Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.
Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).


Novel microbial assemblages inhabiting crustal fluids within mid-ocean ridge flank subsurface basalt.
Although little is known regarding microbial life within our planet's rock-hosted deep subseafloor biosphere, boreholes drilled through deep ocean sediment and into the underlying basaltic crust provide invaluable windows of access that have been used previously to document the presence of microorganisms within fluids percolating through the deep ocean crust. In this study, the analysis of 1.7 million small subunit ribosomal RNA genes amplified and sequenced from marine sediment, bottom seawater and basalt-hosted deep subseafloor fluids that span multiple years and locations on the Juan de Fuca Ridge flank was used to quantitatively delineate a subseafloor microbiome comprised of distinct bacteria and archaea. Hot, anoxic crustal fluids tapped by newly installed seafloor sampling observatories at boreholes U1362A and U1362B contained abundant bacterial lineages of phylogenetically unique Nitrospirae, Aminicenantes, Calescamantes and Chloroflexi. Although less abundant, the domain Archaea was dominated by unique, uncultivated lineages of marine benthic group E, the Terrestrial Hot Spring Crenarchaeotic Group, the Bathyarchaeota and relatives of cultivated, sulfate-reducing Archaeoglobi. Consistent with recent geochemical measurements and bioenergetic predictions, the potential importance of methane cycling and sulfate reduction were imprinted within the basalt-hosted deep subseafloor crustal fluid microbial community. This unique window of access to the deep ocean subsurface basement reveals a microbial landscape that exhibits previously undetected spatial heterogeneity.


Adverse drug reactions induced by cardiovascular drugs in outpatients.
Considering increased use of cardiovascular drugs and limitations in pre-marketing trials for drug safety evaluation, post marketing evaluation of adverse drug reactions (ADRs) induced by this class of medicinal products seems necessary. To determine the rate and seriousness of adverse reactions induced by cardiovascular drugs in outpatients. To compare sex and different age groups in developing ADRs with cardiovascular agents. To assess the relationship between frequencies of ADRs and the number of drugs used. This cross-sectional study was done in cardiovascular clinic at a teaching hospital. All patients during an eight months period were evaluated for cardiovascular drugs induced ADRs. Patient and reaction factors were analyzed in detected ADRs. Patients with or without ADRs were compared in sex and age by using chi-square test. Assessing the relationship between frequencies of ADRs and the number of drugs used was done by using Pearson analysis. The total number of 518 patients was visited at the clinic. ADRs were detected in 105 (20.3%) patients. The most frequent ADRs were occurred in the age group of 51-60. The highest rate of ADRs was recorded to be induced by Diltiazem (23.5%) and the lowest rate with Atenolol (3%). Headache was the most frequent detected ADR (23%). Assessing the severity and preventability of ADRs revealed that 1.1% of ADRs were detected as severe and 1.9% as preventable reactions. Women significantly developed more ADRs in this study (chi square = 3.978, P<0.05). ADRs more frequently occurred with increasing age in this study (chi square = 15.871, P<0.05). With increasing the number of drugs used, the frequency of ADRs increased (Pearson=0.259, P<0.05). Monitoring ADRs in patients using cardiovascular drugs is a matter of importance since this class of medicines is usually used by elderly patients with critical conditions and underlying diseases.


Evaluating predictive relationships between wristbands and urine for assessment of personal PAH exposure.
During events like the COVID-19 pandemic or a disaster, researchers may need to switch from collecting biological samples to personal exposure samplers that are easy and safe to transport and wear, such as silicone wristbands. Previous studies have demonstrated significant correlations between urine biomarker concentrations and chemical levels in wristbands. We build upon those studies and use a novel combination of descriptive statistics and supervised statistical learning to evaluate the relationship between polycyclic aromatic hydrocarbon (PAH) concentrations in silicone wristbands and hydroxy-PAH (OH-PAH) concentrations in urine. In New York City, 109 participants in a longitudinal birth cohort wore one wristband for 48 h and provided a spot urine sample at the end of the 48-hour period during their third trimester of pregnancy. We compared four PAHs with the corresponding seven OH-PAHs using descriptive statistics, a linear regression model, and a linear discriminant analysis model. Five of the seven PAH and OH-PAH pairs had significant correlations (Pearson's r = 0.35-0.64, p ≤ 0.003) and significant chi-square tests of independence for exposure categories (p ≤ 0.009). For these five comparisons, the observed PAH or OH-PAH concentration could predict the other concentration within a factor of 1.47 for 50-80% of the measurements (depending on the pair). Prediction accuracies for high exposure categories were at least 1.5 times higher compared to accuracies based on random chance. These results demonstrate that wristbands and urine provide similar PAH exposure assessment information, which is critical for environmental health researchers looking for the flexibility to switch between biological sample and wristband collection.


Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.
The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 220.127.116.11), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 18.104.22.168) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.


An Investigation of Professional Integrity in Pre-registration Nurse Education: A Modified Grounded Theory Research Study.
Acting with integrity is a central part of nursing practice. However, literature shows that professional integrity can be absent and where this is present it can face challenges. Governmental Inquiries have revealed deficits in the expression of nursing values which underpin professional integrity, in particular caring, compassionate and competent practice that maintains the dignity of patients. Evidence also suggests that it cannot be taken for granted that pre-registration education will have a positive impact on student nurses' ability to practice with integrity. This research explored students', mentors' and lecturers' experiences of professional integrity in pre-registration nurse education. A grounded theory approach was informed by the work of Charmaz (2004, 2006). The study, which took place in a UK university, involved four fields of nursing practice: Adult, Children, Mental Health and Learning Disabilities. 12 student nurses, 5 practice-based mentors and 6 lecturers participated. Semi-structured interviews and focus groups revealed three main themes: meanings, enactment and growth of professional integrity. Pre-registration education can influence the growth of professional integrity by improving students' understanding of the boundaries of nursing practice and potential threats to these, skills to speak up on behalf of patients, and knowledge of the processes involved in raising concerns about practice and potential barriers to this. The proactive development of student nurses' strategies to cope, alongside increasing their understanding of the importance of this is also likely to be beneficial.


Association between the -2548G/A polymorphism of the leptin gene and antipsychotic-induced weight gain: Analysis of the CATIE sample and meta-analysis.
Antipsychotics, especially most of the second-generation antipsychotics, have a high risk for metabolic syndrome and antipsychotic-induced weight gain (AIWG). A promoter variant of the leptin (LEP) gene, -2548G/A (rs7799039), has been associated with AIWG in several studies. The aim of this study was to evaluate this association in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) sample, followed by meta-analysis. We investigated the association between rs7799039 and AIWG in a sub-sample of European (N = 164) individuals from the CATIE study. Body mass index (BMI) change and weight gain (presence or absence) was analyzed using ANCOVA and logistic regression, respectively. For the meta-analysis, a literature search was conducted using MEDLINE, Embase, and PsycINFO up to October 2019. The pooled odds ratio was calculated for presence or absence of weight gain (≥7% weight change) using a random effects model. We did not detect an association between rs7799039 and BMI change or weight gain (presence or absence) in the CATIE sample. As for the meta-analysis, we included 12 studies. No significant associations between the LEP rs7799039 polymorphism and AIWG were observed under the allelic genetic model (allele A vs. allele G) (OR = 1.10 [0.71, 1.70], p = .68). In the subgroup analyses of first-episode schizophrenia patients, a significant association between the A-allele and weight gain was observed, respectively (OR = 2.32 [1.41, 3.82], p = .0009). The present meta-analysis showed no significant effect of rs7799039 on AIWG. However, this variant may influence AIWG in first-episode schizophrenia patients. Further investigation of a larger and more homogenous sample is required to elucidate the role of the LEP gene in AIWG.


Measuring patient-reported outcomes in advanced gastric cancer.
Gastric cancer (GC), one of the most common cancers in the world, is often diagnosed at an advanced stage and associated with a poor prognosis. Quality of life and patient-reported outcomes (PROs) are important considerations when treating GC patients. The aim of this study was to identify existing PRO instruments that would be appropriate for use in GC trials. Data were obtained from a systematic literature review and interviews with clinical experts. A literature search was conducted using OVID (EMBASE and MEDLINE) and yielded 1,008 abstracts; 92 assessed PROs in an advanced GC. Key symptoms and functional impacts identified through the literature and expert input included abdominal pain or pain at the site of distant metastases, dysphagia and other symptoms related to eating, and digestive symptoms. The liver and lungs were the most frequent locations of metastases, leading to dyspnea, abdominal fullness, and jaundice. Symptoms related to changes in bowel habits appeared to be more frequent and pronounced in Asian patients, possibly due to the higher prevalence of GC in the body of the stomach in this population. The five most commonly used PRO instruments were identified, but their validity in advanced-stage GC patients remains unclear. The symptoms and functional impacts identified here should be confirmed with robust input from advanced-stage GC patients. Optimal measurement of PROs in GC should account for patient burden and possible differences between Asian and non-Asian patients.


Cochrane Corner: Extracts from The Cochrane Library: Tonsillectomy or Adenotonsillectomy versus Non-Surgical Management for Obstructive Sleep-Disordered Breathing in Children.
The "Cochrane Corner" is a section in the journal that highlights systematic reviews relevant to otolaryngology-head and neck surgery, with invited commentary to aid clinical decision making. This installment features a Cochrane Review on tonsillectomy for obstructive sleep-disordered breathing (oSDB) in children, which finds moderate-quality evidence that surgery improves symptoms, behavior, and quality of life compared to nonsurgical management. The results apply to nonsyndromic children with SDB confirmed by polysomnography and must be balanced against a favorable natural history in many cases.


Natural History of Barrett's Esophagus.
Barrett's esophagus (BE) is a very common condition. We have obtained fairly profound knowledge of the natural history of this condition. This results from many cross-sectional and cohort studies, many describing patients undergoing long-term surveillance. Their consent to use their clinical data has improved our knowledge to the benefit of these same and other patients. The prevalence of BE increases with age both in men and in women. This increase starts at a younger age in men than in women. The incidence of high-grade dysplasia and cancer in BE depends on segment length, gender, and age. The latter two likely indicate the duration of the presence of BE in an individual patient. Other factors that influence the incidence of dysplasia and cancer are smoking behavior and use of certain medications such as PPIs, statins, and NSAIDs. Surveillance of BE and treatment of dysplasia can impact the incidence of and mortality due to esophageal adenocarcinoma. This is of major benefit to a subgroup of BE patients. The epidemiology and burden of disease ask for further efforts to develop targeted screening, surveillance, and intervention techniques in coming years.


International consensus on allergy immunotherapy.
Allergen immunotherapy (AIT) has been used to treat allergic disease since the early 1900s. Despite numerous clinical trials and meta-analyses proving AIT efficacious, it remains underused and is estimated to be used in less than 10% of patients with allergic rhinitis or asthma worldwide. In addition, there are large differences between regions, which are not only due to socioeconomic status. There is practically no controversy about the use of AIT in the treatment of allergic rhinitis and allergic asthma, but for atopic dermatitis or food allergy, the indications for AIT are not well defined. The elaboration of a wider consensus is of utmost importance because AIT is the only treatment that can change the course of allergic disease by preventing the development of asthma and new allergen sensitizations and by inducing allergen-specific immune tolerance. Safer and more effective AIT strategies are being continuously developed both through elaboration of new allergen preparations and adjuvants and alternate routes of administration. A number of guidelines, consensus documents, or both are available on both the international and national levels. The international community of allergy specialists recognizes the need to develop a comprehensive consensus report to harmonize, disseminate, and implement the best AIT practice. Consequently, the International Collaboration in Asthma, Allergy and Immunology, formed by the European Academy of Allergy and Clinical Immunology; the American Academy of Allergy, Asthma & Immunology; the American College of Allergy, Asthma & Immunology; and the World Allergy Organization, has decided to issue an international consensus on AIT.


Studies on parasitologic and haematologic activities of an enaminone derivative of 4-hydroxyquinolin-2(1H)-one against murine schistosomiasis mansoni.
The activity of a novel enaminone derivative of 4-hydroxyquinoline, BDHQ, was screened for its effectiveness against murine schistosomiasis by electron microscopy and parasitologic studies. The correlation of these studies with serum levels of IFN-gamma and IgE is described. Two groups of 10 mice each were treated with different doses of BDHQ, and their results were correlated with the control and praziquantel (PZQ)-treated groups. Parasitologic study revealed significant reduction in mature worms and tissue egg loads in BDHQ- and PZQ-treated groups, whereas immature worms revealed significant reduction in BDHQ groups only. The group treated with a higher dose of BDHQ showed significant reductions in intestinal ova count when compared with the PZQ-treated group. Ultrastructural examination of the worm revealed significant degeneration of the spines and tegument in all treated groups, while the genital system was affected in BDHQ-treated groups only. BDHQ showed considerable effect on cellular activation where serum levels of IFN-gamma were significantly increased in comparison to control, while anti-soluble worm antigen preparation (SWAP) IgE was significantly increased in comparison to both the control and PZQ-treated groups. Ultrastructural examination revealed cellular activation in buffy coat and the liver in both the BDHQ- and PZQ-treated groups in comparison to the untreated one, whereas in the bone marrow and spleen, evidence of cellular activation was remarkable in the BDHQ-treated groups. In conclusion, BDHQ exhibits high levels of activity against adult and juvenile stages of these parasites, which may be due to its mixed cellular and humoral immunologic mechanisms, as demonstrated by the significant increase of serum levels of IgE and IFN-gamma shown on electron microscopy. Therefore, our results support the comparative advantage that BDHQ has over PZQ.


Dental floss as a possible risk for the development of peri-implant disease: an observational study of 10 cases.
To report on a possible relationship between the use of dental floss or superfloss and the development of peri-implantitis. Ten patients with progressive peri-implantitis with an intensive oral hygiene protocol, which consisted of extensive professional supra- and submucosal cleaning, and not responding to this therapy were scheduled for examination. Plaque and bleeding indices and probing depth measurements were assessed, and radiographic examination was performed every two years. In all ten cases, remnants of dental floss were found around the neck and coronal part of a dental implant. After careful removal of these floss remnants and implant cleansing, a significant improvement in the peri-implant conditions in nine of ten cases was noted. In one case, peri-implant probing depth decreased substantially. However, bleeding on probing was still present. In vitro testing yielded that the application of various types of dental floss on the exposed rough implant surfaces may easily lead to tearing of floss fibers. This may result in the deposition of floss remnants on rough implant surfaces, which, in turn, may lead to the development of plaque-related peri-implant inflammation and, subsequently, bone loss. In case of exposed rough surfaces of the dental implant, the peri-implant conditions may be jeopardized by the application of dental floss, and hence, the utilization of interproximal brushes or toothpicks may be preferred for daily home care practices.


Use of conventional and novel smokeless tobacco products among US adolescents.
To assess the prevalence and correlates of use of conventional and novel smokeless tobacco products among a national sample of US middle and high school students. Data from the 2011 National Youth Tobacco Survey were analyzed to determine national estimates of current use of conventional ("chewing tobacco", "snuff," or "dip"), novel ("snus" and "dissolvable tobacco products"), and any smokeless tobacco products (novel and/or conventional products) within the past 30 days. The overall prevalence of current use of any smokeless tobacco product was 5.6% (n = 960). Among all students, 5.0% used chewing tobacco, snuff, or dip; 1.9% used snus; and 0.3% used dissolvable tobacco products. Among users of any smokeless tobacco, 64.0% used only conventional products, 26.8% were concurrent users of novel plus conventional products, whereas 9.2% exclusively used novel products. Approximately 72.1% of current any smokeless tobacco users concurrently smoked combustible tobacco products, and only 40.1% expressed an intention to quit all tobacco use. Regression analyses indicated that peer (adjusted odds ratio [aOR]: 9.56; 95% confidence interval [CI]: 7.14-12.80) and household (aOR: 3.32; 95% CI: 2.23-4.95) smokeless tobacco use were associated with smokeless tobacco use, whereas believing that all forms of tobacco are harmful was protective (aOR: 0.55; 95% CI: 0.38-0.79). Conventional smokeless tobacco products remain the predominant form of smokeless tobacco use. Most users of novel smokeless tobacco products also concurrently smoked combustible tobacco products. Smokeless tobacco use was associated with lower perception of harm from all tobacco products and protobacco social influences, indicating the need to change youth perceptions about the use of all tobacco products and to engage pediatricians in tobacco use prevention and cessation interventions.


Analysis of in vitro fertilization data with multiple outcomes using discrete time-to-event analysis.
In vitro fertilization (IVF) is an increasingly common method of assisted reproductive technology. Because of the careful observation and follow-up required as part of the procedure, IVF studies provide an ideal opportunity to identify and assess clinical and demographic factors along with environmental exposures that may impact successful reproduction. A major challenge in analyzing data from IVF studies is handling the complexity and multiplicity of outcome, resulting from both multiple opportunities for pregnancy loss within a single IVF cycle in addition to multiple IVF cycles. To date, most evaluations of IVF studies do not make use of full data because of its complex structure. In this paper, we develop statistical methodology for analysis of IVF data with multiple cycles and possibly multiple failure types observed for each individual. We develop a general analysis framework based on a generalized linear modeling formulation that allows implementation of various types of models including shared frailty models, failure-specific frailty models, and transitional models, using standard software. We apply our methodology to data from an IVF study conducted at the Brigham and Women's Hospital, Massachusetts. We also summarize the performance of our proposed methods on the basis of a simulation study.


Deep Learning of Static and Dynamic Brain Functional Networks for Early MCI Detection.
While convolutional neural network (CNN) has been demonstrating powerful ability to learn hierarchical spatial features from medical images, it is still difficult to apply it directly to resting-state functional MRI (rs-fMRI) and the derived brain functional networks (BFNs). We propose a novel CNN framework to simultaneously learn embedded features from BFNs for brain disease diagnosis. Since BFNs can be built by considering both static and dynamic functional connectivity (FC), we first decompose rs-fMRI into multiple static BFNs with modified independent component analysis. Then, the voxel-wise variability in dynamic FC is used to quantify BFN dynamics. A set of paired 3D images representing static/dynamic BFNs can be fed into 3D CNNs, from which we can hierarchically and simultaneously learn static/dynamic BFN features. As a result, the dynamic BFN features can complement static BFN features and, at the meantime, different BFNs can help each other toward a joint and better classification. We validate our method with a publicly accessible, large cohort of rs-fMRI dataset in early-stage mild cognitive impairment (eMCI) diagnosis, which is one of the most challenging problems to the clinicians. By comparing with a conventional method, our method shows significant diagnostic performance improvement by almost 10%. This result demonstrates the effectiveness of deep learning in preclinical Alzheimer's disease diagnosis, based on the complex and high-dimensional voxel-wise spatiotemporal patterns of the resting-state brain functional connectomics. The framework provides a new but intuitive way to fully exploit deeply embedded diagnostic features from rs-fMRI for a better-individualized diagnosis of various neurological diseases.


The link between the West African Ebola outbreak and health systems in Guinea, Liberia and Sierra Leone: a systematic review.
An Ebola outbreak started in December 2013 in Guinea and spread to Liberia and Sierra Leone in 2014. The health systems in place in the three countries lacked the infrastructure and the preparation to respond to the outbreak quickly and the World Health Organisation (WHO) declared a public health emergency of international concern on August 8 2014. The aim of this study was to determine the effects of health systems' organisation and performance on the West African Ebola outbreak in Guinea, Liberia and Sierra Leone and lessons learned. The WHO health system building blocks were used to evaluate the performance of the health systems in these countries. A systematic review of articles published from inception until July 2015 was conducted following the PRISMA guidelines. Electronic databases including Medline, Embase, Global Health, and the Cochrane library were searched for relevant literature. Grey literature was also searched through Google Scholar and Scopus. Articles were exported and selected based on a set of inclusion and exclusion criteria. Data was then extracted into a spreadsheet and a descriptive analysis was performed. Each study was critically appraised using the Crowe Critical Appraisal Tool. The review was supplemented with expert interviews where participants were identified from reference lists and using the snowball method. Thirteen articles were included in the study and six experts from different organisations were interviewed. Findings were analysed based on the WHO health system building blocks. Shortage of health workforce had an important effect on the control of Ebola but also suffered the most from the outbreak. This was followed by information and research, medical products and technologies, health financing and leadership and governance. Poor surveillance and lack of proper communication also contributed to the outbreak. Lack of available funds jeopardised payments and purchase of essential resources and medicines. Leadership and governance had least findings but an overarching consensus that they would have helped prompt response, adequate coordination and management of resources. Ensuring an adequate and efficient health workforce is of the utmost importance to ensure a strong health system and a quick response to new outbreaks. Adequate service delivery results from a collective success of the other blocks. Health financing and its management is crucial to ensure availability of medical products, fund payments to staff and purchase necessary equipment. However, leadership and governance needs to be rigorously explored on their main defects to control the outbreak.


Additive effects of endothelial progenitor cells combined with ACE inhibition and beta-blockade on left ventricular function following acute myocardial infarction.
Animal studies have demonstrated the efficacy of endothelial progenitor cells (EPCs) in preventing left ventricular (LV) remodelling following myocardial infarction (MI). Preliminary human studies are underway, yet no studies have demonstrated efficacy in combination with standard medical therapy, i.e. angiotensin-converting enzyme (ACE) inhibitors and beta-blockers. Nude rats underwent left anterior descending coronary artery ligation to induce MI. Animals were randomised to receive no treatment (MI, n = 5), quinapril 200 mg/L + metoprolol 2 g/L (ACE/BB, n = 5), two million EPCs intravenously (EPC, n = 5)or both (ACE/BB + EPC [n = 5]), then sacrificed after two weeks treatment. ACE/BB resulted in a 75% reduction in fibrosis in the region remote from the MI (p < 0.05), but EPC therapy had little effect here. Conversely, EPC therapy induced neovascularisation at the peri-infarct rim, thereby preventing peri-infarct apoptosis by 81% (p < 0.05). Acting via different but complementary mechanisms, the combination of ACE/BB + EPCs resulted in a greater overall improvement in LV function on echocardiography than either therapy alone. Clinical trials using stem cell therapy in conjunction with standard medical treatment are warranted.


Nickel-vanadium monolayer double hydroxide for efficient electrochemical water oxidation.
Highly active and low-cost electrocatalysts for water oxidation are required due to the demands on sustainable solar fuels; however, developing highly efficient catalysts to meet industrial requirements remains a challenge. Herein, we report a monolayer of nickel-vanadium-layered double hydroxide that shows a current density of 27 mA cm(-2) (57 mA cm(-2) after ohmic-drop correction) at an overpotential of 350 mV for water oxidation. Such performance is comparable to those of the best-performing nickel-iron-layered double hydroxides for water oxidation in alkaline media. Mechanistic studies indicate that the nickel-vanadium-layered double hydroxides can provide high intrinsic catalytic activity, mainly due to enhanced conductivity, facile electron transfer and abundant active sites. This work may expand the scope of cost-effective electrocatalysts for water splitting.


Nivolumab Alone and With Ipilimumab in Previously Treated Metastatic Urothelial Carcinoma: CheckMate 032 Nivolumab 1 mg/kg Plus Ipilimumab 3 mg/kg Expansion Cohort Results.
CheckMate 032 is an open-label, multicohort study that includes patients with unresectable locally advanced or metastatic urothelial carcinoma (mUC) treated with nivolumab 3 mg/kg monotherapy every 2 weeks (NIVO3), nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO3+IPI1), or nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO1+IPI3). We report on the expanded NIVO1+IPI3 cohort and extended follow-up for the NIVO3 and NIVO3+IPI1 cohorts. Patients with platinum-pretreated mUC were enrolled in this phase I/II multicenter study to receive NIVO3, NIVO3+IPI1, or NIVO1+IPI3 until disease progression or unacceptable toxicity. Primary end point was investigator-assessed objective response rate per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, including duration of response. Seventy-eight patients were treated with NIVO3 (minimum follow-up, 37.7 months), 104 with NIVO3+IPI1 (minimum follow-up, 38.8 months), and 92 with NIVO1+IPI3 (minimum follow-up, 7.9 months). Objective response rate was 25.6%, 26.9%, and 38.0% in the NIVO3, NIVO3+IPI1, and NIVO1+IPI3 arms, respectively. Median duration of response was more than 22 months in all arms. Grade 3 or 4 treatment-related adverse events occurred in 21 (26.9%), 32 (30.8%), and 36 (39.1%) patients treated with NIVO3, NIVO3+IPI1, and NIVO1+IPI3, respectively. Grade 5 treatment-related pneumonitis occurred in one patient each in the NIVO3 and NIVO3+IPI1 arms. With longer follow-up, NIVO3 demonstrated sustained antitumor activity alone and in combination with ipilimumab. NIVO1+IPI3 provided the greatest antitumor activity of all regimens, with a manageable safety profile. This result not only supports additional study of NIVO1+IPI3 in mUC, but demonstrates the potential benefit of immunotherapy combinations in this disease.


Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue.
It has been reported that the severe complication of dengue virus infection, dengue hemorrhagic fever (DHF) is much more commonly observed during secondary dengue virus infections than primary infections. In order to elucidate the role of T lymphocytes in the pathogenesis of DHF, we attempted to determine whether T lymphocytes are activated in vivo during dengue virus infections, by examining the levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8), interleukin-2 (IL-2) and interferon-gamma (IFN gamma) in the sera of 59 patients with DHF and 41 patients with dengue fever (DF). The levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma were significantly higher in the acute sera of patients with DHF than in the sera of healthy children (P less than 0.001 for all markers). The acute sera of patients with DF contained higher levels of sIL-2R, sCD4, IL-2, and IFN gamma than the sera of healthy children (P less than 0.001 for sIL-2R, IL-2, and IFN gamma; P less than 0.05 for sCD4), but did not have elevated levels of sCD8. The levels of sIL-2R (P less than 0.05), sCD4 (P less than 0.001), and sCD8 (P less than 0.001) were higher in DHF than in DF on days 3-4 after the onset of fever. The levels of IL-2 and IFN gamma in patients with DHF were highest 1 d before defervescence. There were no significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma among grades 1, 2, and 3 of DHF. These results indicate (a) T lymphocytes are activated and produce IL-2 and IFN gamma in vivo during DHF and DF, (b) CD4+ T lymphocytes are activated in DHF and DF, and the level of activation is higher in DHF than in DF, and (c) activation of CD8+ T lymphocytes is evident in DHF, but not in DF.


Amphiregulin-deficient mice develop spasmolytic polypeptide expressing metaplasia and intestinal metaplasia.
The loss of parietal cells from the fundic mucosa leads to the emergence of metaplastic lineages associated with an increased susceptibility to neoplastic transformation. Both intestinal metaplasia (IM) and spasmolytic polypeptide (TFF2/SP) expressing metaplasia (SPEM) have been identified in human stomach, but only SPEM is present in most mouse models of gastric metaplasia. We previously determined that loss of amphiregulin (AR) promotes SPEM induced by acute oxyntic atrophy. We have now examined whether SPEM in the AR-/- mouse predisposes the stomach to gastric neoplasia. Gross pathology of 18-month-old wild-type, AR-/-, and TGF-alpha-/- mice were examined. Ki-67, beta-catenin, Pdx-1, TFF3, and TFF2/SP expression was analyzed by immunohistochemistry. Metaplastic gastric mucosa was analyzed by dual immunostaining for TFF2/SP with MUC2 or TFF3. By 18 months of age, more than 70% of AR-/- mice developed SPEM while 42% showed goblet cell IM labeled with MUC2, TFF3, and Pdx-1. A total of 28% had invasive gastric lesions in the fundus. No antral abnormalities were observed in AR-/- mice. Metaplastic cell lineages in AR-/- mice showed increases in cell proliferation and cytosolic beta-catenin expression. Dual staining for TFF2/SP with MUC2 or TFF3 showed glands containing both SPEM and IM with intervening cells expressing both TFF2/SP and MUC2 or TFF2/SP and TFF3. AR-/- mice develop SPEM, which gives rise to goblet cell IM and invasive fundic dysplastic lesions. The AR-/- mouse represents the first mouse model for spontaneous development of fundic SPEM with progression to IM.


fog-2, a germ-line-specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans.
This paper describes the isolation and characterization of 16 mutations in the germ-line sex determination gene fog-2 (fog for feminization of the germ line). In the nematode Caenorhabditis elegans there are normally two sexes, self-fertilizing hermaphrodites (XX) and males (XO). Wild-type XX animals are hermaphrodite in the germ line (spermatogenesis followed by oogenesis), and female in the soma. fog-2 loss-of-function mutations transform XX animals into females while XO animals are unaffected. Thus, wild-type fog-2 is necessary for spermatogenesis in hermaphrodites but not males. The fem genes and fog-1 are each essential for specification of spermatogenesis in both XX and XO animals. fog-2 acts as a positive regulator of the fem genes and fog-1. The tra-2 and tra-3 genes act as negative regulators of the fem genes and fog-1 to allow oogenesis. Two models are discussed for how fog-2 might positively regulate the fem genes and fog-1 to permit spermatogenesis; fog-2 may act as a negative regulator of tra-2 and tra-3, or fog-2 may act positively on the fem genes and fog-1 rendering them insensitive to the negative action of tra-2 and tra-3.


Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage.
Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. Animal experimentation. Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT). Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and matrix metal-loproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. VA decreased the levels of TNF-α and IL-1β proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition.


Polyarteritis nodosa associated with hepatitis B virus infection. The role of antiviral treatment and mutations in the hepatitis B virus genome.
Polyarteritis nodosa (PAN) is a systemic inflammatory disease causing vasculitis of medium sized and small arteries. Circulating immune complexes containing viral proteins have been implicated in the pathogenesis of hepatitis B virus (HBV) related PAN and several immunosuppressive and antiviral regimens have been used with varying success. In our hospital seven HBV positive patients with a confirmed diagnosis of PAN could be identified between 1984 and 2001. Most patients had an acute HBV infection and all patients were treated with prednisone. A combination of prednisone and antiviral therapy with alpha-interferon (IFN) was used only in the last four patients. HBV DNA was isolated from serum samples obtained before treatment from the four IFN treated patients and amplified by using the polymerase chain reaction technique. None of the patients without, but two of four with antiviral therapy exhibited HBsAg seroconversion. In three out of four patients HBV DNA decreased rapidly after starting IFN therapy. Clinical remission of PAN was observed in three of the four treated patients, but in none of the three patients who were not receiving antiviral medication. Analysis of the HBV genome revealed no mutations that could be associated with PAN. In one patient a stop codon in the pre-core region and a double mutation A1762T-G1764A were found during antiviral therapy. We did not find HBV heterogeneity predisposing to the development of PAN. In our group of patients it appeared that clinical remission of PAN was primarily related to spontaneous or therapy induced loss of HBV DNA replication. The combined administration of a priming steroid course and IFN appears to be an improvement over prednisone monotherapy and should be considered for every patient with HBV related PAN. The efficacy of new generation nucleoside analogues should be further elucidated in future studies.


Sources of retinal pigment epithelium (RPE) for replacement therapy.
Stem cells, with their capacity to regenerate and replace diseased tissues, have recently been proposed as having great potential in the treatment of age-related macular degeneration (AMD). A stem cell therapeutic approach could operate to replace either the retinal pigment epithelium (RPE), the neurosensory retina or a combination of both. From the scientific perspective, RPE replacement alone is likely to be far more straightforward than rebuilding the complex circuitry of the neurosensory retina. Furthermore, recent advances with induced pluripotent stem cells have raised the real possibility of transplanting healthy 'young' autologous RPE into patients with early signs of AMD. At this stage, however, it is useful to reconsider some of the earlier clinical studies that used suspensions of autologous RPE cells harvested from the peripheral retina. These showed that isolated RPE cell suspensions had little capacity to recreate a monolayer on the diseased Bruch's membrane of AMD. To counter this problem, researchers from Southampton in the UK report the use of a synthetic polymer alternative to Bruch's membrane, which could provide a scaffold for future RPE derived from stem cells or possibly reopen opportunities for autologous RPE cells harvested from the peripheral retina.


Evaluation of metabolic syndrome risk in Korean premenopausal women: not waist circumference but visceral fat.
In clinical practice, using the patient's waist circumference (WC) to evaluate visceral obesity may underestimate disorders with a metabolic origin. This study examined whether or not the WC derived from the cut-off point of the visceral fat area (VFA) can reflect the features of metabolic syndrome (MetS) in premenopausal women. Computed tomography-scanned VFA, MetS components and the concentrations of high-sensitivity C-reactive protein (CRP) and adiponectin were measured in a total of 349 premenopausal women. The VFA at the L1 and the L4 sites was a significant index (p<0.001) of incremental MetS risk. Receiver-operating characteristic curve analysis showed that 75 cm2 of VFA at L4 and 87.5 cm2 at L1 were the optimal thresholds for discrimination of MetS risk. Significant differences in all MetS components, as well as CRP (p<0.05) and adiponectin levels (p<0.005), were observed when subjects were subdivided by the L4 VFA cut-off point (<75/>or=75 cm2), whereas there was a significant difference only in the triglycerides level in the groups divided by WC (WC<88/>or=88 cm). Moreover, subjects with a lower WC-higher VFA showed a similar pattern in MetS components and lower adiponectin than those with a higher WC-higher VFA. This study clarified that VFA rather than WC is a major determinant of MetS risk in premenopausal women.


Postural Stabilization Strategies to Motor Contagion Induced by Action Observation Are Impaired in Parkinson's Disease.
Postural reactions can be influenced by concomitant tasks or different contexts and are modulated by a higher order motor control. Recent studies investigated postural changes determined by motor contagion induced by action observation (chameleon effect) showing that observing a model in postural disequilibrium induces an increase in healthy subjects' body sway. Parkinson's disease (PD) is associated with postural instability and impairments in cognitively controlled balance tasks. However, no studies investigated if viewing postural imbalance might influence postural stability in PD and if patients are able to inhibit a visual postural perturbation. In this study, an action observation paradigm for assessing postural reaction to motor contagion in PD subjects and healthy older adults was used. Postural stability changes were measured during the observation of a static stimulus (control condition) and during a point-light display of a gymnast balancing on a rope (biological stimulus). Our results showed that, during the observation of the biological stimulus, sway area and antero-posterior and medio-lateral displacements of center of pressure significantly increased only in PD participants, whereas correct stabilization reactions were present in elderly subjects. These results demonstrate that PD leads to a decreased capacity to control automatic imitative tendencies induced by motor contagion. This behavior could be the consequence either of an inability to inhibit automatic imitative tendencies or of the cognitive load requested by the task. Whatever the case, the issue about the ability to inhibit automatic imitative tendencies could be crucial for PD patients since it might increase falls risk and injuries.


KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.
Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.


Magnesium dietary manipulation and recovery of function following controlled cortical damage in the rat.
Previous research has shown that dietary magnesium (Mg2+) deficiency prior to injury worsens recovery of function and that systemic administration of Mg2+ pre or post-injury significantly improves functional recovery. The purpose of the present study was to determine if manipulations in dietary Mg2+ would alter functional recovery following unilateral cortical injuries. Two weeks prior to injury, rats were placed on a customized diet enriched with Mg2+, deficient in Mg2+, or on a standard Mg2+ diet. Rats were then prepared with unilateral cortical contusion injuries (CCI) of the sensorimotor cortex. Two days following CCI, rats were tested on a battery of sensorimotor (vibrissae-forelimb placing and bilateral tactile adhesive removal tests), as well as the acquisition of reference memory in the Morris water maze. Serum analysis for Mg2+ prior to injury showed a diet-dependent modulation in levels. The Mg(2+)-enriched diet showed significantly higher levels of serum Mg2+ compared to the normal diet and the Mg(2+)-deficient diet showed significantly lower levels compared to the Mg(2+)-normal diet. On the placing and tactile removal tests Mg2+ deficiency significantly worsened recovery compared to the Mg(2+)-enriched and Mg(2+-)normal diet conditions. There were no statistically significant differences between the Mg(2+)-normal and Mg(2+)-enriched diets on the sensorimotor tests. On the acquisition of reference memory there were no significant difference between diet conditions; however, the Mg(2+)-deficient diet showed a trend toward impaired performance compared to the other diet conditions. The Mg(2+)-deficient diet resulted in a larger lesion cavity compared to the other diet conditions. These findings suggest that dietary Mg2+ modulates recovery of function.


Object features used by humans and monkeys to identify rotated shapes.
Humans and rhesus monkeys can identify shapes that have been rotated in the picture plane. Recognition of rotated shapes can be as efficient as recognition of upright shapes. Here we investigate whether subjects showing view-invariant performance use the same object features to identify upright and rotated versions of a shape. We find marked differences between humans and monkeys. While humans tend to use the same features independent of shape orientation, monkeys use unique features for each orientation. Humans are able to generalize to a greater degree across orientation changes than rhesus monkey observers, who tend to relearn separate problems at each orientation rather than flexibly apply previously learned knowledge to novel problems.


Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman.
Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as 'the Iceman'. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 +/- 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young's modulus of single mummified fibrils was 4.1 +/- 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 +/- 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.


High-definition self-assemblies driven by the hydrophobic effect: synthesis and properties of a supramolecular nanocapsule.
High definition self-assemblies, those that possess order at the molecular level, are most commonly made from subunits possessing metals and metal coordination sites, or groups capable of partaking in hydrogen bonding. In other words, enthalpy is the driving force behind the free energy of assembly. The hydrophobic effect engenders the possibility of (nominally) relying not on enthalpy but entropy to drive assembly. Towards this idea, we describe how template molecules can trigger the dimerization of a cavitand in aqueous solution, and in doing so are encapsulated within the resulting capsule. Although not held together by (enthalpically) strong and directional non-covalent forces, these capsules possess considerable thermodynamic and kinetic stability. As a result, they display unusual and even unique properties. We discuss some of these, including the use of the capsule as a nanoscale reaction chamber and how they can bring about the separation of hydrocarbon gases.


Genome-Scale CRISPR Screening in Human Intestinal Organoids Identifies Drivers of TGF-β Resistance.
Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.


Multiple interferon regulatory factor and NF-κB sites cooperate in mediating cell-type- and maturation-specific activation of the human CD83 promoter in dendritic cells.
CD83 is one of the best-known surface markers for fully mature dendritic cells (mature DCs), and its cell-type- and maturation-specific regulation makes the CD83 promoter an interesting tool for the genetic modulation of DCs. To determine the mechanisms regulating this DC- and maturation-specific CD83 expression, chromatin immunoprecipitation (ChIP)-on-chip microarray, biocomputational, reporter, electrophoretic mobility shift assay (EMSA), and ChIP analyses were performed. These studies led to the identification of a ternary transcriptional activation complex composed of an upstream regulatory element, a minimal promoter, and an enhancer, which have not been reported in this arrangement for any other gene so far. Notably, these DNA regions contain a complex framework of interferon regulatory factor (IRF)- and NF-κB transcription factor-binding sites mediating their arrangement. Mutation of any of the IRF-binding sites resulted in a significant loss of promoter activity, whereas overexpression of NF-κB transcription factors clearly enhanced transcription. We identified IRF-1, IRF-2, IRF-5, p50, p65, and cRel to be involved in regulating maturation-specific CD83 expression in DCs. Therefore, the characterization of this promoter complex not only contributes to the knowledge of DC-specific gene regulation but also suggests the involvement of a transcriptional module with binding sites separated into distinct regions in transcriptional activation as well as cell-type- and maturation-specific transcriptional targeting of DCs.


A Phase I/II Trial to Evaluate the Technical Feasibility of Partial Breast Irradiation with Three-Dimensional Conformal Radiation Therapy in Korean Women with Stage I Breast Carcinoma: An Initial Report of the Korean Radiation Therapy Oncology Group (KROG) Study 0804.
This prospective study was designed to verify the technical feasibility of partial breast irradiation in breast cancer patients with small breasts, which are commonly encountered in Korean women. A total of 40 Gy, administered in 10 fractions on consecutive days (one fraction per day), was prescribed to the isocenters of the fields using three-dimensional conformal radiotherapy (3-DCRT). For all patients, treatment planning and dose parameters strictly adhered to the constraints set forth in the Radiation Therapy Oncology Group (RTOG) 0319 protocol. This study was designed such that if fewer than five of the first 42 evaluable patients received unacceptable scores, the treatment would be considered reproducible. Ten treatment plans (23.8%) were determined to have major variations. There was no major variation in planning target volume (PTV) coverage. The ipsilateral and contralateral breast dose limitations were not met in four (9.5%) and four cases (9.5%), respectively. Major variations in ipsilateral and contralateral lung dose limitations were observed in two cases (4.8%). Major variations in the heart and thyroid dose limitations were observed in one (2.4%) and one case (2.4%), respectively. In multivariate analysis, a ratio of PTV to ipsilateral breast volume (PTV/IB) > 0.16 was the only significant factor that statistically affected major variations. We concluded that partial breast irradiation using 3-DCRT could not be reproduced in Korean breast cancer patients, particularly small-volumed breast surrogated as PTV/IB > 0.16. The dominant cause was the major variation in surrounding normal breast tissues.


The reactive tumor microenvironment: MUC1 signaling directly reprograms transcription of CTGF.
The MUC1 cytoplasmic tail (MUC1.CT) conducts signals from spatial and extracellular cues (growth factor and cytokine stimulation) to evoke a reprogramming of the cellular transcriptional profile. Specific phosphorylated forms of the MUC1.CT achieve this function by differentially associating with transcription factors and redirecting their transcriptional regulatory capabilities at specific gene regulatory elements. The specificity of interaction between MUC1.CT and several transcription factors is dictated by the phosphorylation pattern of the 18 potential phosphorylation motifs within the MUC1.CT. To better appreciate the scope of differential gene expression triggered by MUC1.CT activation, we performed microarray gene expression analysis and chromatin immunoprecipitation (ChIP)-chip promoter analysis and identified the genome-wide transcriptional targets of MUC1.CT signaling in pancreatic cancer. On a global scale, MUC1.CT preferentially targets genes related to invasion, angiogenesis and metastasis, suggesting that MUC1.CT signaling contributes to establishing a reactive tumor microenvironment during tumor progression to metastatic disease. We examined in detail the molecular mechanisms of MUC1.CT signaling that induces the expression of connective tissue growth factor (CTGF/CCN2), a potent mediator of ECM remodeling and angiogenesis. We demonstrate a robust induction of CTGF synthesis and secretion in response to serum factors that is enabled only when MUC1 is highly expressed. We demonstrate the requirement of phosphorylation at distinct tyrosine motifs within the MUC1.CT for MUC1-induced CTGF expression and demonstrate a phosphorylation-specific localization of MUC1.CT to the CTGF promoter. We found that MUC1 reorganizes transcription factor occupancy of genomic regions upstream of the CTGF gene, directing β-catenin and mutant p53 to CTGF gene regulatory elements to promote CTGF expression and destabilizing the interaction at these regions of the transcriptional repressor, c-Jun. With this example we illustrate the capacity of MUC1.CT to mediate transcription factor activity in a context-dependent manner to achieve wide spread and robust changes in gene expression and facilitate creation of the reactive tumor microenvironment.


Black women with polycystic ovary syndrome (PCOS) have increased risk for metabolic syndrome and cardiovascular disease compared with white women with PCOS [corrected].
To determine the prevalence of metabolic syndrome (MetSyn) and Framingham cardiovascular disease (CVD) risk in white and black adolescents and adult women with polycystic ovary syndrome (PCOS) compared with controls. Retrospective cohort study. Center for PCOS. Subjects with PCOS with data on race and cardiometabolic risk (n = 519). Controls were age and race matched from the National Health and Nutrition Examination Survey (NHANES) population (1999-2006). None. MetSyn, coronary heart disease risk, and general CVD risk. Black adolescents and young adults with PCOS had an increased prevalence of MetSyn compared with their white counterparts (adolescents relative risk 2.65 [95% confidence interval 1.29-5.4], adults relative risk 1.44 [95% confidence interval 1.21-2.6]). In contrast, there was no difference in risk of MetSyn between black and white adolescents and adult women in the NHANES dataset. After controlling for age and body mass index, black women with PCOS had a significantly increased prevalence of low high-density lipoprotein and high glucose. The general CVD risk was significantly increased in black adults with PCOS. This is the first study to comprehensively demonstrate increased risk of MetSyn in both black adolescents and adult women with PCOS compared with white subjects with PCOS. This racial disparity was not present in the NHANES controls. Longitudinal studies are needed to assess the independent impact of PCOS and race on CVD risk in women.


Success, clinical performance and patient satisfaction of direct fibre-reinforced composite fixed partial dentures - a two-year clinical study.
To evaluate the success, clinical performance and patient satisfaction of directly placed fibre-reinforced composite (FRC) fixed partial dentures (FPDs) in 2 years. One hundred sixty-seven FRC FPDs (120 subjects) were directly fabricated to restore a single missing tooth by six Advanced Education in General Dentistry (AEGD) residents. The FRC FPDs recipients were randomised into two groups according to the fibre materials (pre-impregnated glass or polyethylene). Clinical performance was evaluated at baseline (2 weeks), 6, 12 and 24 months by two calibrated evaluators for prosthesis adaptation, colour match, marginal discoloration, surface roughness, caries and post-operative sensitivity using modified United State Public Health Service (USPHS) criteria. Prosthesis appearance, colour, chewing ability and overall satisfaction were evaluated by patients using a visual analogue scale (VAS). Kaplan-Meier estimation was used to estimate the prosthesis success. Ninety-four patients with 137 FRC FPDs returned (21·67% attrition rate for study subjects, 17·94% for FRC FPDs). Seventeen FRC FPDs failed, due to one-end (n = 4) or two-ends (n = 4) debonding or pontic fracture (n = 9). The cumulative 2-year success rate was 84·32% and survival rate was 92·7%; there were no statistically significant differences between the groups according to different missing tooth location, retention type or fibre materials (P > 0·05). Patient satisfaction regarding prosthesis appearance, col-our, chewing ability and overall satisfaction was rated high on the VAS (mean >80 mm) for all criteria at all time points. The FRC FPDs (restoring single tooth) fabricated by AEGD residents achieved acceptable success and survival rates in a 2-year follow-up.


Optimization of washing conditions with biogenic mobilizing agents for marine fuel-contaminated beach sands.
Washing is a rapid and effective treatment to remediate contaminated sands impacted by oil spills, although synthetic additives used to increase extraction efficiency may cause additional pollution issues due to their intrinsic toxicity and very often low biodegradability. In this study, different biogenic mobilizing agents (soybean lecithins, cyclodextrins, cholic acids, plant-derived cleaners, rhamnolipids and sophorolipids) were tested in the washing of beach sands artificially contaminated with the Intermediate Fuel Oil IFO-180. Among these, a de-oiled soybean lecithin (SL-1), hydroxypropyl-β-cyclodextrins (HPB-CD) and sophorolipids (SR) achieved hydrocarbon removals close to those attained with the synthetic surfactant Triton™ X-100 (TX) in preliminary washing tests carried out at constant mixing rate, water/sand ratio and IFO-180 contamination level using agents concentrations close to their critical micelle concentration (0.1% and 1% w/v for microbial and non-microbial agents, respectively). The effects of agent concentration, water/sand ratio, mixing rate and IFO-180 contamination on hydrocarbons removal were modelled using face-centred central composite design and ANOVA. Optimal washing parameters for sand contamination levels in the range 0.5-20 g/kg were identified with response surface methodology. While HPB-CD and SR performed equally to TX only at low sand contaminations, SL-1 attained hydrocarbon removal higher or equal to that of TX at any IFO-180 contamination and at lower application rates. SL-1 also outperformed TX when minimizing the water/sand ratio, i.e., the volume of water used. Considering its lower toxicity, higher biodegradability and higher hydrocarbon removal efficiencies, SL-1 is an effective and environmentally sustainable alternative to synthetic surfactants in washing treatments for marine fuel-contaminated sands.


Parameter estimation methods for chaotic intercellular networks.
We have investigated simulation-based techniques for parameter estimation in chaotic intercellular networks. The proposed methodology combines a synchronization-based framework for parameter estimation in coupled chaotic systems with some state-of-the-art computational inference methods borrowed from the field of computational statistics. The first method is a stochastic optimization algorithm, known as accelerated random search method, and the other two techniques are based on approximate Bayesian computation. The latter is a general methodology for non-parametric inference that can be applied to practically any system of interest. The first method based on approximate Bayesian computation is a Markov Chain Monte Carlo scheme that generates a series of random parameter realizations for which a low synchronization error is guaranteed. We show that accurate parameter estimates can be obtained by averaging over these realizations. The second ABC-based technique is a Sequential Monte Carlo scheme. The algorithm generates a sequence of "populations", i.e., sets of randomly generated parameter values, where the members of a certain population attain a synchronization error that is lesser than the error attained by members of the previous population. Again, we show that accurate estimates can be obtained by averaging over the parameter values in the last population of the sequence. We have analysed how effective these methods are from a computational perspective. For the numerical simulations we have considered a network that consists of two modified repressilators with identical parameters, coupled by the fast diffusion of the autoinducer across the cell membranes.


Age at first birth in women is genetically associated with increased risk of schizophrenia.
Previous studies have shown an increased risk for mental health problems in children born to both younger and older parents compared to children of average-aged parents. We previously used a novel design to reveal a latent mechanism of genetic association between schizophrenia and age at first birth in women (AFB). Here, we use independent data from the UK Biobank (N = 38,892) to replicate the finding of an association between predicted genetic risk of schizophrenia and AFB in women, and to estimate the genetic correlation between schizophrenia and AFB in women stratified into younger and older groups. We find evidence for an association between predicted genetic risk of schizophrenia and AFB in women (P-value = 1.12E-05), and we show genetic heterogeneity between younger and older AFB groups (P-value = 3.45E-03). The genetic correlation between schizophrenia and AFB in the younger AFB group is -0.16 (SE = 0.04) while that between schizophrenia and AFB in the older AFB group is 0.14 (SE = 0.08). Our results suggest that early, and perhaps also late, age at first birth in women is associated with increased genetic risk for schizophrenia in the UK Biobank sample. These findings contribute new insights into factors contributing to the complex bio-social risk architecture underpinning the association between parental age and offspring mental health.


An aromatic microdomain at the cannabinoid CB(1) receptor constitutes an agonist/inverse agonist binding region.
The cannabinoid CB(1) receptor transmembrane helix (TMH) 3-4-5-6 region includes an aromatic microdomain comprised of residues F3.25, F3.36, W4.64, Y5.39, W5.43, and W6.48. In previous work, we have demonstrated that aromaticity at position 5.39 in CB(1) is crucial for proper function of CB(1). Modeling studies reported here suggest that in the inactive state of CB(1), the binding site of the CB(1) inverse agonist/antagonist SR141716A is within the TMH3-4-5-6 aromatic microdomain and involves direct aromatic stacking interactions with F3.36, Y5.39, and W5.43, as well as hydrogen bonding with K3.28. Further, modeling studies suggest that in the active state of CB(1), the CB agonist WIN55,212-2 binds in this same aromatic microdomain, with direct aromatic stacking interactions with F3.36, W5.43, and W6.48. In contrast, in the binding pocket model, the CB agonist anandamide binds in the TMH2-3-6-7 region in which hydrogen bonding and C-H.pi interactions appear to be important. Only one TMH3 aromatic residue, F3.25, was found to be part of the anandamide binding pocket. To probe the importance of the TMH3-4-5-6 aromatic microdomain to ligand binding, stable transfected cell lines were created for single-point mutations of each aromatic microdomain residue to alanine. Improper cellular expression of the W4.64A was observed and precluded further characterization of this mutation. The affinity of the cannabinoid agonist CP55,940 was unaffected by the F3.25A, F3.36A, W5.43A, or W6.48A mutations, making CP55,940 an appropriate choice as the radioligand for binding studies. The binding of SR141716A and WIN55,212-2 were found to be affected by the F3.36A, W5.43A, and W6.48A mutations, suggesting that these residues are part of the binding site for these two ligands. Only the F3.25A mutation was found to affect the binding of anandamide, suggesting a divergence in binding site regions for anandamide from WIN55,212-2, as well as SR141716A. Taken together, these results support modeling studies that identify the TMH3-4-5-6 aromatic microdomain as the binding region of SR141716A and WIN55,212-2, but not of anandamide.


Glut1 Deficiency Syndrome (Glut1DS): State of the art in 2020 and recommendations of the international Glut1DS study group.
Glut1 deficiency syndrome (Glut1DS) is a brain energy failure syndrome caused by impaired glucose transport across brain tissue barriers. Glucose diffusion across tissue barriers is facilitated by a family of proteins including glucose transporter type 1 (Glut1). Patients are treated effectively with ketogenic diet therapies (KDT) that provide a supplemental fuel, namely ketone bodies, for brain energy metabolism. The increasing complexity of Glut1DS, since its original description in 1991, now demands an international consensus statement regarding diagnosis and treatment. International experts (n = 23) developed a consensus statement utilizing their collective professional experience, responses to a standardized questionnaire, and serial discussions of wide-ranging issues related to Glut1DS. Key clinical features signaling the onset of Glut1DS are eye-head movement abnormalities, seizures, neurodevelopmental impairment, deceleration of head growth, and movement disorders. Diagnosis is confirmed by the presence of these clinical signs, hypoglycorrhachia documented by lumbar puncture, and genetic analysis showing pathogenic SLC2A1 variants. KDT represent standard choices with Glut1DS-specific recommendations regarding duration, composition, and management. Ongoing research has identified future interventions to restore Glut1 protein content and function. Clinical manifestations are influenced by patient age, genetic complexity, and novel therapeutic interventions. All clinical phenotypes will benefit from a better understanding of Glut1DS natural history throughout the life cycle and from improved guidelines facilitating early diagnosis and prompt treatment. Often, the presenting seizures are treated initially with antiseizure drugs before the cause of the epilepsy is ascertained and appropriate KDT are initiated. Initial drug treatment fails to treat the underlying metabolic disturbance during early brain development, contributing to the long-term disease burden. Impaired development of the brain microvasculature is one such complication of delayed Glut1DS treatment in the postnatal period. This international consensus statement should facilitate prompt diagnosis and guide best standard of care for Glut1DS throughout the life cycle.


Impact of discontinuity in health insurance on resource utilization.
This study sought to describe the incidence of transitions into and out of Medicaid, characterize the populations that transition and determine if health insurance instability is associated with changes in healthcare utilization. 2000-2004 Medical Expenditure Panel Survey (MEPS) was used to identify adults enrolled in Medicaid at any time during the survey period (n = 6,247). We estimate both static and dynamic panel data models to examine the effect of health insurance instability on health care resource utilization. We find that, after controlling for observed factors like employment and health status, and after specifying a dynamic model that attempts to capture time-dependent unobserved effects, individuals who have multiple transitions into and out of Medicaid have higher emergency room utilization, more office visits, more hospitalizations, and refill their prescriptions less often. Individuals with more than one transition in health insurance status over the study period were likely to have higher health care utilization than individuals with one or fewer transitions. If these effects are causal, in addition to individual benefits, there are potentially large benefits for Medicaid programs from reducing avoidable insurance instability. These results suggest the importance of including provisions to facilitate continuous enrollment in public programs as the United States pursues health reform.


Groups and scores: the decline of cooperation.
Cooperation among unrelated individuals in social-dilemma-type situations is a key topic in social and biological sciences. It has been shown that, without suitable mechanisms, high levels of cooperation/contributions in repeated public goods games are not stable in the long run. Reputation, as a driver of indirect reciprocity, is often proposed as a mechanism that leads to cooperation. A simple and prominent reputation dynamic function through scoring: contributing behaviour increases one's score, non-contributing reduces it. Indeed, many experiments have established that scoring can sustain cooperation in two-player prisoner's dilemmas and donation games. However, these prior studies focused on pairwise interactions, with no experiment studying reputation mechanisms in more general group interactions. In this paper, we focus on groups and scores, proposing and testing several scoring rules that could apply to multi-player prisoners' dilemmas played in groups, which we test in a laboratory experiment. Results are unambiguously negative: we observe a steady decline of cooperation for every tested scoring mechanism. All scoring systems suffer from it in much the same way. We conclude that the positive results obtained by scoring in pairwise interactions do not apply to multi-player prisoner's dilemmas, and that alternative mechanisms are needed.


Molecular characterization of fire ants, Solenopsis spp., from Brazil based on analysis of mtDNA gene cytochrome oxidase I.
Species from the Solenopsis saevissima (Smith) (Hymenoptera: Formicidae) species group are native to South America and have a cosmopolitan distribution because they have been accidentally introduced in many countries around the world. In Brazil, they have a wide distribution, including urban areas. The present study was conducted to investigate the characterization of Solenopsis genus populations associated with urban/human interference sites in Brazil by analyzing the mitochondrial gene cytochrome oxidase I and estimating the degree of relatedness of these populations to make inferences about their phylogeny and also observe the patterns of mitochondrial haplotype (mitotype) distribution across their range. The results revealed complete geographical coherence and polyphyly for the Solenopsis invicta Buren and Solenopsis saevissima species groups, which confirms the diversity of the genera. It also suggests the possibility that reproductively-isolated populations occur, resulting in the evolutionary process of speciation. No predominant haplotype was found in the populations analyzed, but some were more prevalent.


Particulate matter exposure during pregnancy is associated with birth weight, but not gestational age, 1962-1992: a cohort study.
Exposure to air pollutants is suggested to adversely affect fetal growth, but the evidence remains inconsistent in relation to specific outcomes and exposure windows. Using birth records from the two major maternity hospitals in Newcastle upon Tyne in northern England between 1961 and 1992, we constructed a database of all births to mothers resident within the city. Weekly black smoke exposure levels from routine data recorded at 20 air pollution monitoring stations were obtained and individual exposures were estimated via a two-stage modeling strategy, incorporating temporally and spatially varying covariates. Regression analyses, including 88,679 births, assessed potential associations between exposure to black smoke and birth weight, gestational age and birth weight standardized for gestational age and sex. Significant associations were seen between black smoke and both standardized and unstandardized birth weight, but not for gestational age when adjusted for potential confounders. Not all associations were linear. For an increase in whole pregnancy black smoke exposure, from the 1(st) (7.4 μg/m(3)) to the 25(th) (17.2 μg/m(3)), 50(th) (33.8 μg/m(3)), 75(th) (108.3 μg/m(3)), and 90(th) (180.8 μg/m(3)) percentiles, the adjusted estimated decreases in birth weight were 33 g (SE 1.05), 62 g (1.63), 98 g (2.26) and 109 g (2.44) respectively. A significant interaction was observed between socio-economic deprivation and black smoke on both standardized and unstandardized birth weight with increasing effects of black smoke in reducing birth weight seen with increasing socio-economic disadvantage. The findings of this study progress the hypothesis that the association between black smoke and birth weight may be mediated through intrauterine growth restriction. The associations between black smoke and birth weight were of the same order of magnitude as those reported for passive smoking. These findings add to the growing evidence of the harmful effects of air pollution on birth outcomes.


Stress history and pubertal development interact to shape hypothalamic-pituitary-adrenal axis plasticity.
Both the magnitude and the duration of the hormonal stress response change dramatically during neonatal development and aging as well as with prior experience with a stressor. However, surprisingly little is known with regard to how pubertal maturation and experience with stress interact to affect hypothalamic-pituitary-adrenal axis responsiveness. Because adolescence is a period of neurodevelopmental vulnerabilities and opportunities that may be especially sensitive to stress, it is imperative to more fully understand these interactions. Thus, we examined hormonal and neural responses in prepubertal (28 d of age) and adult (77 d of age) male rats after exposure to acute (30 min) or more chronic (30 min/d for 7 d) restraint stress. We report here that after acute stress, prepubertal males exhibited a significantly prolonged hormonal stress response (e.g. ACTH and total and free corticosterone) compared with adults. In contrast, after chronic stress, prepubertal males exhibited a higher response immediately after the stressor, but a faster return to baseline, compared with adults. Additionally, we demonstrate that this differential stress reactivity is associated with differential neuronal activation in the paraventricular nucleus of the hypothalamus, as measured by FOS immunohistochemistry. Using triple-label immunofluorescence histochemistry, we found that a larger proportion of CRH, but not arginine vasopressin, cells are activated in the arginine vasopressin in response to both acute and chronic stress in prepubertal animals compared with adults. These data indicate that experience-dependent plasticity of the hypothalamic-pituitary-adrenal neuroendocrine axis is significantly influenced by pubertal maturation.


Management of patients with stroke and a patent foramen ovale.
Observational studies have established a strong association between the presence of patent foramen ovale (PFO) and increased risk of ischemic stroke. The mechanism involved is presumed to be a paradoxical embolism from a venous thrombus that travels via the PFO to the systemic circulation. The best treatment modality to prevent recurrent stroke in patients with PFO remains undefined. There are four major choices: 1) medical therapy with anticoagulants, 2) medical therapy with antiplatelet agents, 3) surgical closure, and 4) percutaneous device closure. The Patent Foramen Ovale in Cryptogenic Stroke Study has demonstrated that antiplatelet and anticoagulant therapies are of equal benefit in preventing recurrent neurologic events in stroke patients with a PFO. Medical therapy should remain as the initial choice of secondary prophylactic therapy. PFO closure, either surgical or percutaneous, may further reduce event rates; however, this remains to be demonstrated because no randomized trial to date has compared PFO closure with medical therapy.


Evaluation of a fentanyl drug checking service for clients of a supervised injection facility, Vancouver, Canada.
British Columbia, Canada, is experiencing a public health emergency related to opioid overdoses driven by consumption of street drugs contaminated with illicitly manufactured fentanyl. This cross-sectional study evaluates a drug checking intervention for the clients of a supervised injection facility (SIF) in Vancouver. Insite is a facility offering supervised injection services in Vancouver's Downtown East Side, a community with high levels of injection drug use and associated harms, including overdose deaths. During July 7, 2016, to June 21, 2017, Insite clients were offered an opportunity to check their drugs for fentanyl using a test strip designed to test urine for fentanyl. Results of the drug check were recorded along with information including the substance checked, whether the client intended to dispose of the drug or reduce the dose and whether they experienced an overdose. Logistic regression models were constructed to assess the associations between drug checking results and dose reduction or drug disposal. Crude odds ratios (OR) and 95% confidence intervals (CI) were reported. About 1% of the visits to Insite during the study resulted in a drug check. Out of 1411 drug checks conducted by clients, 1121 (79.8%) were positive for fentanyl. Although most tests were conducted post-consumption, following a positive pre-consumption drug check, 36.3% (n = 142) of participants reported planning to reduce their drug dose while only 11.4% (n = 50) planned to dispose of their drug. While the odds of intended dose reduction among those with a positive drug check was significantly higher than those with a negative result (OR = 9.36; 95% CI 4.25-20.65), no association was observed between drug check results and intended drug disposal (OR = 1.60; 95% CI 0.79-3.26). Among all participants, intended dose reduction was associated with significantly lower odds of overdose (OR = 0.41; 95% CI 0.18-0.89). Although only a small proportion of visits resulted in a drug check, a high proportion (~ 80%) of the drugs checked were contaminated with fentanyl. Drug checking at harm reduction facilities such as SIFs might be a feasible intervention that could contribute to preventing overdoses in the context of the current overdose emergency.


Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels.
The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2-Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.


National surveillance of asthma: United States, 2001-2010.
Asthma is prevalent but treatable: adherence to evidence-based treatment lessens impairment and lowers the risk of future exacerbations. This report details recent trends in asthma prevalence, health care use, and mortality since 2001 and presents an overview of trends since 1980. Asthma prevalence estimates were obtained from the National Health Interview Survey (2001-2010). Physician office visit data were obtained from the National Ambulatory Medical Care Survey, hospital outpatient department and emergency department (ED) visit data from the National Hospital Ambulatory Medical Care Survey, hospitalization data from the National Hospital Discharge Survey, and death data from the National Vital Statistics System (2001-2009). Two types of rates were calculated: population-based rates based on the total population and risk-based rates based on the population with asthma. Current asthma prevalence increased from 2001 to 2010. There were no significant changes in rates for hospital outpatient department visits, ED visits, or hospitalizations, whereas risk-based rates for private physician office visits declined. Asthma death rates decreased from 2001 to 2009. Over the long term, asthma prevalence rose more slowly after 2001 than during 1980-1996, asthma hospitalizations declined since 1984 and deaths declined since 1999. Disparities by race and sex for adverse outcomes remained high despite these declines. Since 2001, asthma prevalence increased, risk-based rates for visits to private physician offices and deaths declined, and risk-based rates for other types of ambulatory visits and for hospitalizations showed no clear trend.


Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm.
When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize.


Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.
A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an 'autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.


Renal tubular effects of prolonged therapy with the GLP-1 receptor agonist lixisenatide in patients with type 2 diabetes mellitus.
Glucagon-like peptide-1 (GLP-1) receptor agonists (RAs) are well-established glucose-lowering drugs for type 2 diabetes mellitus (T2DM) management. Acute GLP-1RA administration increases urinary excretion of sodium and other electrolytes. However, the renal tubular effects of prolonged GLP-1RA treatment are largely unknown. In this secondary analysis of a randomized trial, we determined the renal tubular effects of 8-wk treatment with 20 μg lixisenatide, a short-acting (prandial) GLP-1RA, versus titrated once-daily insulin glulisine in 35 overweight T2DM-patients on stable insulin glargine background therapy (age: 62 ± 7 yr, glycated hemoglobin: 8.0 ± 0.9%, estimated glomerular filtration rate: >60 ml·min-1·1.73 m-2). After a standardized breakfast, lixisenatide increased absolute and fractional excretions of sodium, chloride, and potassium and increased urinary pH. In contrast, lixisenatide reduced absolute and fractional excretions of magnesium, calcium, and phosphate. At week 8, patients treated with lixisenatide had significantly more phosphorylated sodium-hydrogen exchanger isoform 3 (NHE3) in urinary extracellular vesicles than those on insulin glulisine treatment, which suggested decreased NHE3 activity in the proximal tubule. A rise in postprandial blood pressure with lixisenatide partly explained the changes in the urinary excretion of sodium, potassium, magnesium, and phosphate and the changes in urinary pH. In conclusion, lixisenatide affects postprandial urinary excretion of several electrolytes and increases urinary pH compared with insulin glulisine in T2DM patients after 8 wk of treatment. This is most likely explained by a drug-induced rise in blood pressure or direct inhibitory effects on NHE3 in the proximal tubule.


Diagnostic sensitivity of ultrasound, radiography and computed tomography for gender determination in four species of lizards.
Gender determination is frequently requested by reptile breeders, especially for species with poor or absent sexual dimorphism. The aims of the current study were to describe techniques and diagnostic sensitivities of ultrasound, radiography, and computed tomography for gender determination (identification of hemipenes) in four species of lizards. Nineteen lizards of known sex, belonging to four different species (Pogona vitticeps, Uromastyx aegyptia, Tiliqua scincoides, Gerrhosaurus major) were prospectively enrolled. With informed owner consent, ultrasound, noncontrast CT, contrast radiography, and contrast CT (with contrast medium administered into the cloaca) were performed in conscious animals. Imaging studies were reviewed by three different operators, each unaware of the gender of the animals and of the results of the other techniques. The lizard was classified as a male when hemipenes were identified. Nineteen lizards were included in the study, 10 females and nine males. The hemipenes were seen on ultrasound in only two male lizards, and appeared as oval hypoechoic structures. Radiographically, hemipenes filled with contrast medium appeared as spindle-shaped opacities. Noncontrast CT identified hemipenes in only two lizards, and these appeared as spindle-shaped kinked structures with hyperattenuating content consistent with smegma. Hemipenes were correctly identified in all nine males using contrast CT (accuracy of 100%). Accuracy of contrast radiography was excellent (94.7%). Accuracy of ultrasound and of noncontrast CT was poor (64.3% and 63.1%, respectively). Findings from the current study supported the use of contrast CT or contrast radiography for gender determination in lizards.


Skin nerve misfolded α-synuclein in pure autonomic failure and Parkinson disease.
To characterize the expression in skin nerves of native (n-syn) and misfolded phosphorylated (p-syn) α-synucleins in pure autonomic failure (PAF) and idiopathic Parkinson disease (IPD). The specific aims were to (1) define the importance of n-syn and p-syn as disease biomarkers and (2) ascertain differences in abnormal synuclein skin nerve deposits. We studied 30 patients, including 16 well-characterized IPD patients and 14 patients fulfilling PAF diagnostic criteria, and 15 age-matched controls. Subjects underwent skin biopsy from proximal (ie, cervical) and distal (ie, thigh and leg) sites to study small nerve fiber and intraneural n-syn and p-syn. PAF and IPD showed length-dependent somatic and autonomic small fiber loss, more severely expressed in patients with higher p-syn load. n-syn was similarly expressed in both groups of patients and controls. By contrast, p-syn was not evident in any skin sample of controls but was found in all PAF and IPD patients, although with different skin innervation. In addition, abnormal α-synuclein deposits were found in all analyzed skin samples in PAF but in only 49% of samples with a higher positivity rate at the proximal site in IPD. (1) Intraneural p-syn was a reliable in vivo marker of PAF and IPD; (2) neuritic p-syn inclusions differed in PAF and IPD, suggesting a different underlying pathogenesis; (3) when searching for abnormal p-syn deposits in skin nerves, the site of analysis is irrelevant in PAF but it is critical in IPD.