Two alternative methods for the retrieval of somatic cell populations from the mouse ovary.
Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.


PTV margin determination in conformal SRT of intracranial lesions.
The planning target volume (PTV) includes the clinical target volume (CTV) to be irradiated and a margin to account for uncertainties in the treatment process. Uncertainties in miniature multileaf collimator (mMLC) leaf positioning, CT scanner spatial localization, CT-MRI image fusion spatial localization, and Gill-Thomas-Cosman (GTC) relocatable head frame repositioning were quantified for the purpose of determining a minimum PTV margin that still delivers a satisfactory CTV dose. The measured uncertainties were then incorporated into a simple Monte Carlo calculation for evaluation of various margin and fraction combinations. Satisfactory CTV dosimetric criteria were selected to be a minimum CTV dose of 95% of the PTV dose and at least 95% of the CTV receiving 100% of the PTV dose. The measured uncertainties were assumed to be Gaussian distributions. Systematic errors were added linearly and random errors were added in quadrature assuming no correlation to arrive at the total combined error. The Monte Carlo simulation written for this work examined the distribution of cumulative dose volume histograms for a large patient population using various margin and fraction combinations to determine the smallest margin required to meet the established criteria. The program examined 5 and 30 fraction treatments, since those are the only fractionation schemes currently used at our institution. The fractionation schemes were evaluated using no margin, a margin of just the systematic component of the total uncertainty, and a margin of the systematic component plus one standard deviation of the total uncertainty. It was concluded that (i) a margin of the systematic error plus one standard deviation of the total uncertainty is the smallest PTV margin necessary to achieve the established CTV dose criteria, and (ii) it is necessary to determine the uncertainties introduced by the specific equipment and procedures used at each institution since the uncertainties may vary among locations.


Molecular typing of the recently expanding subtype B HIV-1 epidemic in Romania: evidence for local spread among MSMs in Bucharest area.
HIV-1 subtype B is predominant in Europe except in some countries from Eastern Europe which are characterized by a high prevalence of non-B subtypes and circulating recombinant forms (CRFs). Romania is a particular case: the HIV-1 epidemic started with subtype F1 which is still the most prevalent. Previous studies have shown an increasing prevalence of subtype B which is the second most frequent one among the newly diagnosed individuals, followed by subtype C and several CRFs as well as unique recombinant forms (URFs). Our objective was to analyze in detail the characteristics (way of dispersal, association with transmission risk groups) of the subtype B infections in Romania by means of phylogenetic analysis. Among all the individuals sampled during 2003-2010, 71 out of 1127 patients (6.3%) have been identified to be infected with subtype B strains. The most frequent route of infection identified in HIV-1 subtype B patients in Romania was MSM transmission (39.6%), followed by the heterosexual route (35.2%). Many of the patients acquired the infection abroad, mainly in Western European countries. Phylogenetic analysis indicated the existence of a local transmission network (monophyletic clade) including 14 patients, mainly MSM living in the Bucharest area. We estimate the origin of the local transmission network that dates at the beginning of the 90s; the introduction of the F1 and C subtypes occurred earlier. The rest of the sequences were intermixed with reference strains sampled across Europe suggesting that single infection were not followed by subsequent dispersal within the local population. Although HIV-1 subtype B epidemic in Romania is recent, there is evidence for local spread among the MSMs, in addition to multiple introductions.


Efficacy of topical Rose (Rosa damascena Mill.) oil for migraine headache: A randomized double-blinded placebo-controlled cross-over trial.
To evaluate the effect of topical formulation of Rosa damascena Mill. (R. damascena) oil on migraine headache, applying syndrome diffrentiation model. Forty patients with migraine headache were randomly assigned to 2 groups of this double-blind, placebo-controlled cross-over trial. The patients were treated for the first 2 consecutive migraine headache attacks by topical R. damascena oil or placebo. Then, after one week of washout period, cross-over was done. Pain intensity of the patients' migraine headache was recorded at the beginnig and ten-sequence time schadule of attacks up to 24h. In addition, photophobia, phonophobia, and nausea and/or vomitting (N/V) of the patients were recorded as secondary outcomes. Finally, gathered data were analysed in a syndrome differentiation manner to assess the effect of R. damascena oil on Hot- and Cold-type migraine headache. Mean pain intensity of the patients' migraine headache in the different time-points after R. damascena oil or placebo use, was not significantly different. Additionally, regarding mean scores of N/V, photophobia, and phonophobia severity of the patients, no significant differences between the two groups were observed. Finally, applying syndrome differentiation model, the mean score of migraine headache pain intensity turned out to be significantly lower in patients with "hot" type migraine syndrome at in 30, 45, 60, 90, and 120min after R. damascena oil application compared to "cold" types (P values: 0.001, 0.001, <0.001, <0.001, and 0.02; respectively). It seems that syndrome differentiation can help in selection of patients who may benefit from the topical R. damascena oil in short-term relief of pain intensity in migraine headache. Further studies of longer follow-up and larger study population, however, are necessitated for more scientifically rigorous judgment on efficacy of R. damascena oil for patients with migraine headache.


A Decrease in CD44 on Cell Surfaces (MKN-45 cell line) After RELA Knockout Using CRISPR/Cas9.
The NF-kB signaling pathway was introduced as a key pathway in carcinogenesis that is induced by inflammation in gastrointestinal malignancies. The RelA transcription factor is an important component of this signaling pathway. Furthermore, CD44 is implicated in the tumorigenesis and metastasis of gastric cancer. The aim of this study was to assay the effect of RELA knockout on CD44 expression in MKN45 cells. CRISPR/Cas9 was used to knock out RELA in MKN-45. The median fluorescence intensity (MFI) of CD44 before and after RELA knockout is analyzed in MKN45. The CRISPR/Cas9 vector pSpCas9 (BB)-2A-Puro (PX459) was used for gRNA cloning (two guides). The MKN-45 cell line was co-transfected. The purified co-transfected cells with puromycin were cultured and used for the RELA gene expression assay by real-time PCR. Flow cytometry was used for the analysis of the MFI of CD44+ in MKN45. The results showed that 180 nucleotide sequences between exon 2 and exon 3 of RELA were deleted in MKN45. RELA expression significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells significantly decreased (P <0.001). Knockout of RELA significantly decreased CD44 expression in MKN45 cells. It can be concluded that the NF-kB signaling pathway via RELA is related to CD44 expression and consequently the tumorigenesis of gastric cancer. More studies about this relationship are recommended.


Effect of different human papillomavirus serological and DNA criteria on vaccine efficacy estimates.
Two trials of clinically approved human papillomavirus (HPV) vaccines, Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE I/II) and the Papilloma Trial Against Cancer in Young Adults (PATRICIA), reported a 22% difference in vaccine efficacy (VE) against cervical intraepithelial neoplasia grade 2 or worse in HPV-naïve subcohorts; however, serological testing methods and the HPV DNA criteria used to define HPV-unexposed women differed between the studies. We applied previously described methods to simulate these HPV-naïve subcohorts within the Costa Rica HPV16/18 Vaccine Trial and assessed how these criteria affect the estimation of VE. We applied 2 enzyme-linked immunosorbent assay (ELISA) thresholds for HPV16 and HPV18 seropositivity (8 and 7 ELISA units/mL, respectively, for PATRICIA; 54 and 65 ELISA units/mL, respectively, for FUTURE I/II (to approximate the competitive Luminex immunoassay)) and 2 criteria for HPV DNA positivity (12 oncogenic HPV types, plus HPV66 and 68/73 for PATRICIA; or plus HPV6 and 11 for FUTURE I/II). VE was computed in the 2 naïve subcohorts. Using the FUTURE I/II and PATRICIA criteria, VE estimates against cervical intraepithelial neoplasia grade 2 or worse, regardless of HPV type, were 69.0% (95% confidence interval: 40.3%, 84.9%) and 80.8% (95% confidence interval: 52.6%, 93.5%), respectively (P = 0.1). Although the application of FUTURE I/II criteria to our cohort resulted in the inclusion of more sexually experienced women, methodological differences did not fully explain the VE differences.


alpha2beta1 integrin expression in the tumor microenvironment enhances tumor angiogenesis in a tumor cell-specific manner.
To define the role of the alpha2beta1 integrin in pathologic angiogenesis, we investigated tumor-associated growth and angiogenesis in wild-type and alpha2-null mice. Our findings reveal that the alpha2beta1 integrin plays an important role in angiogenesis via regulation of VEGFR1 expression. When challenged with B16F10 melanoma cells, mice lacking alpha2beta1 integrin ex-pression exhibit increased tumor angiogenesis associated with up-regulated VEGFR1 expression. In contrast, there was no alpha2beta1 integrin-dependent difference in the angiogenic response to Lewis lung carcinoma (LLC) cells. Interestingly, whereas B16F10 cells secrete high levels of placental growth factor (PLGF), LLC cells produce high levels of VEGF, but low levels of PLGF. The alpha2beta1 integrin-dependent difference in angiogenesis was restored to LLC cells by expression of PLGF, strongly suggesting that the angiogenic phenotype and tumor growth in the alpha2-null host is dependent on specific interactions between the tumor cell and the genetically defined integrin repertoire of the host microenvironment. Thus integrin alpha2-null mice represent an example of genetic alterations of "the soil" determining response to the "seed."


Extremely low risk of hepatocellular carcinoma development in patients with chronic hepatitis B in immune-tolerant phase.
Anti-viral therapy is not indicated for patients with chronic hepatitis B (CHB) in the immune-tolerant phase. To investigate the cumulative incidence of phase change and hepatocellular carcinoma (HCC) and independent predictors for phase change in patients with CHB in immune-tolerant phase. In total, 946 patients in immune-tolerant phase, defined as hepatitis B e antigen positivity, HBV-DNA >20 000 IU/mL and alanine aminotransferase (ALT) ≤40 IU/L, between 1989 and 2017 were enrolled from eight institutes. The mean age of study population (429 men and 517 women) was 36.7 years. The mean ALT and HBV-DNA levels were 24.6 IU/L and 8.50 log10 IU/mL, respectively. Of the study population, 476 (50.3%) patients remained in immune-tolerant phase throughout the study period (median: 63.6 months). The cumulative incidence rates of phase change and HCC at 10 years were 70.7% and 1.7%, respectively. Multivariate analyses revealed that HBV-DNA level >107 IU/mL was associated independently with a reduced risk of phase change (hazard ratio [HR] = 0.734, P = 0.008), whereas a high ALT level, above the cut-off recommended in the Korean Association for the Study of the Liver guidelines (34 IU/L for men and 30 IU/L for women), was associated independently with a greater risk of phase change (HR = 1.885, P < 0.001). The criterion of HBV-DNA level > 107 IU/mL may be useful to define immune-tolerant phase. In addition, an extremely low risk of HCC development was observed in patients with CHB in immune-tolerant phase.


Endothelium-dependent contractions induced by angiotensin I and angiotensin II in canine cerebral artery.
Whether angiotensin I and angiotensin II caused endothelium-dependent contraction was examined in canine cerebral arteries. In endothelium-intact preparations, angiotensin I and angiotensin II at 10(-8) to 10(-6) M caused dose-dependent contractions, whereas both angiotensins caused much less contractions in endothelium-removed preparations. The contractions induced by angiotensin I and angiotensin II were strongly attenuated by aspirin (cyclooxygenase inhibitor) (5 x 10(-5) M), OKY-046 [thromboxane (TX) A2 synthetase inhibitor] (10(-5) M) and ONO-3708 (TX)A2 antagonist) (5 X 10(-9) M). Captopril (10(-6) M) significantly attenuated the contractions induced by angiotensin I but not those induced by angiotensin II. Angiotensin I- and angiotensin II- induced contractions were inhibited markedly by Sar1, Ala8-angiotensin II (10(-9) and 10(-8) M). The present experiments demonstrate that angiotensin I and angiotensin II produce endothelium-dependent contraction in canine cerebral artery via a factor which appears to be TXA2. Angiotensin I may be converted by endothelial cells to angiotensin II, which may activate the cells to produce TXA2 in canine cerebral artery.


Functional Connectivity of the Corpus Callosum in Epilepsy Patients with Secondarily Generalized Seizures.
The corpus callosum (CC) plays an important role in generalization of seizure activity. We used resting-state function magnetic resonance imaging (rs-fMRI) to investigate the regional and interregional functional connectivity of CC in patients with magnetic resonance imaging (MRI)-negative and secondarily generalized seizures. We measured the multi-regional coherences of blood oxygen level-dependent (BOLD) signals via rs-fMRI, cortical thickness via high-resolution T1-weighted MRI, and white matter (WM) integrity via diffusion-tensor imaging in 16 epilepsy patients as well as in 16 age- and gender-matched healthy subjects. All patients had non-lesional MRI, medically well-controlled focal epilepsy and history of secondarily generalized convulsions. Individuals with epilepsy had significant differences in regional and interregional hypersynchronization of BOLD signals intrahemispherically and interhemispherically, but no difference in cortical thickness and WM integrity. The only area with increased regional hypersynchrony in WM was over the anterior CC, which also exhibited lower activation of neighboring resting-state networks. The present study revealed abnormal local and distant synchronization of spontaneous neural activities in epileptic patients with secondarily generalized seizures.


Fine-scale variability in harbor seal foraging behavior.
Understanding the variability of foraging behavior within a population of predators is important for determining their role in the ecosystem and how they may respond to future ecosystem changes. However, such variability has seldom been studied in harbor seals on a fine spatial scale (<30 km). We used a combination of standard and Bayesian generalized linear mixed models to explore how environmental variables influenced the dive behavior of harbor seals. Time-depth recorders were deployed on harbor seals from two haul-out sites in the Salish Sea in 2007 (n = 18) and 2008 (n = 11). Three behavioral bout types were classified from six dive types within each bout; however, one of these bout types was related to haul-out activity and was excluded from analyses. Deep foraging bouts (Type I) were the predominant type used throughout the study; however, variation in the use of bout types was observed relative to haul-out site, season, sex, and light (day/night). The proportional use of Type I and Type II (shallow foraging/traveling) bouts differed dramatically between haul-out sites, seasons, sexes, and whether it was day or night; individual variability between seals also contributed to the observed differences. We hypothesize that this variation in dive behavior was related to habitat or prey specialization by seals from different haul-out sites, or individual variability between seals in the study area. The results highlight the potential influence of habitat and specialization on the foraging behavior of harbor seals, and may help explain the variability in diet that is observed between different haul-out site groups in this population.


Circulating CD14+CD16+ monocyte subsets as biomarkers of the severity of coronary artery disease in patients with stable angina pectoris.
Circulating monocytes can be divided into 2 subsets typically identified by the expression of CD14 and CD16. Although previous studies have shown that circulating monocytes contribute to the progression of coronary atherosclerotic lesions, the relationship between the severity of coronary artery disease (CAD) and the 2 distinct monocyte subsets has not previously been evaluated. We investigated the relationship between the monocyte subsets and the severity of CAD assessed by coronary angiography (CAG) in patients with stable angina pectoris (SAP). We enrolled 125 patients who underwent diagnostic CAG. Patients were divided into 3 groups: those without CAD, those with single-vessel disease (SVD), and those with multiple-vessel disease (MVD). In addition, the severity of CAD was evaluated by Gensini score. The 2 monocyte subsets (CD14(+)CD16(-) and CD14(+)CD16(+)) were measured by flow cytometry. Circulating CD14(+)CD16(+) monocytes were more frequently observed in patients with MVD than in those with SVD or without CAD. The proportion of CD14(+)CD16(+) monocytes positively correlated with Gensini score (r=0.618, P<0.001). Multivariate logistic regression analysis revealed that the proportion of CD14(+)CD16(+) monocytes was an independent contributor to MVD (odds ratio: 1.475; 95% confidence interval: 1.273-1.708, P<0.001). A preferential increase in peripheral CD14(+)CD16(+) monocytes may be closely related to the severity of CAD in patients with SAP.


Thyroid hormone transporters--functions and clinical implications.
The cellular influx and efflux of thyroid hormones are facilitated by transmembrane protein transporters. Of these transporters, monocarboxylate transporter 8 (MCT8) is the only one specific for the transport of thyroid hormones and some of their derivatives. Mutations in SLC16A2, the gene that encodes MCT8, lead to an X-linked syndrome with severe neurological impairment and altered concentrations of thyroid hormones. Histopathological analysis of brain tissue from patients who have impaired MCT8 function indicates that brain lesions start prenatally, and are most probably the result of cerebral hypothyroidism. A Slc16a2 knockout mouse model has revealed that Mct8 is an important mediator of thyroid hormone transport, especially T3, through the blood-brain barrier. However, unlike humans with an MCT8 deficiency, these mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood-brain barrier; OATP1C1 is more abundant in rodents than in primates and permits the passage of T4 in the absence of T3 transport, thus preventing full cerebral hypothyroidism. In this Review, we discuss the relevance of thyroid hormone transporters in health and disease, with a particular focus on the pathophysiology of MCT8 mutations.


Garcinone E induces apoptosis and inhibits migration and invasion in ovarian cancer cells.
Ovarian cancer remains the most lethal gynecological malignant tumor. In this study, 24 xanthones were isolated and identified from the pericarps of mangosteen (Garcinia mangostana), and their anti-proliferative activities were tested in ovarian cancer cells. Garcinone E (GE) was found to exhibit excellent anti-proliferative effects among the tested xanthones. It significantly inhibited the proliferation in HEY, A2780, and A2780/Taxol cells as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, Hoechst 33342 staining, annexin V/PI staining, and JC-1 staining. It induced endoplasmic reticulum (ER) stress and activated the protective inositol-requiring kinase (IRE)-1α pathway. Knocking down IRE-1α further activated the caspase cascade and caused an increase in cell death. Moreover, GE eliminated the migratory ability of HEY cells by reducing the expression of RhoA and Rac. It also blocked the invasion, which might be related to downregulation of matrix metalloproteinases (MMPs), i.e., MMP-9 and MMP-2, and upregulation of tissue inhibitors of metalloproteinase (TIMP) -1 and TIMP-2. In summary, GE exerts anticancer activities by inducing apoptosis and suppressing migration and invasion in ovarian cancer cells, which indicates its therapeutic potential for ovarian cancer.


Accounting for epistasis in linkage analysis of general pedigrees.
Complex traits are generally believed to be influenced by multiple loci. Identification of loci involved in complex traits is more difficult for interacting than for additive loci. Here we describe an extension of the program lm_twoqtl in the package MORGAN to handle two quantitative trait loci (QTLs) with gene-gene interaction. We investigate whether parametric linkage analysis that accounts for such epistasis improves prospects for linkage detection and accuracy of localization of QTLs. Through use of simulated data we show that analysis that accounts for epistasis provides higher lod scores and better localization than does analysis without epistasis. In addition, we demonstrate that the difference between lod scores in the presence vs. absence of use of an interaction model in analysis is greater in extended than in nuclear pedigrees.


Low-dose computed tomography versus plain abdominal radiography in the investigation of an acute abdomen.
To compare low-dose abdominal computed tomography (LDCT) with plain abdominal radiography (AR) in the primary investigation of acute abdominal pain to determine if there is a difference in diagnostic yield, the number of additional investigations required and hospital length of stay (LOS). This randomized controlled trial was approved by the institutional review board, and informed consent was obtained. Patients presenting to the emergency department with an acute abdomen and who would normally be investigated with AR were randomized to either AR or LDCT. The estimated radiation dose of the LDCT protocol was 2-3 mSv compared to 1.1 mSv for AR. Pearson's chi-square and the independent samples t-test were used for the statistical analysis. A total of 142 patients were eligible, and after exclusions and omitting those with incomplete data, 55 patients remained for analysis in the AR arm and 53 in the LDCT arm. A diagnosis could be obtained in 12 (21.8%) patients investigated with AR compared to 34 (64.2%) for LDCT (P < 0.001). Twenty-eight (50.9%) patients in the AR group required further imaging during their admission compared to 14 (26.4%) in the LDCT group (P= 0.009). There was no difference in the median hospital LOS (3.84 days for AR versus 4.24 days for LDCT, P= 0.83). LDCT demonstrates a superior diagnostic yield over AR and reduces the number of subsequent imaging tests for a minimal cost in radiation exposure. However, there is no difference in the overall hospital LOS between the two imaging strategies.


Perceptions of the importance of different welfare issues in livestock production.
The opinions of seven respondent groups about the relative importance of different practices pertaining to the welfare of Australian beef cattle, sheep and goats were surveyed. Respondent groups comprised farmers, livestock transportation representatives, veterinarians, meat processors, animal welfare advocates, animal welfare scientists and government officers. The survey consisted of a web-based adaptive conjoint analysis questionnaire, which was administered to a sample population that was selected randomly for large respondent groups and comprehensively for small groups. The hierarchy of opinion concerning the importance of the different beef cattle practices was: stockmanship > ground (road and rail) transport > spaying > food supply > dehorning > stunning > shelter > identification > pretransport food and water deprivation > castration > sea transport > mustering > confinement. For sheep/goat practices the hierarchy was: parasite control > mulesing > shelter > stockmanship > tail docking > ground transport > feeding > predation > stunning > castration > pretransport food and water deprivation > sea transport > mustering. The method of performing invasive procedures was perceived as less important than the provision of pain relief. Differences in opinion were evident between respondent groups, with animal welfare advocates tending to focus on painful procedures more than those with direct involvement in the industry.


Molecular Pathways Mediating Immunosuppression in Response to Prolonged Intensive Physical Training, Low-Energy Availability, and Intensive Weight Loss.
Exercise and exercise-induced weight loss have a beneficial effect on overall health, including positive effects on molecular pathways associated with immune function, especially in overweight individuals. The main aim of our study was to assess how energy deprivation (i.e., "semi-starvation") leading to substantial fat mass loss affects the immune system and immunosuppression in previously normal weight individuals. Thus, to address this hypothesis, we applied a high-throughput systems biology approach to better characterize potential key pathways associated with immune system modulation during intensive weight loss and subsequent weight regain. We examined 42 healthy female physique athletes (age 27.5 ± 4.0 years, body mass index 23.4 ± 1.7 kg/m2) volunteered into either a diet group (n = 25) or a control group (n = 17). For the diet group, the energy intake was reduced and exercise levels were increased to induce loss of fat mass that was subsequently regained during a recovery period. The control group was instructed to maintain their typical lifestyle, exercise levels, and energy intake at a constant level. For quantification of systems biology markers, fasting blood samples were drawn at three time points: baseline (PRE), at the end of the weight loss period (MID 21.1 ± 3.1 weeks after PRE), and at the end of the weight regain period (POST 18.4 ± 2.9 weeks after MID). In contrast to the control group, the diet group showed significant (false discovery rate <0.05) alteration of all measured immune function parameters-white blood cells (WBCs), immunoglobulin G glycome, leukocyte transcriptome, and cytokine profile. Integrative omics suggested effects on multiple levels of immune system as dysregulated hematopoiesis, suppressed immune cell proliferation, attenuated systemic inflammation, and loss of immune cell function by reduced antibody and chemokine secretion was implied after intense weight loss. During the weight regain period, the majority of the measured immune system parameters returned back to the baseline. In summary, this study elucidated a number of molecular pathways presumably explaining immunosuppression in individuals going through prolonged periods of intense training with low-energy availability. Our findings also reinforce the perception that the way in which weight loss is achieved (i.e., dietary restriction, exercise, or both) has a distinct effect on how the immune system is modulated.


The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis.
The establishment and maintenance of polarity is vital for embryonic development and loss of polarity is a frequent characteristic of epithelial cancers, however the underlying molecular mechanisms remain unclear. Here, we identify a novel role for the polarity protein Scrib as a mediator of epidermal permeability barrier acquisition, skeletal morphogenesis, and as a potent tumor suppressor in cutaneous carcinogenesis. To explore the role of Scrib during epidermal development, we compared the permeability of toluidine blue dye in wild-type, Scrib heterozygous and Scrib KO embryonic epidermis at E16.5, E17.5 and E18.5. Mouse embryos were stained with alcian blue and alizarin red for skeletal analysis. To establish whether Scrib plays a tumor suppressive role during skin tumorigenesis and/or progression, we evaluated an autochthonous mouse model of skin carcinogenesis in the context of Scrib loss. We utilised Cre-LoxP technology to conditionally deplete Scrib in adult epidermis, since Scrib KO embryos are neonatal lethal. We establish that Scrib perturbs keratinocyte maturation during embryonic development, causing impaired epidermal barrier formation, and that Scrib is required for skeletal morphogenesis in mice. Analysis of conditional transgenic mice deficient for Scrib specifically within the epidermis revealed no skin pathologies, indicating that Scrib is dispensable for normal adult epidermal homeostasis. Nevertheless, bi-allelic loss of Scrib significantly enhanced tumor multiplicity and progression in an autochthonous model of epidermal carcinogenesis in vivo, demonstrating Scrib is an epidermal tumor suppressor. Mechanistically, we show that apoptosis is the critical effector of Scrib tumor suppressor activity during skin carcinogenesis and provide new insight into the function of polarity proteins during DNA damage repair. For the first time, we provide genetic evidence of a unique link between skin carcinogenesis and loss of the epithelial polarity regulator Scrib, emphasizing that Scrib exerts a wide-spread tumor suppressive function in epithelia.


Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.
A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex.


Personal and parental weight misperception and self-reported attempted weight loss in US children and adolescents, National Health and Nutrition Examination Survey, 2007-2008 and 2009-2010.
The objective of our study was to describe perceptions of child weight status among US children, adolescents, and their parents and to examine the extent to which accurate personal and parental perception of weight status is associated with self-reported attempted weight loss. Our study sample comprised 2,613 participants aged 8 to 15 years in the National Health and Nutrition Examination Survey from the 2 most recent consecutive cycles (2007-2008 and 2009-2010). Categories of weight perception were developed by comparing measured to perceived weight status. Multivariable logistic regression analyses were used to examine the association between weight misperception and self-reported attempted weight loss. Among children and adolescents, 27.3% underestimated and 2.8% overestimated their weight status. Among parents, 25.2% underestimated and 1.1% overestimated their child's weight status. Logistic regression analyses showed that the odds of self-reported attempted weight loss was 9.5 times as high (95% confidence interval [CI]: 3.8-23.6) among healthy-weight children and adolescents who overestimated their weight status as among those who perceived their weight status accurately; the odds of self-reported attempted weight loss were 3.9 (95% CI, 2.4-6.4) and 2.9 (95% CI, 1.8-4.6) times as high among overweight and obese children and adolescents, respectively, who accurately perceived their weight status than among those who underestimated their weight status. Parental misperception of weight was not significantly associated with self-reported attempted weight loss among children and adolescents who were overweight or obese. Efforts to prevent childhood obesity should incorporate education for both children and parents regarding the proper identification and interpretation of actual weight status. Interventions for appropriate weight loss can target children directly because one of the major driving forces to lose weight comes from the child's perception of his or her weight status.


Structural sources of robustness in biochemical reaction networks.
In vivo variations in the concentrations of biomolecular species are inevitable. These variations in turn propagate along networks of chemical reactions and modify the concentrations of still other species, which influence biological activity. Because excessive variations in the amounts of certain active species might hamper cell function, regulation systems have evolved that act to maintain concentrations within tight bounds. We identify simple yet subtle structural attributes that impart concentration robustness to any mass-action network possessing them. We thereby describe a large class of robustness-inducing networks that already embraces two quite different biochemical modules for which concentration robustness has been observed experimentally: the Escherichia coli osmoregulation system EnvZ-OmpR and the glyoxylate bypass control system isocitrate dehydrogenase kinase-phosphatase-isocitrate dehydrogenase. The structural attributes identified here might confer robustness far more broadly.


Evaluation and Minimization of the Pseudohepatic Anisotropy Artifact in Liver Intravoxel Incoherent Motion.
The aim of this study was to evaluate the effect of the pseudohepatic anisotropy artifact on liver intravoxel incoherent motion (IVIM) metrics and whether the use of multiple gradient directions in the IVIM acquisition minimizes the artifact. Multiple breath-holding and forced shallow free-breathing IVIM scans were performed on 8 healthy volunteers using 1 and 6 gradient directions. Cluster analysis was carried out to separate motion-contaminated parenchyma from liver parenchyma and vessels. Nonlinear motion analysis was also performed to look for a possible link between IVIM metrics and nonlinear liver motion. On the basis of the resulted clusters, motion-contaminated parenchyma is often noted in the left liver lobe, where the prominent pseudohepatic artifact has previously been identified. A significant reduction in outliers was obtained with the acquisition of 6 noncoplanar gradient directions and when using forced shallow free-breathing. The pseudohepatic anisotropy artifact can be minimized when using multiple diffusion-encoding gradient directions and forced free-breathing during IVIM acquisition.


Clinicopathologic analysis of intraductal papillary neoplasm of bile duct: Korean multicenter cohort study.
IPNB is very rare disease and most previous studies on IPNB were case series with a small number due to low incidence. The aim of this study is to validate previously known clinicopathologic features of intraductal papillary neoplasm of bile duct (IPNB) based on the first largest multicenter cohort. Among 587 patients previously diagnosed with IPNB and similar diseases from each center in Korea, 387 were included in this study after central pathologic review. We also reviewed all preoperative image data. Of 387 patients, 176 (45.5%) had invasive carcinoma and 21 (6.0%) lymph node metastasis. The 5-year overall survival was 80.9% for all patients, 88.8% for IPNB with mucosal dysplasia, and 70.5% for IPNB with invasive carcinoma. According to the "Jang & Kim's modified anatomical classification," 265 (68.5%) were intrahepatic, 103 (26.6%) extrahepatic, and 16 (4.1%) diffuse type. Multivariate analysis revealed that tumor invasiveness was a unique predictor for survival analysis. (p = 0.047 [hazard ratio = 2.116, 95% confidence interval 1.010-4.433]). This is the first Korean multicenter study on IPNB through central pathologic and radiologic review process. Although IPNB showed good long-term prognosis, relatively aggressive features were also found in invasive carcinoma and extrahepatic/diffuse type.


Lotus Root Extract Stimulates BDNF Gene Expression Through Potential Mechanism Depending on HO-1 Activity in C6 Glioma Cells.
Polyphenolic compounds have been suggested to be involved in the preservation of neural function via the production of neurotrophic factors in the brain. The nonedible joint part of lotus root (a rhizome of Nelumbo nucifera) has been reported to contain large amounts of polyphenolic compounds and, therefore, is expected to improve neural function by stimulating the production of brain-derived neurotrophic factor (BDNF) in glial cells. The effect of the aqueous extract prepared from the joint part of lotus root on BDNF gene expression was examined in C6 glioma cells as an in vitro model. This extract was shown to increase BDNF messenger ribonucleic acid (mRNA) levels to the elevation of HO-1 mRNA levels in the glioma cells, but failed to cause the elevation of BDNF mRNA levels in the cells pretreated with a HO-1 antisense oligodeoxynucleotide (ODN) or an HO-1 enzyme inhibitor zinc protoporphyrin (ZnPP). These findings strongly suggest that the aqueous extract prepared from the nonedible joint part of lotus root might be able to stimulate BDNF gene expression by enhancing HO-1 activity in the glioma cells, proposing the possibility that the joint part of lotus root might potentially improve neural function through the stimulation of BDNF production in glial cells.


Developing approaches for linear mixed modeling in landscape genetics through landscape-directed dispersal simulations.
Dispersal can impact population dynamics and geographic variation, and thus, genetic approaches that can establish which landscape factors influence population connectivity have ecological and evolutionary importance. Mixed models that account for the error structure of pairwise datasets are increasingly used to compare models relating genetic differentiation to pairwise measures of landscape resistance. A model selection framework based on information criteria metrics or explained variance may help disentangle the ecological and landscape factors influencing genetic structure, yet there are currently no consensus for the best protocols. Here, we develop landscape-directed simulations and test a series of replicates that emulate independent empirical datasets of two species with different life history characteristics (greater sage-grouse; eastern foxsnake). We determined that in our simulated scenarios, AIC and BIC were the best model selection indices and that marginal R2 values were biased toward more complex models. The model coefficients for landscape variables generally reflected the underlying dispersal model with confidence intervals that did not overlap with zero across the entire model set. When we controlled for geographic distance, variables not in the underlying dispersal models (i.e., nontrue) typically overlapped zero. Our study helps establish methods for using linear mixed models to identify the features underlying patterns of dispersal across a variety of landscapes.


Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.
New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.


Safety and Short-Term Outcomes of Therapeutic Hypothermia in Preterm Neonates 34-35 Weeks Gestational Age with Hypoxic-Ischemic Encephalopathy.
To evaluate the safety and short-term outcomes of preterm neonates born at 34-35 weeks gestation with hypoxic-ischemic encephalopathy (HIE) treated with therapeutic hypothermia. Medical records of preterm neonates born at 34-35 weeks gestational age with HIE treated with therapeutic hypothermia were retrospectively reviewed. Short-term safety outcomes and the presence, severity (mild, moderate, severe), and patterns of brain injury on magnetic resonance imaging were reviewed using a standard scoring system, and compared with a cohort of term neonates with HIE treated with therapeutic hypothermia. Thirty-one preterm and 32 term neonates were identified. Therapeutic hypothermia-associated complications were seen in 90% of preterm infants and 81.3% of term infants (P = .30). In the preterm infants, hyperglycemia (58.1% vs31.3%, P = .03) and rewarming before completion of therapeutic hypothermia (19.4% vs 0.0%, P = .009) were more likely compared with term infants. All deaths occurred in the preterm group (12.9% vs 0%, P = .04). Neuroimaging showed the presence of injury in 80.6% of preterm infants and 59.4% of term infants (P = .07), with no differences in injury severity. Injury to the white matter was more prevalent in preterm infants compared with term infants (66.7% vs 25.0%, P = .001). Therapeutic hypothermia in infants born at 34-35 weeks gestational age appears feasible. Risks of mortality and side effects warrant caution with use of therapeutic hypothermia in preterm infants.


Imaging precessional motion of the magnetization vector.
We report on imaging of three-dimensional precessional orbits of the magnetization vector in a magnetic field by means of a time-resolved vectorial Kerr experiment that measures all three components of the magnetization vector with picosecond resolution. Images of the precessional mode taken with submicrometer spatial resolution reveal that the dynamical excitation in this time regime roughly mirrors the symmetry of the underlying equilibrium spin configuration and that its propagation has a non-wavelike character. These results should form the basis for realistic models of the magnetization dynamics in a largely unexplored but technologically increasingly relevant time scale.


Comparing functional and morphologic characteristics of lamellar macular holes with and without lamellar hole-associated epiretinal proliferation.
To compare the functional and morphologic characteristics and evolution of lamellar macular holes (LMHs) with and without lamellar hole-associated epiretinal proliferation (LHEP). This was a retrospective observational case review of 145 eyes of 136 patients with LMH seen in a vitreoretinal clinical practice, and the eyes were subdivided into 2 groups based on the presence or absence of LHEP. Main outcome measures were logarithm of minimal angle of resolution (logMAR) visual acuity and morphologic characteristics as seen with spectral domain optical coherence tomography over retrospective follow-up. In 62 eyes (42.7%), LHEP was detected, while 83 eyes (57.3%) had the presence of epiretinal membrane without LHEP. The mean logMAR visual acuity in eyes with LHEP was 0.51 (20/65 Snellen equivalent), which was significantly poorer than that in the eyes without LHEP at 0.33 (20/43 Snellen equivalent, P = 0.002). Multivariate analysis showed that the presence of LHEP was significantly associated with larger LMH diameter at the middle retinal level (P = 0.01) and thinner retinal thickness at the base of the LMH (P < 0.001). A higher proportion of eyes with LHEP (88%) had ellipsoid disruption compared with eyes without LHEP (24%, P = 0.001). Over the mean retrospective follow-up of 26 months, 5% of eyes with LHEP had functional decline of 0.3 logMAR visual acuity compared with 4% of eyes without LHEP (P = 0.99), whereas 18% of eyes with LHEP had morphologic progression compared with 13% of eyes without LHEP (P = 0.49). Eyes with LMH and LHEP were associated with poorer visual acuity, larger LMH diameters, thinner retinal thickness, and higher incidence of ellipsoid disruption compared with eyes without LHEP, suggesting a process involving more severe retinal tissue loss and injury. Both LMH with and without LHEP seemed to be stable configurations over time.


Electrical spin injection and transport in semiconductor nanowires: challenges, progress and perspectives.
Spintronic devices are of fundamental interest for their nonvolatility and great potential for low-power electronics applications. The implementation of those devices usually favors materials with long spin lifetime and spin diffusion length. Recent spin transport studies on semiconductor nanowires have shown much longer spin lifetimes and spin diffusion lengths than those reported in bulk/thin films. In this paper, we have reviewed recent progress in the electrical spin injection and transport in semiconductor nanowires and drawn a comparison with that in bulk/thin films. In particular, the challenges and methods of making high-quality ferromagnetic tunneling and Schottky contacts on semiconductor nanowires as well as thin films are discussed. Besides, commonly used methods for characterizing spin transport have been introduced, and their applicability in nanowire devices are discussed. Moreover, the effect of spin-orbit interaction strength and dimensionality on the spin relaxation and hence the spin lifetime are investigated. Finally, for further device applications, we have examined several proposals of spinFETs and provided a perspective of future studies on semiconductor spintronics.


Titanium(IV) catalysts with ancillary imino-spiroketonato ligands: synthesis, structure and olefin polymerization.
New titanium(IV) complexes having two bidentate β-iminoethyl-spiro[4,5]decan-6-onato ligands with various N-aryl substituents have been synthesized. X-ray crystal structure analysis reveals that these titanium complexes all exhibit a C2-symmetric conformation with a distorted octahedral geometry, although the specific orientation of the ligands around the titanium center varies with the identity of the N-aryl moiety. Upon activation with methylaluminoxane (MAO), these complexes catalyze the polymerization of ethylene and propylene. In the case of ethylene, most complexes exhibit the characteristics of a living polymerization between 0 °C and 25 °C, producing polyethylenes with narrow molecular weight distributions and number average molecular weights up to 100,000 g/mol. Depending on the N-aryl substituents, polymerizations of propylene result in products with tacticity ranging from slightly syndiotactic to slightly isotactic.


Assessing dentin color changes from nightguard vital bleaching.
At-home bleaching with 10 percent carbamide peroxide in a custom-fitted tray has been reported to change the color of dentin. The purpose of this study was to validate the color change of dentin and to determine whether the color change was uniform or occurred from the outside (the dentinoenamel junction) to the inside (the pulpal wall). The authors sectioned 10 extracted human teeth incisogingivally through the midfacial long axis, and sealed their cut surface against glass microscope slides. Identifying marks were placed on the glass over the tooth sections to serve as a color control and in the dentinal areas closest to the dentinoenamel junction and the pulpal wall. Teeth were bleached for 10 days with 10 percent carbamide peroxide. Photographs were taken from the glass-covered side of the teeth, digitized and converted to gray-scale levels (consisting of 256 shades of gray ranging from black to white). Marked areas were measured with a National Institutes of Health Image software program and analyzed statistically for changes in lightness between the control marks and the inner and outer dentinal marks over time. Paired t-tests and analysis of variance indicated a significant increase in lightness (P = .01) for the inner and outer dentinal areas during bleaching compared with the control areas. No significant differences were found in the rate of change for the inner and outer dentinal areas (P = .89). The increase in lightness confirms that a significant color change occurred in the dentin during bleaching with 10 percent carbamide peroxide. This change occurred throughout the dentin at a uniform rate, rather than from the outside inward. The results of this study show that at-home bleaching with 10 percent carbamide peroxide can change the color of dentin, which is important to treat intrinsic stains from tetracycline treatment, trauma and aging or inherited discolorations. The bleaching material easily penetrates the tooth to change the dentin color at the same rate throughout, indicating that the type of stain may be the important factor in determining bleaching success.


Harmonized outcome measures for use in degenerative lumbar spondylolisthesis patient registries and clinical practice.
The development of new treatment approaches for degenerative lumbar spondylolisthesis (DLS) has introduced many questions about comparative effectiveness and long-term outcomes. Patient registries collect robust, longitudinal data that could be combined or aggregated to form a national and potentially international research data infrastructure to address these and other research questions. However, linking data across registries is challenging because registries typically define and capture different outcome measures. Variation in outcome measures occurs in clinical practice and other types of research studies as well, limiting the utility of existing data sources for addressing new research questions. The purpose of this project was to develop a minimum set of patient- and clinician-relevant standardized outcome measures that are feasible for collection in DLS registries and clinical practice. Nineteen DLS registries, observational studies, and quality improvement efforts were invited to participate and submit outcome measures. A stakeholder panel was organized that included representatives from medical specialty societies, health systems, government agencies, payers, industries, health information technology organizations, and patient advocacy groups. The panel categorized the measures using the Agency for Healthcare Research and Quality's Outcome Measures Framework (OMF), identified a minimum set of outcome measures, and developed standardized definitions through a consensus-based process. The panel identified and harmonized 57 outcome measures into a minimum set of 10 core outcome measure areas and 6 supplemental outcome measure areas. The measures are organized into the OMF categories of survival, clinical response, events of interest, patient-reported outcomes, and resource utilization. This effort identified a minimum set of standardized measures that are relevant to patients and clinicians and appropriate for use in DLS registries, other research efforts, and clinical practice. Collection of these measures across registries and clinical practice is an important step for building research data infrastructure, creating learning healthcare systems, and improving patient management and outcomes in DLS.


Beliefs about Pain Control in Patients after Abdominal Aortic Aneurysm Surgery-A Preliminary Study.
Introduction: Pain-control beliefs significantly influence the perception of disease and, therefore, may influence the treatment outcomes of surgical patients. The sense of control is related to the sense of agency and the ability to influence one's own life and environment. This construct may be external or internal. The belief that pain control depends on internal or external factors can depend on many variables. This may be influenced by socio-demographic and clinical characteristics, as well as the source and cause of pain. The aim of the study was the assessment of the relationship between the intensity of postoperative pain and beliefs about pain control in patients after AAA surgery and assessment of the relationship between socio-demographic and clinical variables and beliefs about pain control in patients after AAA surgery. Materials and Methods: The research material consisted of 42 patients aged 57 to 85, hospitalized at the Department of Cardiac Surgery and Vascular Surgery of the University Clinical Center in Gdańsk. The research was conducted from March to September 2020. The study uses a survey technique based on a standardized research tool: the Polish version of the BPCQ (The Beliefs about Pain Control Questionnaire), the NRS (Numerical Rating Scale), and the author's own questionnaire that allows for the collection of socio-demographic data. Results: The highest intensity of pain was observed in subjects with ruptured AAA H (2) = 6.19; p < 0.05 and subjects who underwent classic surgery Z = −2.95; p < 0.05 (non-parametric Mann−Whitney U test). Subjects with ruptured aneurysms are less convinced about the influence of internal factors on pain control H (2) = 5.26; p < 0.05. The respondents' conviction about the influence of doctors on pain control increased together with their age, rHO = 0.38, p < 0.05. Conclusion: Pain intensity after surgery did not significantly correlate with beliefs about pain control. Patients with ruptured AAA are less convinced about the influence of internal factors on pain control. With age, patients have more confidence in their doctors than in themselves to control their pain.


Clinical and Economic Impact of a Potential Switch from 13-Valent to 10-Valent Pneumococcal Conjugate Infant Vaccination in Canada.
Pneumococcal conjugate vaccines (PCVs) have been available in Canada since 2001, with 13-valent PCV (PCV13) added to the infant routine immunization program throughout all Canadian provinces by 2011. The use of PCVs has dramatically reduced the burden of pneumococcal disease in Canada. As a result, decision-makers may consider switching from a more costly, higher-valent vaccine to a lower-cost, lower-valent vaccine in an attempt to allocate funds for other vaccine programs. We assessed the health and economic impact of switching the infant vaccination program from PCV13 to 10-valent PCV (PCV10) in the context of the Canadian health care system. We performed a review of Canadian databases supplemented with published and unpublished data to obtain the historical incidence of pneumococcal disease and direct and indirect medical costs. Observed invasive pneumococcal disease (IPD) trends from surveillance data were used as a basis to forecast the future number of cases of IPD, pneumococcal pneumonia, and acute otitis media given a PCV13- or PCV10-based program. Costs and outcomes over 10 years were then estimated and presented in 2017 Canadian dollars discounted at 3% per year. Switching from PCV13 to PCV10 would result in an additional 762,531 cases of pneumococcal disease over 10 years. Although PCV13 has a higher acquisition cost, switching to PCV10 would increase overall costs by over $500 million. Forecasted overall disease incidence was estimated substantially higher with PCV10 than with PCV13 primarily because of the potential reemergence of serotypes 3 and 19A. PCV13 was also cost saving compared with PCV10, even within a 5-year time horizon. Probabilistic sensitivity analysis showed that a PCV13-based program remained cost saving in all simulations. Although switching to a PCV10-based infant vaccination program in Canada might result in lower acquisition costs, it would also result in higher public health cost and burden because of serotype reemergence. Pfizer Inc.


Self-limiting outbreak of crayfish plague in an Austropotamobius pallipes population of a river basin in the Abruzzi region (central Italy).
Crayfish plague, caused by the oomycete Aphanomyces astaci, is a serious disease of European freshwater crayfish and has eliminated entire populations in several European countries. In September 2011, mortality was observed among the Austropotamobius pallipes population of a river basin in the Abruzzi region (central Italy), and A. astaci DNA was detected by PCR in dead crayfish. A systematic survey was carried out to evaluate the spread and the effects of the plague in the river basin. The source of the outbreak remained unknown since North American crayfish species, which frequently act as subclinical carriers of the infection, were not detected in the area. The A. pallipes population disappeared from a river stretch of ~1 km, where A. astaci infection was detected in dead crayfish. However, apparently unaffected crayfish were still present upstream of that area as well as in a tributary that joined the brook in the apparently depopulated stretch. A. astaci infection was not detected in dead individuals collected in the upstream area and tributary. A follow-up visit conducted in the following season showed the presence of A. pallipes in the river stretch hit by the plague. In this outbreak, the spread of the infection could have been limited by a low density of the crayfish population and by the geographic conformation of the river basin, which includes a dense network of small tributaries, characterized by high flow velocity and low water temperature. In this particular setting, crayfish plague outbreaks can remain undetected. This underlines the importance of active monitoring programs aimed at the prompt recognition of both episodes of mortality and the presence of non-indigenous crayfish species.


Chronic and Acute Ozone Exposure in the Week Prior to Delivery Is Associated with the Risk of Stillbirth.
Chronic and acute air pollution has been studied in relation to stillbirth with inconsistent findings. We examined stillbirth risk in a retrospective cohort of 223,375 singleton deliveries from 12 clinical sites across the United States. Average criteria air pollutant exposure was calculated using modified Community Multiscale Air Quality models for the day of delivery and each of the seven days prior, whole pregnancy, and first trimester. Poisson regression models using generalized estimating equations estimated the relative risk (RR) of stillbirth and 95% confidence intervals (CI) in relation to an interquartile range increase in pollutant with adjustment for temperature, clinical, and demographic factors. Ozone (O₃) was associated with a 13-22% increased risk of stillbirth on days 2, 3, and 5-7 prior to delivery in single pollutant models, and these findings persisted in multi-pollutant models for days 5 (RR = 1.22, CI = 1.07-1.38) and 6 (RR = 1.18, CI = 1.04-1.33). Whole pregnancy and first trimester O₃ increased risk 18-39% in single pollutant models. Maternal asthma increased stillbirth risk associated with chronic PM2.5 and carbon monoxide exposures. Both chronic and acute O₃ exposure consistently increased stillbirth risk, while the role of other pollutants varied. Approximately 8000 stillbirths per year in the US may be attributable to O₃ exposure.


Framing Young Childrens Oral Health: A Participatory Action Research Project.
Despite the widespread acknowledgement of the importance of childhood oral health, little progress has been made in preventing early childhood caries. Limited information exists regarding specific daily-life and community-related factors that impede optimal oral hygiene, diet, care, and ultimately oral health for children. We sought to understand what parents of young children consider important and potentially modifiable factors and resources influencing their children's oral health, within the contexts of the family and the community. This qualitative study employed Photovoice among 10 English-speaking parents of infants and toddlers who were clients of an urban WIC clinic in North Carolina. The primary research question was: "What do you consider as important behaviors, as well as family and community resources to prevent cavities among young children?" Five group sessions were conducted and they were recorded, transcribed verbatim and analyzed using qualitative research methodology. Inductive analyses were based on analytical summaries, double-coding, and summary matrices and were done using Atlas.ti.7.5.9 software. Good oral health was associated with avoidance of problems or restorations for the participants. Financial constraints affected healthy food and beverage choices, as well as access to oral health care. Time constraints and occasional frustration related to children's oral hygiene emerged as additional barriers. Establishment of rules/routines and commitment to them was a successful strategy to promote their children's oral health, as well as modeling of older siblings, cooperation among caregivers and peer support. Community programs and organizations, social hubs including playgrounds, grocery stores and social media emerged as promising avenues for gaining support and sharing resources. Low-income parents of young children are faced with daily life struggles that interfere with oral health and care. Financial constraints are pervasive, but parents identified several strategies involving home care and community agents that can be helpful. Future interventions aimed to improve children's oral health must take into consideration the role of families and the communities in which they live.


Genome fluctuations in cyanobacteria reflect evolutionary, developmental and adaptive traits.
Cyanobacteria belong to an ancient group of photosynthetic prokaryotes with pronounced variations in their cellular differentiation strategies, physiological capacities and choice of habitat. Sequencing efforts have shown that genomes within this phylum are equally diverse in terms of size and protein-coding capacity. To increase our understanding of genomic changes in the lineage, the genomes of 58 contemporary cyanobacteria were analysed for shared and unique orthologs. A total of 404 protein families, present in all cyanobacterial genomes, were identified. Two of these are unique to the phylum, corresponding to an AbrB family transcriptional regulator and a gene that escapes functional annotation although its genomic neighbourhood is conserved among the organisms examined. The evolution of cyanobacterial genome sizes involves a mix of gains and losses in the clade encompassing complex cyanobacteria, while a single event of reduction is evident in a clade dominated by unicellular cyanobacteria. Genome sizes and gene family copy numbers evolve at a higher rate in the former clade, and multi-copy genes were predominant in large genomes. Orthologs unique to cyanobacteria exhibiting specific characteristics, such as filament formation, heterocyst differentiation, diazotrophy and symbiotic competence, were also identified. An ancestral character reconstruction suggests that the most recent common ancestor of cyanobacteria had a genome size of approx. 4.5 Mbp and 1678 to 3291 protein-coding genes, 4%-6% of which are unique to cyanobacteria today. The different rates of genome-size evolution and multi-copy gene abundance suggest two routes of genome development in the history of cyanobacteria. The expansion strategy is driven by gene-family enlargment and generates a broad adaptive potential; while the genome streamlining strategy imposes adaptations to highly specific niches, also reflected in their different functional capacities. A few genomes display extreme proliferation of non-coding nucleotides which is likely to be the result of initial expansion of genomes/gene copy number to gain adaptive potential, followed by a shift to a life-style in a highly specific niche (e.g. symbiosis). This transition results in redundancy of genes and gene families, leading to an increase in junk DNA and eventually to gene loss. A few orthologs can be correlated with specific phenotypes in cyanobacteria, such as filament formation and symbiotic competence; these constitute exciting exploratory targets.


Restoration of genetic connectivity among Northern Rockies wolf populations.
Probably no conservation genetics issue is currently more controversial than the question of whether grey wolves (Canis lupus) in the Northern Rockies have recovered to genetically effective levels. Following the dispersal-based recolonization of Northwestern Montana from Canada, and reintroductions to Yellowstone and Central Idaho, wolves have vastly exceeded population recovery goals of 300 wolves distributed in at least 10 breeding pairs in each of Wyoming, Idaho and Montana. With >1700 wolves currently, efforts to delist wolves from endangered status have become mired in legal battles over the distinct population segment (DPS) clause of the Endangered Species Act (ESA), and whether subpopulations within the DPS were genetically isolated. An earlier study by vonHoldt et al. (2008) suggested Yellowstone National Park wolves were indeed isolated and was used against delisting in 2008. Since then, wolves were temporarily delisted, and a first controversial hunting season occurred in fall of 2009. Yet, concerns over the genetic recovery of wolves in the Northern Rockies remain, and upcoming District court rulings in the summer of 2010 will probably include consideration of gene flow between subpopulations. In this issue of Molecular Ecology, vonHoldt et al. (2010) conduct the largest analysis of gene flow and population structure of the Northern Rockies wolves to date. Using an impressive sampling design and novel analytic methods, vonHoldt et al. (2010) show substantial levels of gene flow between three identified subpopulations of wolves within the Northern Rockies, clarifying previous analyses and convincingly showing genetic recovery.


Complementary and Alternative Medicine in the Context of Earlier Diagnoses of Alzheimer's Disease: Opening the Conversation to Prepare Ethical Responses.
Preclinical Alzheimer's disease (AD), a newly proposed, actively researched, and hotly debated research-only diagnostic category, raises the prospect of an ethical dilemma: whether, and possibly how, to treat a disorder with no target symptoms. This proposed category rests on the detection of a number of biomarkers thought to provide evidence of AD pathophysiology years before any behavioral symptoms manifest. Faced with limited treatment options, patients and their relatives may come to consider complementary and alternative medicine (CAM) a viable option, albeit one with minimal supporting evidence. Accordingly, the hopes and needs of some preclinical patients and their relatives could further fuel market-oriented entrepreneurship for CAM. In this ethics review, we provide background and reflect on some ethical questions related to the roles of key stakeholders arising from the potential for CAM use in the context of a possible preclinical AD diagnosis.


The use of telemedicine in burn care: development of a mobile system for TBSA documentation and remote assessment.
The requirements for accurate documentation within the process of burn assessment have increased dramatically over the years. TBSA (total body surface area) and burn depth are commonly determined by visual inspection, especially in the emergency or acute care setting. However, inexperience often results in incorrect estimation of these factors. In 2001, BurnCase 3D was initiated in order to develop a tool for objective burn assessment and documentation on mobile devices (Apple iPhoneTM). The centerpiece is a 3D model representing the actual patient. At two international burn meetings, a survey containing three pictures of patients was conducted and this data was collected. A patient-specific 3D model adapted to the height and weight of the real patient was created and the digital picture was superimposed in the computer system. The burns were transferred to the model and the TBSA in % was calculated by the software BurnCase 3D. The preferred methods of the 80 respondents for burn extent estimation were: the Rule of Nines (38%), the Rule of Palm (37%) and the Lund-Browder chart (18%). Analysis showed very high deviations of TBSA within the participants, even among the group of experts. In comparison to a computer-aided method we found massive overestimation of up to 230%. The use of BurnCase 3D could have a true impact on the quality of treatment in burns. In the acute care setting for burn injuries, telemedicine has great potential to help guide decisions regarding triage and transfer based on TBSA, burn depth, patient age and injury mechanism.


How I do it: Apalutamide use in non-metastatic castrate resistant prostate cancer.
Urologists have been using oral nonsteroidal antiandrogens (AA) for 30 years as a component of combined androgen blockade. In February 2018, a new third generation AA, apalutamide, became available for the first time for non-metastatic (M0) castrate resistant prostate cancer (CRPC). Apalutamide was found to delay the presence of metastases (metastases free survival-MFS) by approximately 2 years versus placebo in M0 CRPC. While overall survival benefit has yet to be established, the MFS benefit is clinically meaningful and urology practices should be equipped to manage patients using this new oral agent. Since the majority of patients remain under urologic care when this disease stage develops and because the drug is straightforward to administer, urology practices are ideal to identify and treat. The objective of this brief article is to discuss the typical patient profile for use of apalutamide and to review the pros and cons of use and common side effects and management.


The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production.
Methyl jasmonate (MeJA) elicits stomatal closing similar to abscisic acid (ABA), but whether the two compounds use similar or different signaling mechanisms in guard cells remains to be clarified. We investigated the effects of MeJA and ABA on second messenger production and ion channel activation in guard cells of wild-type Arabidopsis (Arabidopsis thaliana) and MeJA-insensitive coronatine-insensitive 1 (coi1) mutants. The coi1 mutation impaired MeJA-induced stomatal closing but not ABA-induced stomatal closing. MeJA as well as ABA induced production of reactive oxygen species (ROS) and nitric oxide (NO) in wild-type guard cells, whereas MeJA did not induce production of ROS and NO in coi1 guard cells. The experiments using an inhibitor and scavengers demonstrated that both ROS and NO are involved in MeJA-induced stomatal closing as well as ABA-induced stomatal closing. Not only ABA but also MeJA activated slow anion channels and Ca(2+) permeable cation channels in the plasma membrane of wild-type guard cell protoplasts. However, in coi1 guard cell protoplasts, MeJA did not elicit either slow anion currents or Ca(2+) permeable cation currents, but ABA activated both types of ion channels. Furthermore, to elucidate signaling interaction between ABA and MeJA in guard cells, we examined MeJA signaling in ABA-insensitive mutant ABA-insensitive 2 (abi2-1), whose ABA signal transduction cascade has some disruption downstream of ROS production and NO production. MeJA also did not induce stomatal closing but stimulated production of ROS and NO in abi2-1. These results suggest that MeJA triggers stomatal closing via a receptor distinct from the ABA receptor and that the coi1 mutation disrupts MeJA signaling upstream of the blanch point of ABA signaling and MeJA signaling in Arabidopsis guard cells.


PROGRESS: the PROMISE governance framework to decrease coercion in mental healthcare.
Reducing physical intervention in mental health inpatient care is a global priority. It is extremely distressing both to patients and staff. PROactive Management of Integrated Services and Environments (PROMISE) was developed within Cambridgeshire and Peterborough NHS Foundation Trust (CPFT) to bring about culture change to decrease coercion in care. This study evaluates the changes in physical intervention numbers and patient experience metrics and proposes an easy-to-adopt and adapt governance framework for complex interventions. PROMISE was based on three core values of: providing a caring response to all distress; courage to challenge the status quo; and coproduction of novel solutions. It sought to transform daily front-line interactions related to risk-based restrictive practice that often leads to physical interventions. PROactive Governance of Recovery Settings and Services, a five-step governance framework (Report, Reflect, Review, Rethink and Refresh), was developed in an iterative and organic fashion to oversee the improvement journey and effectively translate information into knowledge, learning and actions. Overall physical interventions reduced from 328 to 241and210 across consecutive years (2014, 2015-2016 and 2016-2017, respectively). Indeed, the 2016-2017 total would have been further reduced to 126 were it not for the perceived substantial care needs of one patient. Prone restraints reduced from 82 to 32 (2015-2016 and 2016-2017, respectively). During 2016-2017, each ward had a continuous 3-month period of no restraints and 4 months without prone restrains. Patient experience surveys (n=4591) for 2014-2017 rated overall satisfaction with care at 87%. CPFT reported fewer physical interventions and maintained high patient experience scores when using a five-pronged governance approach. It has a summative function to define where a team or an organisation is relative to goals and is formative in setting up the next steps relating to action, learning and future planning.


Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter.
The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.


Toxicity of methyl tert butyl ether to soil invertebrates (springtails: Folsomia candida, Proisotoma minuta, and Onychiurus folsomi) and lettuce (Lactuca sativa).
Experiments were conducted to assess the toxicity of methyl tert butyl ether (MTBE) to three species of Collembola (Proisotoma minuta, Folsomia candida, and Onychiurus folsomi) and lettuce (Lactuca sativa L.) using an artificial Organization for Economic Cooperation and Development (OECD) soil and field-collected sandy loam and silt loam soil samples. Soil invertebrate tests were carried out in airtight vials to prevent volatilization of MTBE out of the test units and to allow for direct head-space sampling and gas chromatography-mass spectrometry (GC-MS) analysis for residual MTBE. The use of the airtight vial protocol proved to be very successful, in that the measured MTBE concentrations at the beginning of the experiments were within 95% of nominal concentrations. The test methods used in this study could be used to test the toxicity of other volatile organic compounds to Collembola. The soil invertebrates tested had inhibitory concentration (ICx) and lethal concentration (LCx) values that ranged from 242 to 844 mg MTBE/kg dry soil. When the three test species of Collembola were tested under identical conditions in the artificial OECD soil, O. folsomi was the most sensitive collembolan, with a median inhibitory concentration (IC50; reproduction) of 296 mg MTBE/kg dry soil. The most sensitive endpoint for lettuce was an IC50 for root length of 81 mg MTBE/kg dry soil after 5 d of germination in OECD soil. Data on the loss of MTBE from the three test soils over time indicated that MTBE was retained in the silt loam soil longer than in either the sandy loam or the artificial OECD soil.


The specificity of cellular immune responses in guinea pigs. I. T cells specific for 2,4-dinitrophenyl-o-tyrosyl residues.
Guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP) coupled directly to Mycobacterium tuberculosis of strain H37Ra (DNP-H37) show a variety of cell-mediated immune responses to DNP coupled to protein carriers. The cells responsible for this specific response are thought to be T lymphocytes for the following reasons: Guinea pigs immunized with DNP-H37 displayed delayed hypersensitivity reactions to several DNP-proteins and contact sensitivity to dinitrofluorobenzene. Peritoneal exudate lymphocytes (PELs) obtained from DNP-H37 immune animals respond to DNP-proteins with DNA systhesis and cause inhibition of macrophage migration. PELs are highly enriched in T lymphocytes and contain few immunoglobulin-bearing cells. Further depletion of immunoglobulin-bearing cells from this population does not diminish the in vitro proliferative response to antigen. Nitrophenyl conjugates of proteins lacking a paranitro group stimulated DNA synthesis poorly or not at all, indicating the importance of the paranitro group of DNP in antigen recognition by T cells in this system. In this respect, the specificity of T cells resembles that of DNP-specific antibody from the same animals. On the other hand, DNP conjugates of copolymers of glutamic acid and lysine and DNP conjugated to proteins via an interposed beta-alanyl-glycyl-glycyl spacer failed to stimulate DNA synthesis, although such compounds bind very efficiently to anti-DNP antibody. By contrast, DNP conjugates of synthetic polypeptide carriers containing as little as 7% tyrosine strongly stimulated DNA synthesis in DNP-H37 immune PELs. That the determinant responsible for this stimulation was DNP coupled to the hydroxyl group of tyrosine was shown by selective removal of DNP from tyrosine by thiolysis with 2-mercaptoethanol, which abolished their ability to stimulate T cells.


Trans-equatorial migration routes, staging sites and wintering areas of a high-Arctic avian predator: the long-tailed Skua (Stercorarius longicaudus).
The Long-tailed Skua, a small (<300 g) Arctic-breeding predator and seabird, is a functionally very important component of the Arctic vertebrate communities in summer, but little is known about its migration and winter distribution. We used light-level geolocators to track the annual movements of eight adult birds breeding in north-east Greenland (n = 3) and Svalbard (n = 5). All birds wintered in the Southern Hemisphere (mean arrival-departure dates on wintering grounds: 24 October-21 March): five along the south-west coast of Africa (0-40°S, 0-15°E), in the productive Benguela upwelling, and three further south (30-40°S, 0-50°E), in an area extending into the south-west Indian Ocean. Different migratory routes and rates of travel were documented during post-breeding (345 km d(-1) in late August-early September) and spring migrations (235 km d(-1) in late April) when most birds used a more westerly flyway. Among the different staging areas, a large region off the Grand Banks of Newfoundland appears to be the most important. It was used in autumn by all but one of the tracked birds (from a few days to three weeks) and in spring by five out of eight birds (from one to more than six weeks). Two other staging sites, off the Iberian coast and near the Azores, were used by two birds in spring for five to six weeks. Over one year, individuals travelled between 43,900 and 54,200 km (36,600-45,700 when excluding staging periods) and went as far as 10,500-13,700 km (mean 12,800 km) from their breeding sites. This study has revealed important marine areas in both the south and north Atlantic Ocean. Sustainable management of these ocean basins will benefit Long-tailed Skuas as well as other trans-equatorial migrants from the Arctic.


Building trust with people receiving supported employment and housing first services.
The developing literature on supported employment for people who have a mental illness and recent history of homelessness has yet to explore the relationship between clients and their employment specialists. The objective of the present article is to explore and understand the way in which service users experience supported employment services and how these experiences differ from those receiving usual services. Semistructured qualitative interviews were conducted with 27 people from a randomized controlled trial of supported employment, 14 receiving supported employment, and 13 receiving usual services. Thematic content analysis was used to generate themes and compare experiences between the 2 groups. Trust emerged as an important facilitator to development of a collaborative relationship. It developed with time and featured in the narratives of participants who found jobs. Lack of trust and communication was associated with greater difficulty finding work. People receiving usual services rarely had repeated contact with service providers and therefore did not develop working alliances to the same extent as people receiving supported employment. Without the support of an employment specialist, participants receiving usual services relied more on internal motivation to search for employment opportunities. Programs assisting people to reach their employment goals must be sensitive to homelessness-specific experiences that may make establishing trust difficult. Vocational services should be designed to allow clients to deal exclusively with 1 service provider to permit the development of a working alliance.


Effectiveness of local antibiotic delivery with an osteoinductive and osteoconductive bone-graft substitute.
The morbidity associated with open fractures and open fracture treatment is well established. An osteoinductive and osteoconductive bone-graft substitute that prevents infection would decrease the number of procedures required to treat contaminated fractures by eliminating the need for surgical removal of cement beads and perhaps autograft harvest. We hypothesized that the combination of tobramycin-impregnated calcium sulfate pellets and demineralized bone matrix would prevent the establishment of infection in a contaminated fracture model. A unicortical 12-mm-diameter defect was created in the proximal tibial metaphysis of twenty-nine Spanish goats. After contaminating the wounds with an infective dose of Staphylococcus aureus, we divided the animals into four groups. The negative control group received no treatment, the positive control group received tobramycin-impregnated polymethylmethacrylate beads, the demineralized bone matrix group received 2.5 mL of demineralized bone matrix, and the experimental group received tobramycin-impregnated calcium sulfate pellets with 2.5 mL of demineralized bone matrix. Radiographs were made and intraosseous tissue cultures were performed on postoperative day 21. The cultures showed no evidence of intramedullary infection in the experimental or the positive control group, but they were positive for Staphylococcus aureus in six of the seven goats in the negative control group and seven of the eight goats in the demineralized bone matrix group. The combination of tobramycin-impregnated calcium sulfate pellets and demineralized bone matrix was effective in preventing intramedullary Staphylococcus aureus infection in a contaminated goat fracture model.


Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis.
To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.


Extended overview techniques for outdoor augmented reality.
In this paper, we explore techniques that aim to improve site understanding for outdoor Augmented Reality (AR) applications. While the first person perspective in AR is a direct way of filtering and zooming on a portion of the data set, it severely narrows overview of the situation, particularly over large areas. We present two interactive techniques to overcome this problem: multi-view AR and variable perspective view. We describe in details the conceptual, visualization and interaction aspects of these techniques and their evaluation through a comparative user study. The results we have obtained strengthen the validity of our approach and the applicability of our methods to a large range of application domains.


Subsurface cation vacancy stabilization of the magnetite (001) surface.
Iron oxides play an increasingly prominent role in heterogeneous catalysis, hydrogen production, spintronics, and drug delivery. The surface or material interface can be performance-limiting in these applications, so it is vital to determine accurate atomic-scale structures for iron oxides and understand why they form. Using a combination of quantitative low-energy electron diffraction, scanning tunneling microscopy, and density functional theory calculations, we show that an ordered array of subsurface iron vacancies and interstitials underlies the well-known (√2 × √2)R45° reconstruction of Fe3O4(001). This hitherto unobserved stabilization mechanism occurs because the iron oxides prefer to redistribute cations in the lattice in response to oxidizing or reducing environments. Many other metal oxides also achieve stoichiometry variation in this way, so such surface structures are likely commonplace.


Loperamide binding to opiate receptor sites of brain and myenteric plexus.
Loperamide, a new antidiarrheal agent, was tested to determine whether its biological activity involves binding to opiate receptor sites. Loperamide and morphine competitively inhibited 3H-naloxone binding to homogenates a guinea-pig brain and myenteric plexus. The Kp values obtain in the presence of Na+ were: morphine, 9.60-10(-9) M (brain), 1.66-10(-7) M (myenteric plexus); loperamide, 7.20-10(-9) M (brain), 1.33-10(-7) M (myenteric plexus); naloxone, 4.78-10(-10) M (brain), 1.27-10(-9) M (myenteric plexus. In the absence of Na+, binding a loperamide and morphine to brain homogenate was enhanced while the binding of naloxone was reduced. Morphine (IC50 = 7.5-10(-8) M) and loperamide (IC50 = 6.9-10(-9) M) inhibited the electrically induced contractions of longitudinal muscle from guinea-pig ileum, and naloxone competitively antagonized these effects. The Kd value calculated for the interaction of naloxone with binding sites associated with the contracting muscle was between 0.98-10(-9) M and 1.85-10(-9) M. In the mouse hot plate test, subcutaneous administration of morphine (minimal effective dose = 6.6 mugmol/kg) and loperamide (minimal effective dose = 78 mugmol/kg) delayed the response to heat stimuli and this effect was completely blocked by prior administration of naloxone. In the anesthetixed dog, intravenous administration of morphine (100 mug/kg) and loperamide (100 mug/kg) enhanced the contractile activity of circular muscle in proximal and distal duodenum, distal ileum and proximal colon but duodenal longitudinal muscle was relaxed; these effects were completely reversed by subsequent administration of naloxone. It is concluded that loperamide binds to opiate receptor sites and possesses opiate agonist activity both in vivo and in vitro.


Social determinants: taking the social context of asthma seriously.
Although asthma has emerged as a major contributor to disease and disability among US children, the burden of this disease is unevenly distributed within the population. This article provides a brief overview of social-status variables that predict variations in asthma risks and social exposures, such as stress and violence, that are emerging as important risk factors. The central focus of the article is on the distal social variables that have given rise to unhealthy residential environments in which the risk factors for asthma and other diseases are clustered. Effective initiatives for the prevention and treatment of childhood asthma need to address these nonmedical determinants of the prevalence of asthma.


Self-reported symptoms and bronchial hyperresponsiveness in elite cross-country skiers.
Respiratory symptoms in relationship to exercise, bronchial hyperresponsiveness (BHR), and exercise-induced asthma (EIA) are very common in elite winter athletes. Symptom-based screening for BHR would facilitate selection of athletes with possible EIA. The aim of the present study was to evaluate the diagnostic accuracy of self-reported symptoms as predictors of BHR in an unselected population of adult elite cross-country skiers. Forty-six Swedish adult skiers competing at national or international level were included. They had a mean (SD) training volume in the past 12 months of 593 (122) hours. Twenty-four subjects had previous physician-diagnosed asthma. The European Community Respiratory Health Survey questionnaire was used to evaluate the presence of respiratory symptoms. BHR was defined as bronchoconstriction to either eucapnic voluntary hyperventilation, dry powder mannitol or methacholine provocation. The "classical" EIA symptom of shortness of breath post-exercise was reported by 17% of all skiers. Eight subjects (17%) had BHR. None of the self-reported respiratory symptoms had high positive predictive values. However, symptoms caused by grass or pollen had high negative predictive values. EIA in elite winter athletes cannot accurately be based only on self-reported symptoms but requires verification with objective testing of BHR. Bronchoprovocation of elite winter athletes reporting respiratory symptoms in rest or because of exercise will probably reveal a high proportion of athletes without BHR. EUDRA-CT number 2006-005822-21.


Isolation and characterization of the UBASH3A gene on 21q22.3 encoding a potential nuclear protein with a novel combination of domains.
In order to identify candidate genes for Down syndrome phenotypes or monogenic disorders that map to human chromosome 21q22.3, we have used genomic sequence and expressed sequence tags mapping to an autosomal recessive deafness (DFNB10) critical region to isolate a novel 2.5-kb cDNA that maps between TFF1 and D21S49. A semi-quantitative reverse transcription/polymerase chain reaction method revealed that UBASH3A gene expression is limited to only a few tissues, with its highest expression in spleen, peripheral blood leukocytes, and bone marrow. The putative 661-amino-acid protein shows considerable homology to a hypothetical protein from Drosophila melanogaster but only domain homologies to other organisms. Both the human and D. melanogaster proteins contain protein-protein interaction domains, viz., SH3 and ubiquitin-associated (UBA) domains, in addition to a novel domain also containing a nuclear localization signal. This is the first protein described containing both UBA and SH3 domains. The gene, thus called UBASH3A, spans 40 kb and is divided into 15 exons. Mutation analysis excluded UBASH3A as being responsible for DFNB10.


Heterogeneity of severe asthma in childhood: confirmation by cluster analysis of children in the National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program.
Asthma in children is a heterogeneous disorder with many phenotypes. Although unsupervised cluster analysis is a useful tool for identifying phenotypes, it has not been applied to school-age children with persistent asthma across a wide range of severities. This study determined how children with severe asthma are distributed across a cluster analysis and how well these clusters conform to current definitions of asthma severity. Cluster analysis was applied to 12 continuous and composite variables from 161 children at 5 centers enrolled in the Severe Asthma Research Program. Four clusters of asthma were identified. Children in cluster 1 (n = 48) had relatively normal lung function and less atopy. Children in cluster 2 (n = 52) had slightly lower lung function, more atopy, and increased symptoms and medication use. Cluster 3 (n = 32) had greater comorbidity, increased bronchial responsiveness, and lower lung function. Cluster 4 (n = 29) had the lowest lung function and the greatest symptoms and medication use. Predictors of cluster assignment were asthma duration, the number of asthma controller medications, and baseline lung function. Children with severe asthma were present in all clusters, and no cluster corresponded to definitions of asthma severity provided in asthma treatment guidelines. Severe asthma in children is highly heterogeneous. Unique phenotypic clusters previously identified in adults can also be identified in children, but with important differences. Larger validation and longitudinal studies are needed to determine the baseline and predictive validity of these phenotypic clusters in the larger clinical setting.