrived immigrant population.
This study aims to establish whether children of an immigrant maternal population presented with a higher rate of autism than the indigenous population and to explore their presentation with regard to severity of symptoms, demographics and ethnicity. It is a retrospective case note analysis of 366 children who presented to the paediatric developmental service in the Adelaide and Meath incorporating the National Children's Hospital, Tallaght, Ireland between 2007 and 2009. During the study period, 366 children presented. Fifty-eight children (16 %) had mothers who were born in Africa and 53 (14 %) were born to mothers originating from a wider variety of countries. Two hundred and forty-eight children (68 %) had mothers born in Ireland. Maternal origin was not identified for seven children (2 %). An autistic spectrum disorder (ASD) was diagnosed in 131 children and speech and language delay in 132. Of the children with an ASD diagnosis, a higher proportion of the African cohort 13/18 (72.2 %) presented with moderate/severe cognitive disability compared to the Irish group 9/55(16.3 %), and the children in the African cohort showed a higher heritability with 36.9 % having a positive family history of autism reported compared to 26.3 % of the Irish cohort with an ASD diagnosis. This study highlights an observation of increased rates of ASD among a migrant population derived particularly from children born to mothers originating in Sub-Saharan Africa. This cohort is more severely affected. Further validation in an epidemiological sample is warranted, which if replicated, may help to identify possible aetiological risk factors.


Impact of Porosity and Electrolyte Composition on the Surface Charge of Hydroxyapatite Biomaterials.
The success or failure of a material when implanted in the body is greatly determined by the surface properties of the material and the host tissue reactions. The very first event that takes place after implantation is the interaction of soluble ions, molecules and proteins from the biological environment with the material surface leading to the formation of an adsorbed protein layer that will later influence cell attachment. In this context, the particular topography and surface charge of a material become critical as they influence the nature of the proteins that will adsorb. However, very limited information is available on the surface charge of porous substrates. Only until very recently was the determination of the zeta potential on porous membranes accurately determined. The goal of this work was to implement the previous findings for the determination of the zeta potential of a series of porous hydroxyapatite (HA) substrates and to assess how porosity affects the measurements. In addition, studies using various electrolytes were also performed to prove how the specific affinity of certain ions for HA can further impact surface charge. The results showed that all materials exhibited very similar external surface charge (approximately -23 mV), consistent with their almost identical topographies. However, the presence of interconnected pores underneath the sample surface resulted in an additional internal zeta potential that varied with the porosity content. Measurements with different electrolytes confirmed the selectivity of divalent ions for HA underlying the importance of testing biomaterials using relevant electrolytes.


Visual Triggers of Skin Picking Episodes: An Experimental Study in Self-Reported Skin Picking Disorder and Atopic Dermatitis.
Skin Picking Disorder (SPD) is a new diagnosis with limited information available about triggers of picking episodes. Itch can be induced via audio-visual stimuli and the effect of contagious itch is stronger for those affected by atopic dermatitis. We examined if picking-related visual stimuli can trigger the urge to pick skin in self-reported SPD. We compared itch and the urge to pick in a sample of AD and/or SPD-affected to controls without either. Urge to pick skin and/or scratch when viewing 24 itch-related, picking-related or neutral online pictures was assessed in adult females, who self-report skin-picking (SPD-only, n = 147) and/or atopic dermatitis (AD-only, n = 47; AD+SPD, n = 46) as well as in skin healthy controls (HC, n = 361). All participants reported a stronger urge to pick for picking-related pictures compared to neutral content (F[1, 597] = 533.96, p < .001, ηp2 = .472) and more itch for itch-related pictures compared to neutral stimuli (F[1, 597] = 518.73, p < .001, ηp2 = .465). SPD-all (SPD-only & AD+SPD) reported stronger urges to pick for picking-related vs. other stimuli compared to the AD-only and HC group (p < .001, ηp2 = .047). Likewise, AD-all (AD-only & AD+SPD) reported significantly stronger itching for itch-related vs. other stimuli compared to SPD-only and HC (p = .001, ηp2 = .019). Analog to visual provocation of itch, the urge to pick can be triggered by visual stimuli. Treatments for SPD and AD may profit from addressing visual stimuli.


Achieving sequential therapy in advanced gastric cancer: the importance of appropriate patient management for the elderly and/or those with ascites.
Treatment options for patients with advanced gastric cancer (AGC) are limited. One approach to improving survival in patients with AGC is to optimize the available agents via sequential therapy. However, clinical trial reports of first-line chemotherapy indicate that elderly patients and patients with massive ascites are less likely to receive subsequent lines of therapy. In addition, clinical trials of second- and third-line chemotherapy generally exclude these two patient populations because they are likely to have poor performance status and additional issues that are difficult to manage. Good patient management is likely to be key to the successful use of sequential therapy in these two patient populations by minimizing adverse effects to allow patients to derive benefit from the additional treatment. This narrative review summarizes the available information on AGC treatment and patient management in elderly patients and patients with massive ascites. The available data suggest that elderly patients benefit from chemotherapy; however, monitoring toxicity is essential to avoid chemotherapy-related toxicities. Important aspects of patient management for elderly patients include symptom monitoring, nutritional support, and fall prevention. The available data for patients with massive ascites show limited success for a range of treatment approaches, including systemic chemotherapy. The management of ascites is also challenging, with no clear guidance on the preferred strategies. To address these gaps in knowledge, future clinical trials should incorporate more inclusive eligibility criteria to enroll populations of patients with AGC that are more reflective of the real-world population with respect to age, complications, and overall health status.


Estimating a mosquito repellent's potential to reduce malaria in communities.
Probability models for assessing a mosquito repellent's potential to reduce malaria transmission are not readily available to public health researchers. To provide a means for estimating the epidemiological efficacy of mosquito repellents in communities, we developed a simple mathematical model. A static probability model is presented to simulate malaria infection in a community during a single transmission season. The model includes five parameters- sporozoite rate, human infection rate, biting pressure, repellent efficacy, and product-acceptance rate. The model assumes that a certain percentage of the population uses a personal mosquito repellent over the course of a seven-month transmission season and that this repellent maintains a constant rate of protective efficacy against the bites of malaria vectors. This model measures the probability of evading infection in circumstances where vector biting pressure, repellent efficacy, and product acceptance may vary. [corrected] Absolute protection using mosquito repellents alone requires high rates of repellent efficacy and product acceptance. [corrected] Using performance data from a highly effective repellent, the model estimates an 88.9% reduction of infections over a seven- month transmission season. A corresponding reduction in the incidence of super-infection in community members not completely evading infection can also be presumed. Thus, the model shows that mass distribution of a repellent with >98% efficacy and >98% product acceptance would suppress new malaria infections to levels lower than those achieved with insecticide treated nets (ITNs). A combination of both interventions could create synergies that result in reductions of disease burden significantly greater than with the use of ITNs alone.


Psychological Responses to High-Intensity Interval Training: A Comparison of Graded Walking and Ungraded Running at Equivalent Metabolic Loads.
High-intensity interval training (HIIT) is highly beneficial for health and fitness and is well tolerated. Treadmill-based HIIT normally includes running interspersed with walking. The purpose of this study was to compare ungraded running and graded walking HIIT on perceived exertion, affective valence, and enjoyment. Thirty-four active, healthy adults completed maximal testing and two 20-min HIIT trials alternating between 85% of VO2peak and a comfortable walking speed. Affective valence, enjoyment, and perceived exertion, both overall (ratings of perceived exertion [RPE]-O) and legs only (RPE-L), were measured. RPE-O and affective valence were similar between HIIT trials (p > .05), RPE-L was higher for walk HIIT (p < .05), and enjoyment was higher for run HIIT (p < .05). Findings indicate that both walk and run HIIT produce exertion, affective, and enjoyment responses that are positive and possibly supportive of exercise behavior. Walk HIIT may be desirable for individuals who are unable or do not want to run.


Acute intermittent hypoxia drives hepatic de novo lipogenesis in humans and rodents.
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition. It is tightly associated with an adverse metabolic phenotype (including obesity and type 2 diabetes) as well as with obstructive sleep apnoea (OSA) of which intermittent hypoxia is a critical component. Hepatic de novo lipogenesis (DNL) is a significant contributor to hepatic lipid content and the pathogenesis of NAFLD and has been proposed as a key pathway to target in the development of pharmacotherapies to treat NAFLD. Our aim is to use experimental models to investigate the impact of hypoxia on hepatic lipid metabolism independent of obesity and metabolic disease. Human and rodent studies incorporating stable isotopes and hyperinsulinaemic euglycaemic clamp studies were performed to assess the regulation of DNL and broader metabolic phenotype by intermittent hypoxia. Cell-based studies, including pharmacological and genetic manipulation of hypoxia-inducible factors (HIF), were used to examine the underlying mechanisms. Hepatic DNL increased in response to acute intermittent hypoxia in humans, without alteration in glucose production or disposal. These observations were endorsed in a prolonged model of intermittent hypoxia in rodents using stable isotopic assessment of lipid metabolism. Changes in DNL were paralleled by increases in hepatic gene expression of acetyl CoA carboxylase 1 and fatty acid synthase. In human hepatoma cell lines, hypoxia increased both DNL and fatty acid uptake through HIF-1α and -2α dependent mechanisms. These studies provide robust evidence linking intermittent hypoxia and the regulation of DNL in both acute and sustained in vivo models of intermittent hypoxia, providing an important mechanistic link between hypoxia and NAFLD.


Systems level analysis of two-component signal transduction systems in Erwinia amylovora: role in virulence, regulation of amylovoran biosynthesis and swarming motility.
Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied. We used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified. Our results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.


The activation mechanism of Hsp26 does not require dissociation of the oligomer.
Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.


Becoming a written word: eye movements reveal order of acquisition effects following incidental exposure to new words during silent reading.
We know that from mid-childhood onwards most new words are learned implicitly via reading; however, most word learning studies have taught novel items explicitly. We examined incidental word learning during reading by focusing on the well-documented finding that words which are acquired early in life are processed more quickly than those acquired later. Novel words were embedded in meaningful sentences and were presented to adult readers early (day 1) or later (day 2) during a five-day exposure phase. At test adults read the novel words in semantically neutral sentences. Participants' eye movements were monitored throughout exposure and test. Adults also completed a surprise memory test in which they had to match each novel word with its definition. Results showed a decrease in reading times for all novel words over exposure, and significantly shorter [corrected] total reading times at test for early than late novel words. Early-presented novel words were also remembered better in the offline test. Our results show that order of presentation influences processing time early in the course of acquiring a new word, consistent with partial and incremental growth in knowledge occurring as a function of an individual's experience with each word.


Correction for specimen movement and rotation errors for in-vivo Optical Projection Tomography.
The application of optical projection tomography to in-vivo experiments is limited by specimen movement during the acquisition. We present a set of mathematical correction methods applied to the acquired data stacks to correct for movement in both directions of the image plane. These methods have been applied to correct experimental data taken from in-vivo optical projection tomography experiments in Caenorhabditis elegans. Successful reconstructions for both fluorescence and white light (absorption) measurements are shown. Since no difference between movement of the animal and movement of the rotation axis is made, this approach at the same time removes artifacts due to mechanical drifts and errors in the assumed center of rotation.


Genetic background of carcinogenesis in the thyroid gland.
The process of carcinogenesis is permanently one of the most interesting and significant issues for researchers in different fields of medicine. Therefore, we attempted to bring closer the problem of neoplastic transformation in the thyroid gland. This article covers the latest data about genetic factors, involved in thyroid carcinogenesis. We have presented results of the most recent studies referred to molecular biology of thyroid neoplasms. We have demonstrated not only the genetic background of cancers, derived from the thyroid follicular cell, but also genetic aspects related to medullary thyroid carcinoma and some benign thyroid lesions. The review describes DNA methylation disturbances and the mutations in thyrotropin receptor and G protein genes. Furthermore, we introduce the results of studies performed at our laboratory, concerning mutations in the following protooncogenes: RAS, RET, Trk, MET, and BRAF. Also, we present our data, regarding the loss of heterozygosity (LOH) in the short arm of chromosome 3. Additionally, we discuss overexpression of cyclin D1 gene in benign and malignant thyroid lesions. Previous studies performed at our laboratory indicate the role of IGF-I in the pathogenesis and invasiveness of thyroid cancers. The review indicates that progress in genetics of the thyroid cancer is extremely rapid.


Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide.
The therapeutic potential of glucagon-like peptide-1 (GLP-1) in improving glycaemic control in diabetes has been widely studied, but the potential beneficial effects of glucose-dependent insulinotropic polypeptide (GIP) have until recently been almost overlooked. One of the major problems, however, in exploiting either GIP or GLP-1 as potential therapeutic agents is their short duration of action, due to enzymatic degradation in vivo by dipeptidylpeptidase IV (DPP IV). Therefore, this study examined the plasma stability, biological activity and antidiabetic potential of two novel NH2-terminal Ala2-substituted analogues of GIP, containing glycine (Gly) or serine (Ser). Following incubation in plasma, (Ser2)GIP had a reduced hydrolysis rate compared with native GIP, while (Gly2)GIP was completely stable. In Chinese hamster lung fibroblasts stably transfected with the human GIP receptor, GIP, (Gly2)GIP and (Ser2)GIP stimulated cAMP production with EC(50) values of 18.2, 14.9 and 15.0 nM respectively. In the pancreatic BRIN-BD11 beta-cell line, (Gly2)GIP and (Ser2)GIP (10(-8) M) evoked significant increases (1.2- and 1.5-fold respectively; P<0.01 to P<0.001) in insulinotropic activity compared with GIP. In obese diabetic ob/ob mice, both analogues significantly lowered (P<0.001) the glycaemic excursion in response to i.p. glucose. This enhanced glucose-lowering ability was coupled to a significantly raised (P<0.01) and more protracted insulin response compared with GIP. These data indicate that substitution of the penultimate Ala2 in GIP by Gly or Ser confers resistance to plasma DPP IV degradation, resulting in enhanced biological activity, therefore raising the possibility of their use in the treatment of type 2 diabetes.


Comparison of peripheral metabolic effects of insulin and metformin following severe burn injury.
Both insulin and metformin have been shown to attenuate hyperglycemia and reduce net muscle protein catabolism following burn injury. The purpose of this study was to compare the peripheral metabolic effects of insulin and metformin in severe burn patients. Six adult patients with burns greater than 40% of their body surface underwent metabolic evaluation utilizing isotopic dilution of phenylalanine, femoral arterial and venous blood sampling, and sequential biopsies of leg muscle. Following baseline measurements, insulin was infused into the femoral artery at 0.45 mIU/min 100 mL leg volume. Patients were then given metformin (850 mg every 8 hours) for seven days with repeat metabolic evaluation before and during intra-arterial infusion of insulin. Intra-arterial administration of insulin significantly increased insulin concentrations within the femoral vein, creating hyperinsulinemia localized to the extremity. Metformin had no significant effect on either peripheral glucose clearance or the rate of glucose oxidation. Furthermore, the availability of ATP and energy charge within muscle was not overtly affected by either insulin or metformin. Metformin did significantly increase the fractional synthetic rate of muscle protein which increased even further with insulin administration. Both metformin and insulin separately increased the rate of muscle protein synthesis as calculated using three compartment modeling. This study demonstrates a significant anabolic effect on muscle protein with metformin and a modest response with insulin. Findings also suggest that metformin and insulin may work synergistically to further improve muscle protein kinetics.


Expression of bcl-2 protein predicts efficacy of adjuvant treatments in operable node-positive breast cancer.
The proto-oncogene bcl-2 encodes a protein that inhibits apoptosis, a common mechanism of cell death caused by hormone and chemotherapy. We have analyzed bcl-2 protein expression by immuno-cytochemistry in primary node-positive breast cancers in two groups of patients (for a total of 180 cases). One group received adjuvant hormone therapy, the other chemotherapy (cyclophosphamide, methotrexate, and fluorouracil), and both groups were followed for a median time of 63 months. We compared our findings with conventional clinicopathological indicators [menopausal status, number of axillary nodes, histological grade, tumor size and type, estrogen receptor (ER), and progesterone receptor] and with p53 protein expression. bcl-2 protein was present in 65% of the carcinomas (117/180) and it was significantly associated with ER and progesterone receptor and inversely associated with p53 in both the groups of patients treated with adjuvant chemotherapy and tamoxifen. In patients treated either with adjuvant chemotherapy or tamoxifen, relapse-free survival at 5 years was significantly better among patients with bcl-2-positive tumors than in those with bcl-2 negative ones (P = 0.05 and 0.02, respectively). As far as overall survival is concerned, patients with bcl-2-positive tumors had a significantly better outcome in the group treated with adjuvant chemotherapy (P = 0.03). Multivariate analyses were performed for the two treatment groups. In the group treated with tamoxifen, lack of expression of ER and of bcl-2 was the only significant and independent predictor for poor relapse-free survival (P < 0.01). A number of nodes above 3 was the only significant and independent predictor for poor overall survival (P < 0.01). In the cyclophosphamide-methotrexate-fluorouracil-treated group, bcl-2 absence was significant for poor overall survival (P = 0.02) as well as a number of nodes above 3 (P = 0.04) and a tumor size above 2 cm (P = 0.05). For poor relapse-free survival only a number of nodes above 3 (P < 0.01) and progesterone negativity (P = 0.02) were significant and independent predictors of a higher probability of relapse. Thus, in contrast to in vitro data on drug resistance, bcl-2 expression was associated with better outcomes in patients treated with hormone and chemotherapy. Overall, these results suggest that expression of bcl-2 protein and the number of metastatic lymph nodes are independent features predictive of clinical outcome in patients with node-positive breast cancer, irrespective of the type of adjuvant treatment. The determination of bcl-2 protein may prove to be a useful tool to distinguish patients for whom conventional forms of adjuvant therapy are beneficial from those with bcl-2 negative and ER-negative tumors for whom novel therapeutic strategies are needed.


Inducibility of drug-metabolizing enzymes by xenobiotics in mice with liver-specific knockout of Ctnnb1.
Basal as well as xenobiotic-induced expression of the main enzymes from phase I and phase II of drug metabolism is confined to the perivenous areas of the mammalian liver lobule. Whereas signal transduction pathways that govern xenobiotic-induced expression of these enzymes via ligand-activated transcription factors such as constitutive androstane receptor (CAR) or the aryl hydrocarbon receptor (AhR) have been intensively studied, the mechanisms regulating zone-specific basal expression of genes related to drug metabolism and preferential response of perivenous hepatocytes to xenobiotic inducers are still largely unknown. Recent publications by our and other groups point to an important role for the Wnt/beta-catenin pathway in the maintenance of the perivenous hepatocyte gene expression profile including the main hepatic detoxification enzymes, and beta-catenin signaling was recently implicated in the expression of several cytochrome P450 isoenzymes. To analyze, whether the beta-catenin pathway would also affect inducible expression of drug-metabolizing enzymes, mice with liver-specific knockout of the Ctnnb1 gene (encoding beta-catenin) were treated with different model inducers of xenobiotic metabolism. Knockout of beta-catenin led to alterations in basal expression of most drug metabolism-related genes analyzed and resulted in strongly diminished responses to agonists of CAR-, AhR-, and nuclear factor erythroid-related factor 2-dependent transcription. Taken together, the data presented in this study indicate that beta-catenin not only regulates basal expression of drug-metabolizing enzymes but also determines the magnitude and hepatic localization of response to xenobiotic inducers in vivo.


Transient receptor potential canonical-3 channel-dependent fibroblast regulation in atrial fibrillation.
Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca(2+)-permeable transient receptor potential canonical-3 (TRPC3) channels. Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (I(NSC)) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca(2+) influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca(2+) removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5' promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca(2+) influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.


Toxic Effects of the Mixture of Phthalates and Bisphenol A-Subacute Oral Toxicity Study in Wistar Rats.
Phthalates and bisphenol A, classified as endocrine disruptors, have weak estrogenic, anti-androgenic properties, and affect thyroid hormone regulation. The aim of this study on male rats was to compare the subacute toxic effects of low doses of single compounds (bis (2 -ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and bisphenol A (BPA)) with the effects of their mixture through different biochemical, hormonal, and hematological parameters. Rats were divided into five experimental groups: Control (corn oil), DEHP (50 mg/kg b.w./day), DBP (50 mg/kg b.w./day), BPA (25 mg/kg b.w./day), and MIX (50 mg/kg b.w./day DEHP + 50 mg/kg b.w/day DBP + 25 mg/kg b.w./day BPA). Animals were sacrificed after 28 days of oral treatment and blood was collected for further analysis. The results demonstrated that the mixture produced significant changes in lipid profile, liver-related biochemical parameters, and glucose level. Furthermore, the opposite effects of single substances on the thyroxine level have been shown in comparison with the mixture, as well as a more pronounced effect of the mixture on testosterone level. This study contributes to the body of knowledge on the toxicology of mixtures and gives one more evidence of the paramount importance of mixture toxicity studies, especially in assessing the endocrine disruptive effects of chemicals.