The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.
The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.


Seasonal changes in circulating gonadal steroid levels and physiological evidence for the presence of intrinsic circannual reproductive cycles in captive finless porpoises Neophocaena asiaeorientalis from the western Inland Sea, Japan.
We monitored annual fluctuations of gonadal steroid levels in three sexually mature captive finless porpoises (Neophocaena asiaeorientalis; two males and one female) from two different facilities over 56-91 months. Two animals (one male and one female) were held in an indoor tank with a sunroof (facility A) and the other male was held in an indoor tank without a sunroof (facility B). Water temperatures in both facilities reflected seasonal changes during the study period with a minor difference in the fluctuation pattern. Testosterone levels of the male in facility A were higher from spring to summer every year and exhibited a 12-month cycle. The female showed estrus cycles in 1-month intervals from summer to winter, excluding 2 anestrus years. In contrast, the period of higher testosterone levels of the male in facility B gradually initiated earlier over the years under a constant photoperiod (11.5L:12.5D) and exhibited a 9-month cycle during the first 52 months. After changing the light conditions to a natural photoperiod, its testosterone levels were high from early spring to summer for 3 consecutive years and exhibited a 12-month cycle. Our results showed that under a constant artificial photoperiod, the male in facility B failed to recognize the seasonal changes of a natural external environment, resulting in a 9-month, free-running hormone cycle.


Mutational analysis of conserved residues in HhaI DNA methyltransferase.
HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.


The Perioperative Nurse Surgeon's Assistant: Issues and challenges associated with this emerging advanced practice nursing role in Australia.
The aim of this study was to obtain Perioperative Nurse Surgeon's Assistants' views about their emerging new role in contemporary nursing practice in Australia. Internationally advanced practice nursing has led to a range of specialist roles aimed at delivering higher quality, efficient nursing care. In 2005 an Australian university developed the Perioperative Nurse Surgeon's Assistant graduate education and training program to provide nurses with an opportunity to gain advanced practice knowledge and extended skills specifically in the perioperative setting. This study was a qualitative research design that used online surveys and in-depth interviews to explore the issues and challenges associated with the introduction of the (currently non-accredited) Perioperative Nurse Surgeon's Assistant role in Australia. Experienced Australia perioperative nurses who had undertaken graduate education and training in this field were recruited for this study. Data were collected between August and October 2011. An inductive thematic analysis was used to interpret the findings. Eighteen nurses completed the online survey and six were interviewed (n = 24). Nurses cited their commitment to professional development and the delivery of high quality patient care, along with surgeons' encouragement for them to complete specialist clinical training, as key reasons for undertaking Perioperative Nurse Surgeon's Assistant education and training. The Perioperative Nurse Surgeon's Assistant role led to greater job satisfaction and autonomy, and assisted nurses to better meet the needs of patients, surgeons and clinical perioperative teams. Without formal recognition of the Perioperative Nurse Surgeon's Assistant role its future in the Australian health care system is under threat.


Whole-body cryotherapy - promising add-on treatment of depressive disorders.
New, effective biological interventions for treatment of depressive episodes and recurrent depression are still needed. Whole-body cryotherapy (WBC), which is a treatment using cryogenic temperature, is a novel therapeutic modality in neurology and rheumatology. The objective of this study was to determine the efficacy and safety of WBC as an add-on treatment for depressive episode. 30 adults diagnosed with depressive episode were recruited to an observational, prospective study. 21 participants (17 women, 81%), mean age 46.1 (±16.7), completed the whole study procedure. The Hamilton Depression Rating Scale and the Beck Depression Inventory were used to assess the severity of depressive symptoms. Additionally, quality of life and anhedonia were assessed with the WHOQoL-BREF and the SHAPS. Participants undertook 10, 2-minute (from - 110 C to - 135 degrees C) WBC sessions within two weeks. Patients after WBC sessions showed significant improvement in the form of a reduction in total scores in scales assessing depressive symptoms: the HDRS (p< 0.00001) between T1 (16.94±4.3) and T4 (4.50±4.2) and the BDI-II (T1: 13.48±4.6; T4: 6.14±6.7, p<0.03), lower anhedonialevelon SHAPS (p =0.011) and higher quality of lifein thefollowing domains: physical health (p =0.024), psychological health (p =0.016) and environmental domain (p =0.003). Pre/post comparison of self-report well-being measured by the VAS scale showed a significant increase (p< 0.00001). It was shown that WBC have no effect on the level of cytokines, NO, hsCRP, ESR and TAS in blood (p> 0.05). WBC proved to be an effective, safe, and tolerable add-on intervention in patients with depressive episode. Further randomized controlled trials should be conducted.


Community-level effect of the reproductive health vouchers program on out-of-pocket spending on family planning and safe motherhood services in Kenya.
Although vouchers can protect individuals in low-income countries from financial catastrophe and impoverishment arising from out-of-pocket expenditures on healthcare, their effectiveness in achieving this goal depends on whether both service and transport costs are subsidized as well as other factors such as service availability in a given locality and community perceptions about the quality of care. This paper examines the community-level effect of the reproductive health vouchers program on out-of-pocket expenditure on family planning, antenatal, delivery and postnatal care services in Kenya. Data are from two rounds of cross-sectional household surveys in voucher and non-voucher sites. The first survey was conducted between May 2010 and July 2011 among 2,933 women aged 15-49 years while the second survey took place between July and October 2012 among 3,094 women of similar age groups. The effect of the program on out-of-pocket expenditure is determined by difference-in-differences estimation. Analysis entails comparison of changes in proportions, means and medians as well as estimation of multivariate linear regression models with interaction terms between indicators for study site (voucher or non-voucher) and period of study (2010-2011 or 2012). There were significantly greater declines in the proportions of women from voucher sites that paid for antenatal, delivery and postnatal care services at health facilities compared to those from non-voucher sites. The changes were also consistent with increased uptake of the safe motherhood voucher in intervention sites over time. There was, however, no significant difference in changes in the proportions of women from voucher and non-voucher sites that paid for family planning services. The results further show that there were significant differences in changes in the amount paid for family planning and antenatal care services by women from voucher compared to those from non-voucher sites. Although there were greater declines in the average amount paid for delivery and postnatal care services by women from voucher compared to those from non-voucher sites, the difference-in-differences estimates were not statistically significant. The reproductive health vouchers program in Kenya significantly contributed to reductions in the proportions of women in the community that paid out-of-pocket for safe motherhood services at health facilities.


Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9.
We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.


Light waves in thin films and integrated optics.
Integrated optics is a far-reaching attempt to apply thin-film technology to optical circuits and devices, and, by using methods of integrated circuitry, to achieve a better and more economical optical system. The specific topics discussed here are physics of light waves in thin films, materials and losses involved, methods of couplings light beam into and out of a thin film, and nonlinear interactions in waveguide structures. The purpose of this paper is to review in some detail the important development of this new and fascinating field, and to caution the reader that the technology involved is difficult because of the smallness and perfection demanded by thin-film optical devices.


Leiomyosarcoma: Principles of management.
The term soft-tissue sarcomas (STS) embraces more than 50 different sub-types that are often associated with poor prognosis. Only a very limited number of agents are active against STS. Doxorubicin and ifosfamide are widely accepted as the most effective compounds. However, their low response rates and poor impact on the overall survival of the patients illustrate the need for new treatment options. Among them, leiomyosarcomas are one of the most frequently occurring subtypes. In spite of the relatively high incidence of leiomyosarcomas, the overall effectiveness of the currently available systemic treatments is still poor. The heterogeneity of its biological origin, clinical behavior and responsiveness to chemotherapy, together with the scarcity of successful clinical trials, makes the treatment of leiomyosarcoma especially challenging. In addition, the evidence-based treatment for leiomyosarcoma comes from trials in which, in the majority of cases, no distinctions have been made among the different STS sub-types. As a result, every therapeutic decision should be made on an individual basis in collaboration with the patient. The results of new specific histology-designed clinical trials should aid decision making in this complex field.


From phenyl chlorides to α,n-didehydrotoluenes via phenyl cations. A CPCM-CASMP2 investigation.
Calculations with the complete active space self-consistent field (CASSCF) method were carried out for rationalizing the photochemical generation of the three isomeric didehydrotoluenes (DHTs) from the corresponding (n-chlorobenzyl)trimethylsilanes. Moreover, the original CASSCF energies were corrected through the introduction of the dynamic electron correlation term (at the MP2 level) and of an appropriate solvent model (CPCM). The work demonstrated the viability of intersystem crossing (conical intersection located) leading to the lowest lying triplet state of the silanes that fragments to give the corresponding triplet phenyl cations. The para- and ortho-isomers desilylate directly from such states of radical/radical cation character and yield the corresponding DHTs in their triplet state. Different from the other isomers, the meta-cation has a radical/radical cation structure in both spin states and thus two potential accesses to the different spin states of the corresponding DHT.


Combining multi-modality data for searching biomarkers in schizophrenia.
Identification of imaging biomarkers for schizophrenia is an important but still challenging problem. Even though considerable efforts have been made over the past decades, quantitative alterations between patients and healthy subjects have not yet provided a diagnostic measure with sufficient high sensitivity and specificity. One of the most important reasons is the lack of consistent findings, which is in part due to single-mode study, which only detects single dimensional information by each modality, and thus misses the most crucial differences between groups. Here, we hypothesize that multimodal integration of functional MRI (fMRI), structural MRI (sMRI), and diffusion tensor imaging (DTI) might yield more power for the diagnosis of schizophrenia. A novel multivariate data fusion method for combining these modalities is introduced without reducing the dimension or using the priors from 161 schizophrenia patients and 168 matched healthy controls. The multi-index feature for each ROI is constructed and summarized with Wilk's lambda by performing multivariate analysis of variance to calculate the significant difference between different groups. Our results show that, among these modalities, fMRI has the most significant featureby calculating the Jaccard similarity coefficient (0.7416) and Kappa index (0.4833). Furthermore, fusion of these modalities provides the most plentiful information and the highest predictive accuracy of 86.52%. This work indicates that multimodal integration can improve the ability of distinguishing differences between groups and might be assisting in further diagnosis of schizophrenia.


The Reach of the Gastrocnemius Musculocutaneous Flap: How High Is High?
Gastrocnemius muscle and musculocutaneous flaps are very versatile and one of the commonly used flaps for lower extremity reconstruction. There is significant literature available on the use of these flaps. However, we feel that the potential of the gastrocnemius musculocutaneous (GMC) flaps has not yet been fully explored in terms of increasing their reach, viability and arc of rotation. An attempt is made to refine the technique of flap harvestation to optimize outcomes of this versatile flap. Six patients of complex lower limb defects were managed using the GMC flaps. Harvesting of the flap was always initiated from the posterior midline to include the proximal sural pedicle, sural nerve, short saphenous vein and the muscle belly of either the medial or the lateral gastrocnemius muscle along with the cutaneous paddle. All the flaps were islanded and denervated. The origin of the gastrocnemius muscle was detached in all cases to increase the reach of this flap. The flap can reliably and comfortably cover defects from middle third-lower third junction of thigh and the entire posterior aspect of the thigh. Such a local option offers relatively simple but more cost-effective approach to complex clinical problem with tolerable impairment of the donor site. The GMC flap can be considered as a worthwhile alternative to free-tissue transfer for limb salvage.


Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma.
Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.


Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays.
Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.


Muscular strength after total hip arthroplasty. A prospective comparison of 3 surgical approaches.
Minimizing the decrease in muscular strength after total hip arthroplasty (THA) might allow patients to recover faster. We evaluated muscular strength in patients who were operated on using 3 surgical approaches. PATIENTS AND METHODs: In a prospective cohort study, 60 patients scheduled for primary THA were allocated to the direct lateral, posterior, or anterior approach. Leg press and abduction strength were evaluated 2 weeks or less preoperatively, 2 and 8 days postoperatively, and at 6-week and 3-month follow-up. Differences in maximal strength change were greatest after 2 and 8 days. The posterior and anterior approaches produced less decrease in muscular strength than the direct lateral approach. 6 weeks postoperatively, the posterior approach produced greater increase in muscular strength than the direct lateral approach, and resulted in a greater increase in abduction strength than the anterior approach. At 3-month follow-up, no statistically significant differences between the groups were found. The operated legs were 18% weaker in leg press and 15% weaker in abduction than the unoperated legs, and the results were similar between groups. The posterior and anterior approaches appeared to have the least negative effect on abduction and leg press muscular strength in the first postoperative week; the posterior approach had the least negative effect, even up to 6 weeks postoperatively. THA patients have reduced muscle strength in the operated leg (compared to the unoperated leg) 3 months after surgery.


Effects of glutamine alone or in combination with zinc and vitamin A on growth, intestinal barrier function, stress and satiety-related hormones in Brazilian shantytown children.
To determine the impact of supplemental zinc, vitamin A, and glutamine alone or in combination on growth, intestinal barrier function, stress and satiety-related hormones among Brazilian shantytown children with low median height-for-age z-scores. A randomized, double-blind, placebo-controlled trial was conducted in children aged two months to nine years from the urban shanty compound community of Fortaleza, Brazil. Demographic and anthropometric information was assessed. The random treatment groups available for testing (a total of 120 children) were as follows: (1) glutamine alone, n = 38; (2) glutamine plus vitamin A plus zinc, n = 37; and a placebo (zinc plus vitamin A vehicle) plus glycine (isonitrogenous to glutamine) control treatment, n = 38. Leptin, adiponectin, insulin-like growth factor (IGF-1), and plasma levels of cortisol were measured with immune-enzymatic assays; urinary lactulose/mannitol and serum amino acids were measured with high-performance liquid chromatography. ClinicalTrials.gov: NCT00133406. Glutamine treatment significantly improved weight-for-height z-scores compared to the placebo-glycine control treatment. Either glutamine alone or all nutrients combined prevented disruption of the intestinal barrier function, as measured by the percentage of lactulose urinary excretion and the lactulose:mannitol absorption ratio. Plasma leptin was negatively correlated with plasma glutamine (p = 0.002) and arginine (p = 0.001) levels at baseline. After glutamine treatment, leptin was correlated with weight-for-age (WAZ) and weight-for-height z-scores (WHZ) (p≤0.002) at a 4-month follow-up. In addition, glutamine and all combined nutrients (glutamine, vitamin A, and zinc) improved the intestinal barrier function in these children. Taken together, these findings reveal the benefits of glutamine alone or in combination with other gut-trophic nutrients in growing children via interactions with leptin.


Discrete solution of the electrokinetic equations.
We present a robust scheme for solving the electrokinetic equations. This goal is achieved by combining the lattice-Boltzmann method with a discrete solution of the convection-diffusion equation for the different charged and neutral species that compose the fluid. The method is based on identifying the elementary fluxes between nodes, which ensures the absence of spurious fluxes in equilibrium. We show how the model is suitable to study electro-osmotic flows. As an illustration, we show that, by introducing appropriate dynamic rules in the presence of solid interfaces, we can compute the sedimentation velocity (and hence the sedimentation potential) of a charged sphere. Our approach does not assume linearization of the Poisson-Boltzmann equation and allows us for a wide variation of the Peclet number.


Exercise rehabilitation following intensive care unit discharge for recovery from critical illness: executive summary of a Cochrane Collaboration systematic review.
Skeletal muscle wasting and weakness are major complications of critical illness and underlie the profound physical and functional impairments experienced by survivors after discharge from the intensive care unit (ICU). Exercise-based rehabilitation has been shown to be beneficial when delivered during ICU admission. This review aimed to determine the effectiveness of exercise rehabilitation initiated after ICU discharge on primary outcomes of functional exercise capacity and health-related quality of life. We sought randomized controlled trials, quasi-randomized controlled trials, and controlled clinical trials comparing an exercise intervention commenced after ICU discharge vs. any other intervention or a control or 'usual care' programme in adult survivors of critical illness. Cochrane Central Register of Controlled Trials, Medical Literature Analysis and Retrieval System Online (MEDLINE), Excerpta Medica Database, and Cumulative Index to Nursing and Allied Health Literature databases were searched up to February 2015. Dual, independent screening of results, data extraction, and quality appraisal were performed. We included six trials involving 483 patients. Overall quality of evidence for both outcomes was very low. All studies evaluated functional exercise capacity, with three reporting positive effects in favour of the intervention. Only two studies evaluated health-related quality of life and neither reported differences between intervention and control groups. Meta-analyses of data were precluded due to variation in study design, types of interventions, and selection and reporting of outcome measurements. We were unable to determine an overall effect on functional exercise capacity or health-related quality of life of interventions initiated after ICU discharge for survivors of critical illness. Findings from ongoing studies are awaited. Future studies need to address methodological aspects of study design and conduct to enhance rigour, quality, and synthesis.


From Pioneer to Repressor: Bimodal foxd3 Activity Dynamically Remodels Neural Crest Regulatory Landscape In Vivo.
The neural crest (NC) is a transient embryonic stem cell-like population characterized by its multipotency and broad developmental potential. Here, we perform NC-specific transcriptional and epigenomic profiling of foxd3-mutant cells in vivo to define the gene regulatory circuits controlling NC specification. Together with global binding analysis obtained by foxd3 biotin-ChIP and single cell profiles of foxd3-expressing premigratory NC, our analysis shows that, during early steps of NC formation, foxd3 acts globally as a pioneer factor to prime the onset of genes regulating NC specification and migration by re-arranging the chromatin landscape, opening cis-regulatory elements and reshuffling nucleosomes. Strikingly, foxd3 then gradually switches from an activator to its well-described role as a transcriptional repressor and potentially uses differential partners for each role. Taken together, these results demonstrate that foxd3 acts bimodally in the neural crest as a switch from "permissive" to "repressive" nucleosome and chromatin organization to maintain multipotency and define cell fates.


Types of Erythropoietin-Stimulating Agents and Mortality among Patients Undergoing Hemodialysis.
Despite the widespread use of erythropoietin-stimulating agents (ESAs) to treat anemia in patients undergoing hemodialysis, the relative mortality risks associated with use of different types of ESAs are unknown. To compare the mortality risk associated with use of short-acting ESAs versus long-acting ESAs, we conducted a nationwide cohort study of 194,698 hemodialysis patients in Japan who received either a short-acting (epoetin α/β or epoetin κ) or a long-acting (darbepoetin or epoetin β pegol) ESA. Study outcomes were 2-year all-cause and cause-specific mortality. In addition to Cox proportional hazards models, we performed an instrumental variable analysis in which facility-level long-acting ESA prescription rates were taken as the instrumental variable. During the 2-year follow-up period, 31,557 deaths occurred. In a multivariable Cox model, long-acting ESA users had a 13% higher rate of deaths compared with short-acting ESA users, a significant difference (P<0.001). Similar results were obtained in other analyses. This difference in risk was pronounced among patients receiving high doses of ESA (for whom the adjusted 2-year number needed to harm for death was 30.8). Long-acting ESA use was associated with an increased rate of death from cardiovascular diseases, infection, and malignancies. In the instrumental variable analysis, long-acting ESA users remained at a significantly higher risk of death. Compared with anemic (hemoglobin 9.0-9.9 g/dl) short-acting ESA users, long-acting ESA users who achieved more optimal hemoglobin levels (10.0-10.9 g/dl) showed a higher mortality rate. Among patients undergoing hemodialysis, use of long-acting ESAs might be associated with a higher risk of death than use of short-acting ESAs.


Effect of formalin toxoiding on Pseudomonas aeruginosa toxin A: biological, chemical, and immunochemical studies.
We investigated the effect of Formalin toxoiding on the biological, chemical, and immunological activities of Pseudomonas aeruginosa toxin A. Formalin treatment alone resulted in a 1,000-fold decrease in toxin-induced cell cytotoxicity and altered the antigenicity of the toxin A molecule without adversely affecting enzymatic activity. Competitive blocking experiments indicated that Formalin-mediated detoxification proceeded via alterations in a region of the toxin molecule other than the active site of the enzyme. The addition of lysine to Formalin-toxin mixtures not only increased the rate and extent of detoxification and antigenic alteration, but also completely destroyed enzymatic activity. The immunogenicities of different toxoids varied substantially. Upon dialysis and storage, Formalin-derived toxoids underwent partial toxic reversion, whereas a Formalin-lysine-derived toxoid did not. The sodium dodecyl sulfate-polyacrylamide gel patterns of Formalin- and Formalin-lysine-treated toxins indicated that these treatments caused the formation of a heterogenous group of high-molecular-weight species and produced substantial changes in the electrophoretic mobility of toxin A.


Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.
Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.


Exploring the fundamental dynamics of error-based motor learning using a stationary predictive-saccade task.
The maintenance of movement accuracy uses prior performance errors to correct future motor plans; this motor-learning process ensures that movements remain quick and accurate. The control of predictive saccades, in which anticipatory movements are made to future targets before visual stimulus information becomes available, serves as an ideal paradigm to analyze how the motor system utilizes prior errors to drive movements to a desired goal. Predictive saccades constitute a stationary process (the mean and to a rough approximation the variability of the data do not vary over time, unlike a typical motor adaptation paradigm). This enables us to study inter-trial correlations, both on a trial-by-trial basis and across long blocks of trials. Saccade errors are found to be corrected on a trial-by-trial basis in a direction-specific manner (the next saccade made in the same direction will reflect a correction for errors made on the current saccade). Additionally, there is evidence for a second, modulating process that exhibits long memory. That is, performance information, as measured via inter-trial correlations, is strongly retained across a large number of saccades (about 100 trials). Together, this evidence indicates that the dynamics of motor learning exhibit complexities that must be carefully considered, as they cannot be fully described with current state-space (ARMA) modeling efforts.


Randomized controlled trial comparing rubber band ligation with stapled haemorrhoidopexy for Grade II circumferential haemorrhoids: long-term results.
An improved understanding of the pathophysiology of haemorrhoids has resulted in the introduction of new surgical techniques including stapled haemorrhoidopexy (SH). This randomized controlled trial compared the long-term effectiveness of SH with rubber band ligation (RBL) in the treatment of grade II circumferential symptomatic haemorrhoids. A consecutive cohort of patients was randomly allocated to either SH or RBL. Data on haemorrhoidal symptoms, Cleveland continence scores, sphincter assessment, SF-36, EQ-5D, HAD score and prior treatment history were assessed at enrollment and reassessed by long-term postal questionnaire. The details were analysed using spss 12.0 from Microsoft Access. Sixty patients were allocated by computer block randomization. Both groups were balanced for age, sex and symptoms. Recurrence favoured SH [3 vs 11; OR 0.18, 95% CI (0.03 to 0.86), P = 0.028] at 1 year and, at a mean of 40.67 (31-47) months [4 vs 12; OR 0.23, 95% CI (0.05, 0.95); P = 0.039]. SH patients experienced prolonged pain [Median (IQR) = 7 (5,7) vs 3 (1,7), P = 0.008] and took a longer time to return to work [6 (3,7) vs 3 (1,6) days, P = 0.018]. This was no significant difference in quality of life. Stapled haemorrhoidopexy achieved better disease control at 1 year without any major complication. This was sustained in the long-term. Further studies with greater patient numbers are needed to confirm this study.


Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance.
S6K1, like other serine and threonine kinases activated by insulin (such as mTOR and PKCzeta), has recently been shown to participate in negative feedback mechanisms aimed at terminating insulin signaling through IRS (insulin receptor substrate) phosphorylation. Such homeostatic mechanisms can also be activated by excess nutrients or inducers of insulin resistance (such as fatty acids and proinflammatory cytokines) to produce an insulin-resistant state that often leads to the development of diabetes. Identification of the specific kinases involved in such insulin resistance pathways can help lead to the rational design of novel therapeutic agents for treating insulin resistance and type 2 diabetes.


Spatial integration in mouse primary visual cortex.
Responses of many neurons in primary visual cortex (V1) are suppressed by stimuli exceeding the classical receptive field (RF), an important property that might underlie the computation of visual saliency. Traditionally, it has proven difficult to disentangle the underlying neural circuits, including feedforward, horizontal intracortical, and feedback connectivity. Since circuit-level analysis is particularly feasible in the mouse, we asked whether neural signatures of spatial integration in mouse V1 are similar to those of higher-order mammals and investigated the role of parvalbumin-expressing (PV+) inhibitory interneurons. Analogous to what is known from primates and carnivores, we demonstrate that, in awake mice, surround suppression is present in the majority of V1 neurons and is strongest in superficial cortical layers. Anesthesia with isoflurane-urethane, however, profoundly affects spatial integration: it reduces the laminar dependency, decreases overall suppression strength, and alters the temporal dynamics of responses. We show that these effects of brain state can be parsimoniously explained by assuming that anesthesia affects contrast normalization. Hence, the full impact of suppressive influences in mouse V1 cannot be studied under anesthesia with isoflurane-urethane. To assess the neural circuits of spatial integration, we targeted PV+ interneurons using optogenetics. Optogenetic depolarization of PV+ interneurons was associated with increased RF size and decreased suppression in the recorded population, similar to effects of lowering stimulus contrast, suggesting that PV+ interneurons contribute to spatial integration by affecting overall stimulus drive. We conclude that the mouse is a promising model for circuit-level mechanisms of spatial integration, which relies on the combined activity of different types of inhibitory interneurons.


Always look on both sides: phylogenetic information conveyed by simple sequence repeat allele sequences.
Simple sequence repeat (SSR) markers are widely used tools for inferences about genetic diversity, phylogeography and spatial genetic structure. Their applications assume that variation among alleles is essentially caused by an expansion or contraction of the number of repeats and that, accessorily, mutations in the target sequences follow the stepwise mutation model (SMM). Generally speaking, PCR amplicon sizes are used as direct indicators of the number of SSR repeats composing an allele with the data analysis either ignoring the extent of allele size differences or assuming that there is a direct correlation between differences in amplicon size and evolutionary distance. However, without precisely knowing the kind and distribution of polymorphism within an allele (SSR and the associated flanking region (FR) sequences), it is hard to say what kind of evolutionary message is conveyed by such a synthetic descriptor of polymorphism as DNA amplicon size. In this study, we sequenced several SSR alleles in multiple populations of three divergent tree genera and disentangled the types of polymorphisms contained in each portion of the DNA amplicon containing an SSR. The patterns of diversity provided by amplicon size variation, SSR variation itself, insertions/deletions (indels), and single nucleotide polymorphisms (SNPs) observed in the FRs were compared. Amplicon size variation largely reflected SSR repeat number. The amount of variation was as large in FRs as in the SSR itself. The former contributed significantly to the phylogenetic information and sometimes was the main source of differentiation among individuals and populations contained by FR and SSR regions of SSR markers. The presence of mutations occurring at different rates within a marker's sequence offers the opportunity to analyse evolutionary events occurring on various timescales, but at the same time calls for caution in the interpretation of SSR marker data when the distribution of within-locus polymorphism is not known.


Specific activation of pro-Infliximab enhances selectivity and safety of rheumatoid arthritis therapy.
During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.


Variation in mitochondrial genotype has substantial lifespan effects which may be modulated by nuclear background.
Mitochondria are thought to play a central role in aging. In humans, specific naturally occurring mitochondrial genetic variants are overrepresented among centenarians, but only in certain populations; therefore, we cannot tell whether this effect is due solely to mitochondrial genetics or to nuclear-mitochondrial gene complexes, nor do we know the magnitude of the effect in terms we can relate to, such as mean lifespan differences. To examine the effects of natural mitochondrial DNA (mtDNA) variation on lifespan, we need to vary the mitochondrial genotype while controlling the nuclear genotype. Here, nuclear genome replacement is achieved using strains of Drosophila melanogaster bearing multiply inverted 'balancer' chromosomes that suppress recombination, and an isogenic donor strain, thus forcing replacement of entire chromosomes in a single cross while suppressing recombination. Lifespans of wild-type mtDNA variants on the chromosome replacement background vary substantially, and sequencing of the entire protein coding mitochondrial genomes indicates that these lifespan differences are sometimes associated with single amino acid differences. On other nuclear genetic backgrounds, the magnitude and direction of these lifespan effects can change dramatically, and this can be due to changes in baseline mortality risk, rate of aging and/or time of onset of aging. The limited mtDNA variation in D. melanogaster makes it an ideal organism for biochemical studies to link genotype and aging phenotype.


Early data from Project Engage: a program to identify and transition medically hospitalized patients into addictions treatment.
Patients with untreated substance use disorders (SUDs) are at risk for frequent emergency department visits and repeated hospitalizations. Project Engage, a US pilot program at Wilmington Hospital in Delaware, was conducted to facilitate entry of these patients to SUD treatment after discharge. Patients identified as having hazardous or harmful alcohol consumption based on results of the Alcohol Use Disorders Identification Test-Primary Care (AUDIT-PC), administered to all patients at admission, received bedside assessment with motivational interviewing and facilitated referral to treatment by a patient engagement specialist (PES). This program evaluation provides descriptive information on self-reported rates of SUD treatment initiation of all patients and health-care utilization and costs for a subset of patients. Program-level data on treatment entry after discharge were examined retrospectively. Insurance claims data for two small cohorts who entered treatment after discharge (2009, n = 18, and 2010, n = 25) were reviewed over a six-month period in 2009 (three months pre- and post-Project Engage), or over a 12-month period in 2010 (six months pre- and post-Project Engage). These data provided descriptive information on health-care utilization and costs. (Data on those who participated in Project Engage but did not enter treatment were unavailable). Between September 1, 2008, and December 30, 2010, 415 patients participated in Project Engage, and 180 (43%) were admitted for SUD treatment. For a small cohort who participated between June 1, 2009, and November 30, 2009 (n = 18), insurance claims demonstrated a 33% ($35,938) decrease in inpatient medical admissions, a 38% ($4,248) decrease in emergency department visits, a 42% ($1,579) increase in behavioral health/substance abuse (BH/SA) inpatient admissions, and a 33% ($847) increase in outpatient BH/SA admissions, for an overall decrease of $37,760. For a small cohort who participated between June 1, 2010, and November 30, 2010 (n = 25), claims demonstrated a 58% ($68,422) decrease in inpatient medical admissions; a 13% ($3,308) decrease in emergency department visits; a 32% ($18,119) decrease in BH/SA inpatient admissions, and a 32% ($963) increase in outpatient BH/SA admissions, for an overall decrease of $88,886. These findings demonstrate that a large percentage of patients entered SUD treatment after participating in Project Engage, a novel intervention with facilitated referral to treatment. Although the findings are limited by the retrospective nature of the data and the small sample sizes, they do suggest a potentially cost-effective addition to existing hospital services if replicated in prospective studies with larger samples and controls.


The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb.
In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.


Pregnancy complicated by haemorrhagic ascites in a woman with newly diagnosed HIV.
A young pregnant Zambian woman was referred from a district hospital in South Zambia to the university teaching hospital, Lusaka with severe anaemia and ascites. The ascites had developed over a month and the woman was currently 15 weeks pregnant. Further workup revealed that the patient was HIV-positive and the ascitic tap showed haemorrhagic fluid. After being reviewed by multiple doctors, the cause of the haemorrhagic ascites remained unclear; therefore, the decision was made to do a laparotomy. The laparotomy revealed haemoperitoneum and a large cyst attached to the liver containing 5 L of bloodstained fluid. The histopathology report revealed features consistent with a giant haemangioma. There were many barriers to accessing optimum healthcare in this case. These included limited access to blood, poor communication resulting in the patient being unaware of her HIV status and lack of patient education about HIV.


Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli.
SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.


A pilot quality improvement intervention in patients with diabetes and hypertension in primary care settings of Cyprus.
The achievement of quality of care constitutes a priority for modern health care systems. The objective of our study was to evaluate a quality improvement intervention in primary care of Cyprus. In a two-arm non-randomized controlled study in primary care centres in Cyprus, all patients with hypertension (HTN) and diabetes (n = 539) were invited. In one urban and one rural centre, a quality improvement programme was implemented; two other centres (one urban and one rural) served as control practices. The intervention mainly consisted of the introduction of clinical disease management guidelines and an electronic medical record system. The primary outcome measurement was improvement of specific clinical indicators for HTN and diabetes. Patients' satisfaction was evaluated using the European Task Force on Patient Evaluations of General Practice (EUROPEP) questionnaire over an 18-month follow-up period. Five hundred and four patients completed the study, 278 patients in the intervention practices and 226 patients in the control practices. Mean results for blood pressure, total cholesterol and low density lipoprotein-cholesterol and three annual performance measures (urine protein testing, dilated eye and foot examination) had improved at 18-month follow-up in the intervention as compared to the control group. There was no improvement of HbA1c levels. Patients' satisfaction improved in the intervention practices (improvement of 10/23 EUROPEP items) but decreased in the control group (decline of 20/23 items). A pilot multifaceted quality improvement intervention programme for patients with diabetes and HTN implemented in primary care settings in Cyprus showed promising results. Future studies need to involve a broader number of practices and patient populations.


Cardiovascular Disease Risk Factors and Physical Fitness in Volunteer Firefighters.
Forty-seven percent of volunteer firefighter line of duty deaths are caused by cardiovascular events. Aggressive cardiovascular disease (CVD) risk factor reduction and improved physical fitness could reduce CVD mortality within this population. We assessed CVD risk factors and physical fitness in a large cohort of volunteer firefighters to help establish a health and fitness profile of this population, which may serve as evidence for the need to initiate programs aimed at reducing morbidity and mortality caused by CVD in the volunteer fire service. Seventy-four male volunteer firefighters were assessed for eight CVD risk factors and anthropometric characteristics. Physical fitness was assessed via push-ups, sit-ups, and the YMCA step test. Sixty-eight percent of the firefighters had two or more CVD risk factors. The sample was considered obese via body fat percentage (25.3 ± 5.7%), 27% were hypertensive, 30% had hypercholesterolemia, and 46% were sedentary. The average number of sit-ups performed was 27.3 ± 10.5, which was ranked in the 25th percentile. The average heart rate after the YMCA step test was 160.2 ± 14.6 bpm, which was ranked very poor. The number of CVD risk factors and poor physical fitness in this cohort of volunteer firefighters was noteworthy. Most volunteer firefighters in our sample were at elevated risk for CVD and had inadequate physical fitness. This evidence conveys the need to initiate physical activity and nutrition outreach programs, led by health and fitness professionals, aimed at reducing firefighter morbidity and mortality within the volunteer fire service.


Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells.
Human embryonic stem cells (hESCs) may provide a cell source for functional hepatocytes for clinical applications and drug development. Initially, the hESC population was enriched to be more than 85% definitive endoderm (DE) as assessed by the expression of CXCR4, SOX17, and FOXA2. We then successfully converted DE into hepatic progenitors with 93% of the cells being positive for alpha-feto protein within 9 days. The percentage of albumin positive cells gradually increased to 90% at days 20-22 after differentiation. Moreover, our hESC-derived hepatocytes (hEH) developed a complete biotransformation system including phase I and II metabolizing enyzmes and phase III transporters. Nuclear receptors, which are critical in regulating the expression of metabolizing enzymes, were also expressed by our hEH. Using ultraperformance liquid chromatography-tandem mass spectrometry technology, we identified seven metabolic pathways of the drug bufuralol including four newly-reported ones in our hEH, which are the same as those in freshly isolated human primary hepatocytes (hPH). In addition, the results of the metabolism of four drugs indicate that our hEH have the capacity to metabolize these drugs at levels that are comparable to hPH. In conclusion, we have generated a relatively homogenous population of hepatocytes from hESCs, which appear to have complete metabolic function that is comparable to primary liver cells. These results represent a significant step towards the efficient differentiation of mature hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies.


[Digital image processing of screening mammographies: application to masses].
A method based on image processing of tumoral neighbourhood is described for studying masses. By using polar and pseudopolar representations for the tumor and its neighbourhood, it is possible to detect divergent structures around the lesion and to evidence fuzzy areas in the boundary of the tumor, which constitute two significant features of breast cancer. The degree of spiculation is deduced from a shape parameter that characterizes the irregularity of the boundary; the radial component of the gradient on the boundary provides a measurement of fuzzy appearance.


Helicobacter pylori Multidrug Resistance Due to Misuse of Antibiotics in Iran.
Helicobacter pylori might become highly resistant to antibiotics taken through the life time of patients. This study examined the change in antibiotic resistance of H. pylori by time. Out of 985 dyspeptic patients who were referred to the endoscopy unit of Shariati hospital during 2010-2017, 218 patients with gastric biopsies positive for rapid urease test (RUT) and H. pylori culture were recruited in the study. H. pylori isolates were examined for resistance to 8 currently used antibiotics by the disc diffusion method. Results were compared with those from our three previous studies. The frequency of multidrug resistance (MDR) was also assessed. The highest resistance rate was to metronidazole (MTZ) (79.4%) followed by ofloxacin (OFX) (58.7%), ciprofloxacin (CIP) (46.8%), levofloxacin (LVX) (45%), tetracycline (TET) (38.5%), clarithromycin (CLR) (34.4%), amoxicillin (AMX) (27.1%) and furazolidone (FRZ) (23.9%). No significant difference was found between resistance of H. pylori isolates from male and female <40 and >40 years old and patients with gastritis and peptic ulcer. The highest rates of MDR were to MTZ+OFX (4.6%), MTZ+OFX+TET (2.8%), MTZ+OFX+CIP+LVX (6.4%) and MTZ+OFX+TET+ CIP+LVX (5%). Resistance to MTZ increased from 33%-55.6% in previous studies to 79.4% by time, to CLR increased from 1.4-7.3% to 34.4%, to TET increased from 0-38.1% to 38.5%, to AMX increased from 1.4%-7.3% to 27.1% and to FRZ increased from 0%-4.5% to 23.9%. Resistance to FQs was 45%-58.7%. Increase in H. pylori antibiotic resistance indicates antibiotic misuse. In Iran, with a considerable number of H. pylori- infected patients, antibiotic therapy should be saved for high risk patients and according to local antibiotic resistance patterns.


Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.
Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.


(--)-Xanthatin selectively induces GADD45γ and stimulates caspase-independent cell death in human breast cancer MDA-MB-231 cells.
exo-Methylene lactone group-containing compounds, such as (--)-xanthatin, are present in a large variety of biologically active natural products, including extracts of Xanthium strumarium (Cocklebur). These substances are reported to possess diverse functional activities, exhibiting anti-inflammatory, antimalarial, and anticancer potential. In this study, we synthesized six structurally related xanthanolides containing exo-methylene lactone moieties, including (--)-xanthatin and (+)-8-epi-xanthatin, and examined the effects of these chemically defined substances on the highly aggressive and farnesyltransferase inhibitor (FTI)-resistant MDA-MB-231 cancer cell line. The results obtained demonstrate that (--)-xanthatin was a highly effective inhibitor of MDA-MB-231 cell growth, inducing caspase-independent cell death, and that these effects were independent of FTase inhibition. Further, our results show that among the GADD45 isoforms, GADD45γ was selectively induced by (--)-xanthatin and that GADD45γ-primed JNK and p38 signaling pathways are, at least in part, involved in mediating the growth inhibition and potential anticancer activities of this agent. Given that GADD45γ is becoming increasingly recognized for its tumor suppressor function, the results presented here suggest the novel possibility that (--)-xanthatin may have therapeutic value as a selective inducer of GADD45γ in human cancer cells, in particular in FTI-resistant aggressive breast cancers.


Domestic wastewater treatment by forward osmosis-membrane distillation (FO-MD) integrated system.
In this study, real domestic wastewater treatment by forward osmosis-membrane distillation (FO-MD) integrated system was investigated in laboratory scale. The integrated membrane system presented a good separation performance and the removal efficiency of most contaminants in the domestic wastewater was higher than 90%. High molecular weight contaminants were completely removed, while a few low molecular weight contaminants permeated through the membrane. The FO membrane fouling layer mainly consisted of organic substances like polysaccharides and proteins, and was very loose and could be effectively removed by rinsing the membrane surface with tap water. By comparison, the MD membrane fouling was mainly induced by inorganic salts and was not as severe as that of the FO membrane. During 120 h continuous operation, the FO-MD integrated system exhibited satisfying performance stability and maintained a high water yield and high product water quality. The results indicated the potential of the FO-MD integrated system for municipal wastewater treatment in coastal cities, water purification and desalination.


Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg(2+) functions as a voltage-dependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg(2+) is in the physiological range, with Mg(2+) binding with an IC(50) of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at +50 mV. The effective electrical distance for the Mg(2+) binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg(2+), suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg(2+) acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg(2+) may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg(2+) could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.


World Health Organization cancer priorities in developing countries.
The burden of cancer in developing countries is growing and threatens to exact a heavy morbidity, mortality, and economic cost in these countries in the next 20 years. The unfolding global public health dimensions of the cancer pandemic demand a widespread effective international response. The good news is that the majority of cancers in developing countries are preventable, and the efficacy of treatment can be improved with early detection. Currently, the knowledge exists to implement sound, evidence-based practices in cancer prevention, screening/early detection, treatment, and palliation. It is estimated that the information at hand could prevent up to one-third of new cancers and increase survival for another one-third of cancers detected at an early stage. To achieve this, knowledge must be translated into action. To facilitate the call to action in the fight against cancer, the World Health Organization (WHO) has developed a comprehensive approach to cancer control. The WHO has produced many valuable guidelines and resources for the effective implementation of national cancer control programs. Several milestones in the WHO's efforts include the Framework Convention for Tobacco Control, and global strategies for diet and exercise, reproductive health, and cervical cancer. This review examines the strategies and approaches that have successfully resulted into global action to confront the rising global burden of cancer in the developing world.


Functional characterization of cell-wall-associated protein WapA in Streptococcus mutans.
Streptococcus mutans is known as a primary pathogen responsible for dental caries. One of the virulence factors of S. mutans in cariogenicity is its ability to attach to the tooth surface and form a biofilm. Several surface proteins have been shown to be involved in this process. A 29 kDa surface protein named wall-associated protein A (WapA, antigen A or antigen III), was previously used as a vaccine in animal studies for immunization against dental caries. However, the function of WapA in S. mutans is still not clear. This study characterized the function of WapA in cell surface structure and biofilm formation. Compared to the wild-type, the wapA mutant had much-reduced cell chain length, diminished cell-cell aggregation, altered cell surface ultrastructure, and unstructured biofilm architecture. Furthermore, in vivo force spectroscopy revealed that the cell surface of the wapA mutant was less sticky than that of the wild-type cells. More interestingly, these phenotypic differences diminished as sucrose concentration in the medium was increased to 0.5 %. Real-time RT-PCR analysis demonstrated that sucrose strongly repressed wapA gene expression in both planktonic and biofilm cells. These results suggest that the WapA protein plays an important structural role on the cell surface, which ultimately affects sucrose-independent cell-cell aggregation and biofilm architecture.


Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies.
Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds. In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. STATEMENT OF SIGNIFICANCE: Articular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term. Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.


In vitro and in vivo evaluation of water-soluble iminophosphorane ruthenium(II) compounds. A potential chemotherapeutic agent for triple negative breast cancer.
A series of organometallic ruthenium(II) complexes containing iminophosphorane ligands have been synthesized and characterized. Cationic compounds with chloride as counterion are soluble in water (70-100 mg/mL). Most compounds (especially highly water-soluble 2) are more cytotoxic to a number of human cancer cell lines than cisplatin. Initial mechanistic studies indicate that the cell death type for these compounds is mainly through canonical or caspase-dependent apoptosis, nondependent on p53, and that the compounds do not interact with DNA or inhibit protease cathepsin B. In vivo experiments of 2 on MDA-MB-231 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (shrinkage) of 56% after 28 days of treatment (14 doses of 5 mg/kg every other day) with low systemic toxicity. Pharmacokinetic studies showed a quick absorption of 2 in plasma with preferential accumulation in the breast tumor tissues when compared to kidney and liver, which may explain its high efficacy in vivo.


Wastewater irrigation and environmental health: implications for water governance and public policy.
Climate change is a large-scale and emerging environmental risk. It challenges environmental health and the sustainability of global development. Wastewater irrigation can make a sterling contribution to reducing water demand, recycling nutrients, improving soil health and cutting the amount of pollutants discharged into the waterways. However, the resource must be carefully managed to protect the environment and public health. Actions promoting wastewater reuse are every where, yet the frameworks for the protection of human health and the environment are lacking in most developing countries. Global change drivers including climate change, population growth, urbanization, income growth, improvements in living standard, industrialization, and energy intensive lifestyle will all heighten water management challenges. Slowing productivity growth, falling investment in irrigation, loss of biodiversity, risks to public health, environmental health issues such as soil salinity, land degradation, land cover change and water quality issues add an additional layer of complexity. Against this backdrop, the potential for wastewater irrigation and its benefits and risks are examined. These include crop productivity, aquaculture, soil health, groundwater quality, environmental health, public health, infrastructure constraints, social concerns and risks, property values, social equity, and poverty reduction. It is argued that, wastewater reuse and nutrient capture can contribute towards climate change adaptation and mitigation. Benefits such as avoided freshwater pumping and energy savings, fertilizer savings, phosphorous capture and prevention of mineral fertilizer extraction from mines can reduce carbon footprint and earn carbon credits. Wastewater reuse in agriculture reduces the water footprint of food production on the environment; it also entails activities such as higher crop yields and changes in cropping patterns, which also reduce carbon footprint. However, there is a need to better integrate water reuse into core water governance frameworks in order to effectively address the challenges and harness the potential of this vital resource for environmental health protection. The paper also presents a blueprint for future water governance and public policies for the protection of environmental health.


Genetic interaction implicates iRhom2 in the regulation of EGF receptor signalling in mice.
iRhoms are closely related to rhomboid intramembrane proteases but lack catalytic activity. In mammals iRhoms are known to regulate the trafficking of TACE, the protease that cleaves the membrane bound inflammatory cytokine TNF. We have mapped a spontaneously occurring mouse mutation with a loss of hair phenotype, curly bare (cub), to the Rhbdf2 locus, which encodes the iRhom2 protein. The cub deletion removes the first 268 amino acids of the iRhom2 protein but is not a loss of function. We have also identified a previously reported suppressor of cub, called Mcub (modifier of curly bare), and find it to be a loss of function allele of the amphiregulin gene (Areg). Amphiregulin is an activating ligand of the epidermal growth factor receptor (EGFR) that, like TNF, is released by TACE. Our results therefore imply a regulatory link between iRhoms and EGFR signalling in mammals. We have tested the model that the cub mutation leads to iRhom2 hyperactivity and consequently excess TACE processing of amphiregulin and elevated EGFR signalling. Our results do not support this hypothesis: we find that, compared to wild-type cells, cub mutant embryonic fibroblasts release less amphiregulin, and that the cub mutant form of iRhom2 is less able than wild type to bind to TACE and promote its maturation.


A conserved domain is present in different families of vesicular fusion proteins: a new superfamily.
We have analyzed conserved domains in t-SNAREs [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors in the target membrane], proteins that are believed to be involved in the fusion of transport vesicles with their target membrane. By using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain that is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) families, which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain was also identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins were identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins.


Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4.
Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that GRK2, a critical regulator of myocardial β-AR signaling, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (Iso) caused increased oxidative stress and apoptosis. There was also increased Nox4 expression, but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. Adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with Iso stimulation. These increases in oxidative stress were abolished by pre-treatment with the non-specific Nox inhibitor, apocynin, or siRNA knockdown of Nox4. Adenoviral-mediated expression of a GRK2 inhibitor prevented ROS production and apoptosis in response to Iso stimulation. β-Arrestins are signaling proteins that function downstream of GRK2 in β-AR uncoupling. Adenoviral-mediated overexpression of β-arrestins increased ROS production and Nox4 expression. Chronic β-agonist stimulation in mice increased Nox4 expression and apoptosis compared to PBS or AngII treatment. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in cardiac myocytes and provides an additional novel mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition to prevent the development of HF.


Mexico's "ley de narcomenudeo" drug policy reform and the international drug control regime.
It has been over half a century since the landmark Single Convention on Narcotic Drugs was adopted, for the first time unifying international drug policy under a single treaty aimed at limiting use, manufacture, trade, possession, and trafficking of opiates, cannabis, and other narcotics. Since then, other international drug policy measures have been adopted, largely emphasizing enforcement-based approaches to reducing drug supply and use. Recently, in response to concerns that the historic focus on criminalization and enforcement has had limited effectiveness, international drug policies have begun to undergo a paradigm shift as countries seek to enact their own reforms to partially depenalize or deregulate personal drug use and possession. This includes Mexico, which in 2009 enacted national drug policy reform partially decriminalizing possession of small quantities of narcotics for personal consumption while also requiring drug treatment for repeat offenders. As countries move forward with their own reform models, critical assessment of their legal compatibility and effectiveness is necessary. In this commentary we conduct a critical assessment of the compatibility of Mexico's reform policy to the international drug policy regime and describe its role in the current evolving drug policy environment. We argue that Mexico's reform is consistent with flexibilities allowed under international drug treaty instruments and related commentaries. We also advocate that drug policy reforms and future governance efforts should be based on empirical evidence, emphasize harm reduction practices, and integrate evidence-based evaluation and implementation of drug reform measures.


Nonsense-mediated decay: paving the road for genome diversification.
The expression of protein-encoding genes is a complex process culminating in the production of mature mRNA and its translation by the ribosomes. The production of a mature mRNA involves an intricate series of processing steps. The majority of eukaryotic protein-encoding genes contain intron sequences that disrupt the protein-encoding frame, and hence have to be removed from immature mRNA prior to translation into protein. The mechanism involved in the selection of correct splice sites is incompletely understood. A considerable body of evidence suggests that the splicing machinery has suboptimal efficiency and fidelity leading to substantial processing inaccuracy. Here we discuss a recently published article that extends observations that cells rely on nonsense-mediated mRNA decay (NMD) to compensate for such suboptimal processing accuracy. Intriguingly these authors provide evidence for a strong selective pressure in favour of premature termination of mRNA translation in the event of intron retention. The analysis presented implies a positive role of NMD in transcript diversification through alternative splicing and suggest that this ancient surveillance mechanism may have co-evolved with intron acquisition born from the need for quality control of splicing patterns.


Cryoablation of renal fossa recurrence after radical nephrectomy.
Renal fossa recurrence of renal-cell carcinoma (RCC) after radical nephrectomy historically necessitates open surgical resection. Cryoablation provides a minimally invasive alternative to open resection for local recurrence. We describe our experience with percutaneous cryoablation in three patients who were experiencing fossa recurrence. We retrospectively reviewed medical records of three patients with renal fossa recurrence of RCC who were treated with percutaneous cryoablation. Percutaneous cryoablation was well tolerated; no complications were noted, and patients were discharged within 23 hours. One patient needed an additional cryoablation procedure, and one patient underwent subsequent open extirpation. Metastatic disease developed in one patient, while the other two patients remain negative for disease by axial imaging and percutaneous biopsy. All patients are alive at 43 months (range 22-54 months). Percutaneous cryoablation, a minimally invasive intervention, has low morbidity and may be offered as an alternative to extirpation, although long-term oncologic data are lacking.


Multidisciplinary perceptions of the role of nurses and healthcare assistants in rehabilitation of older adults in acute health care.
This paper describes the perceptions of nurses, healthcare assistants, doctors and therapists of rehabilitation and the role of nurses and healthcare assistants on an acute older adults ward in a London teaching hospital. The role perception, education and training and attitudes towards the older adults have been identified as barriers that have an impact upon the nurses' role within rehabilitation. However, little is known about the role of nurses and healthcare assistants in rehabilitation of older adults in acute health care. Action research study. Twenty-four semi-structured interviews were conducted and audio recorded about healthcare professionals' understanding of rehabilitation, the type of skills needed and their perceptions of the role of nurses and healthcare assistants in rehabilitation. The interview transcripts were analysed using the thematic content analysis. The findings suggest that the therapists relied on nurses and healthcare assistants for therapy carry-over. Healthcare assistants were perceived as the professional group who could deliver therapy carry-over. There was an evidence of role hierarchy as healthcare assistants perceived that they were not actively involved in decision-making or discharge planning. This paper suggests that healthcare assistants and nurses are viewed as the professional group best placed to deliver therapy carry-over. However, whilst there is an acknowledgement of their role, there remains a reluctance to acknowledge healthcare assistants as a professional group and to involve them within decision-making and discharge planning. Employers must be seen to advocate, support and implement education and training programs for healthcare assistants. However, whilst nurses and healthcare assistants have an integral role in rehabilitation, there needs to be more research into the how they are supported by therapy professionals.


Amphetamine induces a calcium/calmodulin-dependent protein kinase II-dependent reduction in norepinephrine transporter surface expression linked to changes in syntaxin 1A/transporter complexes.
Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH(2) terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CAD-hNETDelta(28-47) cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNETDelta(28-47) cells, AMPH induces a rapid and substantial reduction in surface hNETDelta(28-47) accompanied by a large increase in plasma membrane hNETDelta(28-47)/SYN1A complexes. We also found that AMPH in CAD-hNETDelta(28-47) cells induces a robust increase in cytosolic Ca2+ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca2+ or CaMKII activity blocks AMPH-stimulated hNETDelta(28-47) trafficking and the formation of hNETDelta(28-47)/SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH2 terminus are eliminated.


Non-Hodgkin's lymphoma in the elderly. Part 1: Overview and treatment of follicular lymphoma.
Non-Hodgkin's lymphoma is one of a few malignancies that have been increasing in incidence over the past several decades. Likewise, these disorders are more common in elderly patients, with a median age of occurrence of 65 years. Therapy in elderly patients may be affected by multiple factors, especially attendant comorbidities. The approaches to management of these patients, with either indolent or aggressive disease processes, have been based on prospective clinical trial results, many of which have included a younger patient population. Fortunately, over the past decade, results of treatment trials that have targeted an older patient population have emerged. The disease incidence and treatment approaches for both follicular (part 1 of this article) and diffuse aggressive (part 2) histologies in elderly patients are reviewed, as well as the impact of aging on the care of these patients.


Clinical challenges and ongoing role changes for primary health-care nurses.
To investigate the perceptions of primary health-care nurses (PHNs) about clinical demands and future challenges. Qualitative content-analysis of open questions from a questionnaire. A total of 121 PHNs from health-care districts in northern Sweden filled in a questionnaire during a mandatory educational day. Key issues that were raised included: defending the specific professional role of the PHNs; strengthening their self-governance and authority; and ensuring adequate care in the future through education that focuses more on nursing practice than on academic work. To ensure that the role of the PHN continues to develop, it is necessary to enable them to articulate, discuss, and evaluate their profession and to learn, in concrete forms, how to implement science in clinical practice.


Entropy-based clustering of embryonic stem cells using digital holographic microscopy.
Embryonic stem (ES) cells are an important factor in the development of cell-based therapeutic strategies. In this work, the use of digital holographic interferometric microscopy and statistical identification for automatic discrimination of ES cells and fibroblast (FB) cells is discussed in detail. The proposed algorithm first reduces the complex data structure to lower dimensions. Then, based on asymptotic normality, model-based clustering and linear discriminant analysis are applied to the transformed data to obtain the classification between ES and FB cells. The proposed algorithm is robust because it does not depend on parametric assumptions and can be extended to the classification of other cell image data. Experimental results are presented to demonstrate the performance of the system.


Noncoding RNAs in Wound Healing: A New and Vast Frontier.
Significance: Wound healing requires a highly orchestrated coordination of processes that are not yet fully understood. Therefore, available clinical therapies are thus far limited in their efficacy in preventing and treating both chronic wounds and scars. Current gene-based therapeutics is largely based on our understanding of the protein-coding genome and proteins involved in known wound healing pathways. Recent Advances: Noncoding RNAs such as microRNAs and long noncoding RNAs have recently been found to be significant modulators of gene expression in diverse cellular pathways. Research has now implicated noncoding RNAs in nearly every stage of the wound healing process, suggesting that they may serve as clinical therapeutic targets. Noncoding RNAs are critical regulators in processes such as angiogenesis and cutaneous cell migration and proliferation, including classically described biological pathways previously attributed to mostly protein constituents. Critical Issues: The complexity and diversity of the interactions of noncoding RNAs with their targets and other binding partners require thorough characterization and understanding of their functions before they may be altered to modulate human wound healing pathways. Future Directions: Research in the area of noncoding RNAs continues to rapidly expand our understanding of their potential roles in physiological and pathological wound healing. Coupled with improving technologies to enhance or suppress target noncoding RNA in vivo, these advances hold great promise in the development of new therapies for wound healing.


How Should We Respond to Non-Dominant Healing Practices, the Example of Homeopathy.
The debate around the ethics of homeopathy in recent issues of the journal has been approached as a binary question; is homeopathy ethical or not? This paper suggests that this is an unhelpful question and instead discusses a framework to establish the extent to which the dominant (medical) culture should tolerate non-dominant health practices such as homeopathy. This requires a sophisticated understanding of the placebo effect, a critical evaluation of what evidence is available, a consideration of the harm that the non-dominant practice might cause, and a consideration of how this might be affected by the culture of the patient. This is presented as a matter of cultural competence. At a clinical level clinicians need to respect the values and beliefs of their patients and communicate with all the practitioners involved in a patient's care. At a societal level there are a number of factors to be considered when a community decides which practices to tolerate and to what extent.


Genetic alteration of stemness factors and p53 in mouse forestomach by chemical carcinogen-induced carcinogenesis.
The expression profiles of genes normally enriched in embryonic stem (ES) cells (stemness factors) are associated with poor clinical outcome in solid tumors. However, whether such gene expression is responsible for tumor initiation and progression remains to be determined. The tumor suppressor gene p53 is known to attenuate the expression of Nanog, which is essential for maintaining stem cells in response to DNA damage. On the basis of these findings, we hypothesized that stemness factors and p53 closely correlate with each other and form a network in response to genomic damage in the early phase of carcinogenesis and in the process of tumor progression. In this study, we applied the N-methylbenzylnitrosoamine (NMBA)-induced carcinogenesis model to the mouse forestomach to clarify the role of the stemness factors, c-Myc, Klf4, Sox2, Oct3/4 and Nanog, in cancer development using p53(+/+) (n=26) and p53(+/-) (n=11) C57BL/6J mice. Thirty weeks following NMBA administration, histologically evident squamous cell carcinoma was detected in the forestomachs of p53(+/+) mice, and the percentage of p53-positive nuclei in the forestomach epithelium gradually increased during carcinogenesis. Tumor development in p53(+/-) mice occurred significantly earlier than in p53(+/+) mice. Quantitative real-time PCR analyses revealed a reduced c-Myc and Klf4 expression before evident morphological changes were observed, and an increased expression with the development of squamous cell carcinoma. Sox2 expression remained unchanged until tumor development and increased with the appearance of squamous cell carcinomas. The expression of Oct3/4 and Nanog increased at the early stages following NMBA administration, and Nanog expression in situ was not positively affected by the deficiency of p53. Findings of the present study suggested that Oct3/4 may be involved in the progression of carcinogenesis from normal epithelial cells at early stages, suggesting the potential use of Oct3/4 as a biomarker in forestomach tumor formation at early stages of chemical carcinogenesis.


morePhyML: improving the phylogenetic tree space exploration with PhyML 3.
PhyML is a widely used Maximum Likelihood (ML) phylogenetic tree inference software based on a standard hill-climbing method. Starting from an initial tree, the version 3 of PhyML explores the tree space by using "Nearest Neighbor Interchange" (NNI) or "Subtree Pruning and Regrafting" (SPR) tree swapping techniques in order to find the ML phylogenetic tree. NNI-based local searches are fast but can often get trapped in local optima, whereas it is expected that the larger (but slower to cover) SPR-based neighborhoods will lead to trees with higher likelihood. Here, I verify that PhyML infers more likely trees with SPRs than with NNIs in almost all cases. However, I also show that the SPR-based local search of PhyML often does not succeed at locating the ML tree. To improve the tree space exploration, I deliver a script, named morePhyML, which allows escaping from local optima by performing character reweighting. This ML tree search strategy, named ratchet, often leads to higher likelihood estimates. Based on the analysis of a large number of amino acid and nucleotide data, I show that morePhyML allows inferring more accurate phylogenetic trees than several other recently developed ML tree inference softwares in many cases.


Wnt5a does not support hematopoiesis in stroma-free, serum-free cultures.
Previously we reported that Wnt5a is highly expressed in the murine urogenital ridge-derived UG26-1B6 cells but not embryonic liver-derived EL08-1D2 cells. Mouse long-term repopulating hematopoietic stem cells (LTR-HSC) were maintained in non-contact UG26-1B6 cultures but not EL08-1D2 non-contact cultures, unless Wnt5a was also added to the cultures, suggesting a role for Wnt5a in the in vitro maintenance of LTR-HSC. Here, we investigated if the effect of Wnt5a on adult LTR-HSC activity is HSC-autonomous. To test the effect of Wnt5a on maintenance of LTR-HSC, we performed limiting dilution competitive transplantation assays of murine Lin-Sca1(+) c-kit(+) (LSK) cells cultured for 5 days with TPO and SCF with and without Wnt5a. The effect of Wnt5a on the generation of colony forming units (CFU) and the homing ability of LSK progeny was also tested. No effects were found of Wnt5a on total cell expansion, the number of CFU, or homing ability of day 5 LSK progeny. Furthermore, addition of Wnt5a did not improve, but may have impeded maintenance of LTR-HSC. In conclusion, our data indicate that Wnt5a does not enhance the maintenance and expansion of adult murine LTR-HSCs or committed progenitors cultured in vitro in serum- and stroma-free conditions.