NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11.
NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8-11 for all 122 alleles. artificial neural network predictions are given as actual IC(50) values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75-80% confirmed MHC binders. Here, the performance is further validated and benchmarked using a large set of newly published affinity data, non-redundant to the training set. The server is free of use and available at: http://www.cbs.dtu.dk/services/NetMHC.


Feasibility, acceptability, and effectiveness of non-pharmaceutical interventions against infectious diseases among crisis-affected populations: a scoping review.
Non-pharmaceutical interventions (NPIs) are a crucial suite of measures to prevent and control infectious disease outbreaks. Despite being particularly important for crisis-affected populations and those living in informal settlements, who typically reside in overcrowded and resource limited settings with inadequate access to healthcare, guidance on NPI implementation rarely takes the specific needs of such populations into account. We therefore conducted a systematic scoping review of the published evidence to describe the landscape of research and identify evidence gaps concerning the acceptability, feasibility, and effectiveness of NPIs among crisis-affected populations and informal settlements. We systematically reviewed peer-reviewed articles published between 1970 and 2020 to collate available evidence on the feasibility, acceptability, and effectiveness of NPIs in crisis-affected populations and informal settlements. We performed quality assessments of each study using a standardised questionnaire. We analysed the data to produce descriptive summaries according to a number of categories: date of publication; geographical region of intervention; typology of crisis, shelter, modes of transmission, NPI, research design; study design; and study quality. Our review included 158 studies published in 85 peer-reviewed articles. Most research used low quality study designs. The acceptability, feasibility, and effectiveness of NPIs was highly context dependent. In general, simple and cost-effective interventions such as community-level environmental cleaning and provision of water, sanitation and hygiene services, and distribution of items for personal protection such as insecticide-treated nets, were both highly feasible and acceptable. Logistical, financial, and human resource constraints affected both the implementation and sustainability of measures. Community engagement emerged as a strong factor contributing to the effectiveness of NPIs. Conversely, measures that involve potential restriction on personal liberty such as case isolation and patient care and burial restrictions were found to be less acceptable, despite apparent effectiveness. Overall, the evidence base was variable, with substantial knowledge gaps which varied between settings and pathogens. Based on the current landscape, robust evidence-based guidance is not possible, and a research agenda is urgently required that focusses on these specific vulnerable populations. Although implementation of NPIs presents unique practical challenges in these settings, it is critical that such an agenda is put in place, and that the lessons learned from historical and present experiences are documented to build a firm evidence base.


The metabolism of diclofenac--enzymology and toxicology perspectives.
Diclofenac is a nonsteroidal anti-inflammatory drug bearing a carboxylic acid functional group. As a result, the metabolism of diclofenac in humans partitions between acyl glucuronidation and phenyl hydroxylation, with the former reaction catalyzed primarily by uridine 5'-diphosphoglucuronosyl transferase 2B7 while the latter is catalyzed by cytochrome P450 (CYP)2C9 and 3A4. Further hydroxylation of diclofenac glucuronide was shown to occur in vitro with recombinant CYP2C8, which may be of clinical significance in terms of defining major metabolic routes involved in the elimination of diclofenac in humans. The 4'-hydroxylation of the drug appears to represent a feature reaction for CYP2C9 catalysis, and this regioselective oxidation is presumably dictated by interactions of the carboxylate moiety of the substrate with a putative cationic residue of the enzyme. Several other residues of CYP2C9 were identified in studies with site-directed mutants that influence substrate binding affinity and specificity, including Arg97, Phe114, Asn289 and Ser286. The 5-hydroxylation of diclofenac is subject to CYP3A4 cooperativity elicited by quinidine. In this case, enhancement by quinidine of diclofenac metabolism in vitro was attributed to increases in the V(max) with little contribution from changes in the K(m) value. These cooperative interactions in recombinant systems, however, appeared to be influenced by enzyme host membranes of various cDNA-directed expressing CYP3A4. Nevertheless, the in vivo significance of CYP3A cooperativity was demonstrated in a pharmacokinetic study in monkeys, wherein the hepatic clearance of diclofenac increased 2-fold when quinidine was co-administered. Therapeutic use of diclofenac is associated with rare but sometimes fatal hepatotoxicity characterized by delayed onset of symptoms and lack of a clear dose-response relationship. The toxicity has consequently been categorized as metabolic idiosyncrasy. In this regard, the acyl glucuronide of the drug was demonstrated to be reactive and capable of covalent modification of cellular proteins, with covalent binding to liver proteins in rats depending on the activity of multidrug resistance protein 2, a hepatic canalicular transporter. One of the modified proteins was identified as dipeptidyl peptidase IV. Formation of protein adducts also was evident following the oxidative metabolism of diclofenac catalyzed by CYP enzymes. The reactive intermediates in this case were presumably diclofenac 1',4'- and 2,5-quinone imines, both of which were trapped by conjugation with glutathione and identified as glutathione adducts. These same glutathione adducts were detected in rats as well as in human hepatocytes treated with diclofenac, and a corresponding mercapturic acid derivative was identified in urine from patients administered the drug. It is conceivable that the acyl glucuronide and benzoquinone imines derived from diclofenac modify proteins covalently and thereby produce toxicity in susceptible patients via either direct disruption of critical cellular functions or elicitation of immunological responses.


The Relationship Between Images Posted by New Mothers on WeChat Moments and Postpartum Depression: Cohort Study.
As social media posts reflect users' emotions, WeChat Moments, the most popular social media platform in China, may offer a glimpse into postpartum depression in the population. This study aimed to investigate the features of the images that mothers posted on WeChat Moments after childbirth and to explore the correlation between these features and the mothers' risk of postpartum depression. We collected the data of 419 mothers after delivery, including their demographics, factors associated with postpartum depression, and images posted on WeChat Moments. Postpartum depression was measured using the Edinburgh Postnatal Depression Scale. Descriptive analyses were performed to assess the following: content of the images, presence of people, the people's facial expressions, and whether or not memes were posted on WeChat Moments. Logistic regression analyses were used to identify the image features associated with postpartum depression. Compared with pictures of other people, we found that pictures of their children comprised the majority (3909/6887, 56.8%) of the pictures posted by the mothers on WeChat Moments. Among the posts showing facial expressions or memes, more positive than negative emotions were expressed. Women who posted selfies during the postpartum period were more likely to have postpartum depression (P=.003; odds ratio 2.27, 95% CI 1.33-3.87). The vast majority of mothers posted images conveying positive emotions during the postpartum period, but these images may have masked their depression. New mothers who have posted selfies may be at a higher risk of postpartum depression. International Clinical Trials Registry Platform ChiCTR-ROC-16009255; http://www.chictr.org.cn/showproj.aspx?proj=15699.


A Systematic Review of Nutraceuticals for the Treatment of Bipolar Disorder.
Certain nutrient supplements (nutraceuticals) may target neurobiological pathways perturbed in bipolar disorder (BD) such as inflammation, oxidative stress, and mitochondrial dysfunction. Nutraceuticals thus may have a potential role as adjunctive treatments for BD. A search of Embase via embase.com, PubMed via PubMed, Cumulated index to nursing and allied health literature (CINAHL) Complete via EBSCO, and Cochrane Central Register of Controlled Clinical Trials via cochranelibrary.com was conducted to identify published randomized controlled trials assessing the efficacy of nutraceuticals on mood symptomatology in adults with BD. Search terms for BD, nutraceuticals, and clinical trials (total search terms = 75) were used to search from inception to February 20, 2020. The Cochrane Collaboration's tool for assessing the risk of bias in randomized trials was used to assess the risk of bias. A total of 1,712 studies were identified through the search. After rigorous screening, 22 studies were included in the review. There was large variability across the studies with 15 different nutraceutical agents assessed and as such insufficient homogeneity for a meta-analysis to be conducted (I2 > 50%). Studies revealed promising, albeit conflicting, evidence for omega-3 fatty acids and N-acetylcysteine. Isolated positive results were reported for coenzyme Q10. Given nutraceuticals are tolerable and accessible, they may be useful as potential adjunctive treatments for BD. Nutraceuticals targeting neuroinflammation or mitochondrial activity may have the most potential for the depressive phase. However, further studies are required to determine efficacy.


Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis.
CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440-669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440-669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.


Acute Vertebrobasilar Artery Occlusion with Underlying Atherosclerosis: Balloon Angioplasty Combined with Tirofiban as Initial Salvage Therapy.
The optimal rescue endovascular treatment for patients with intracranial atherosclerotic stenosis in acute vertebrobasilar artery occlusion is not well established. We investigated the safety and efficacy of balloon angioplasty combined with tirofiban as the initial rescue strategy in these patients. We retrospectively analyzed the records of 41 patients admitted between January 2014 and September 2022, with vertebrobasilar artery atherosclerotic occlusion. Balloon angioplasty in combination with tirofiban was used as the first-line salvage therapy after the failure of mechanical thrombectomy. The technical success rate, recanalization outcome, procedure-related complications, symptomatic intracranial hemorrhage, and functional outcome at 90 days were reviewed. Recanalization with a modified Thrombolysis in Cerebral Infarction grade of 2b-3 was achieved in 38 of the 41 patients (92.7%). Acute stents were deployed in 5 patients who did not achieve successful reperfusion after balloon angioplasty. Six patients (14.6%, 6/41) underwent stent angioplasty in the stable stage for severe residual stenosis detected on follow-up imaging. There was no procedure-related complication. Hemorrhagic transformation was detected on follow-up imaging in 11 patients (26.8%), while no symptomatic intracranial hemorrhage was recorded. Good functional outcome rate was 31.7% (13/41). Balloon angioplasty combined with intravenous tirofiban administration is a safe and effective salvage therapy in patients with acute atherosclerotic occlusion of the vertebrobasilar artery.


Characterisation of and polysaccharide production by amoxycillin-resistant streptococci.
Small numbers of bacteria capable of growing on agar supplemented with amoxycillin 40 mg/L were isolated from the saliva of 9 out of 20 adult volunteers in a previous study. All the bacteria were identified as Streptococcus sanguis although no strains produced dextran in conventional tests. However, using a specific assay, all the antibiotic-resistant strains were found to secrete glucosyltransferases (GTF), the enzymes that synthesise these extracellular polysaccharides; the production of GTF-S, the enzyme that synthesizes dextran, was 22-43% less than that of an antibiotic-sensitive control strain. Enzyme production by both antibiotic-resistant and sensitive bacteria was markedly inhibited by dextran primer. The amoxycillin-resistant bacteria were resistant to other penicillins; their resistance to erythromycin was variable but they were uniformly sensitive to cephalothin and clindamycin. As dextran production has been proposed as a key factor in the colonisation of damaged heart valves by bacteria such as S. sanguis, these highly resistant bacteria may not pose a threat to the susceptible individual.


[Seroepidemiological study of coronavirus infection in children and adults in St. Petersburg].
As the result of prolonged (17 years) observations of patients with acute respiratory infections hospitalized in basic departments of clinics of the Research Institute of Influenza, coronavirus infection was found to be the cause of respiratory diseases, on the average, in 12% of cases (in some years in 6.8% to 28.6% of cases). The analysis of extensive morbidity rates among different age groups of the population showed that children were affected by coronavirus infection 5-7 times more often than adults. Three year cycles of this infection were established. The periods of coronaviruses activation were accompanied by their detection in patient material by electron-microscopy, a sharp increase of immune response of patients as well as in the number of nosocomial infections and the proportion of the monoinfection of the coronavirus nature. Coronaviruses played the leading role among other viruses in the etiology of hospital respiratory infections. Mucosal antibodies to coronaviruses in the secretions of the nasal cavity proved to be more important than serum antibodies not only in protection from infection, but also in the pattern of clinical manifestations of the disease.


Outpatient mental health care, self-help groups, and patients' one-year treatment outcomes.
To examine the association between the duration and amount of outpatient mental health care, participation in self-help groups, and patients' casemix-adjusted one-year outcomes. A total of 2,376 patients with substance use disorders, 35% of whom also had psychiatric disorders, were assessed at entry to treatment and at a one-year follow-up. Information about the duration and amount of outpatient mental health care was obtained from a centralized health services utilization database. Patients who obtained regular outpatient mental health care over a longer interval and patients who attended more self-help group meetings had better one-year substance use and social functioning outcomes than did patients who were less involved in formal and informal care. The amount of outpatient mental health care did not independently predict one-year outcomes. The duration of outpatient mental health care and the level of self-help involvement are independently associated with less substance use and more positive social functioning. The provision of low intensity treatment for a longer time interval may be a cost-effective way to enhance substance abuse and psychiatric patients' long-term outcomes.


Predictors of low cardiac output in decompensated severe heart failure.
To identify predictors of low cardiac output and mortality in decompensated heart failure. Introduction: Patients with decompensated heart failure have a high mortality rate, especially those patients with low cardiac output. However, this clinical presentation is uncommon, and its management is controversial. We studied a cohort of 452 patients hospitalized with decompensated heart failure with an ejection fraction of <0.45. Patients underwent clinical-hemodynamic assessment and Chagas disease immunoenzymatic assay. Low cardiac output was defined according to L and C clinical-hemodynamic profiles. Multivariate analyses assessed clinical outcomes. P<0.05 was considered significant. The mean age was 60.1 years; 245 (54.2%) patients were >60 years, and 64.6% were men. Low cardiac output was present in 281 (63%) patients on admission. Chagas disease was the cause of heart failure in 92 (20.4%) patients who had higher B type natriuretic peptide levels (1,978.38 vs. 1,697.64 pg/mL; P = 0.015). Predictors of low cardiac output were Chagas disease (RR: 3.655, P<0.001), lower ejection fraction (RR: 2.414, P<0.001), hyponatremia (RR: 1.618, P = 0.036), and renal dysfunction (RR: 1.916, P = 0.007). Elderly patients were inversely associated with low cardiac output (RR: 0.436, P = 0.001). Predictors of mortality were Chagas disease (RR: 2.286, P<0.001), ischemic etiology (RR: 1.449, P = 0.035), and low cardiac output (RR: 1.419, P = 0.047). In severe decompensated heart failure, predictors of low cardiac output are Chagas disease, lower ejection fraction, hyponatremia, and renal dysfunction. Additionally, Chagas disease patients have higher B type natriuretic peptide levels and a worse prognosis independent of lower ejection fraction.


Establishment and functional characterization of immortalized rabbit dermal papilla cell lines.
Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFβ2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.


Psychosocial stress and ovarian function in adolescent and young adult cancer survivors.
Is psychosocial stress associated with ovarian function in reproductive-aged survivors of cancer diagnosed as adolescents and young adults (AYA survivors)? We observed no association between self-reported and biomarkers of psychosocial stress and ovarian function in AYA survivors. Psychosocial stress suppresses hypothalamic-pituitary-ovarian axis, resulting in ovulatory dysfunction, decreased sex steroidogenesis and lower fertility in reproductive-aged women. Many cancer survivors experience high psychosocial stress and hypothalamic-pituitary-adrenal axis dysregulation. The menstrual pattern disturbances and infertility they experience have been attributed to ovarian follicle destruction, but the contribution of psychosocial stress to these phenotypes is unknown. A cross-sectional study was conducted estimating the association between perceived stress, measured by self-report and saliva cortisol, and ovarian function, measured by bleeding pattern, dried blood spot (DBS) FSH and LH, and saliva estradiol. We included 377 AYA survivor participants. AYA survivor participants were ages 15-35 at cancer diagnosis and ages 18-40 at study enrollment, had completed primary cancer treatment, had a uterus and at least one ovary, did not have uncontrolled endocrinopathy and were not on hormone therapy. Recruited from cancer registries, physician referrals and cancer advocacy groups, participants provided self-reported information on psychosocial stress (Perceived Stress Scale-10 (PSS-10)) and on cancer and reproductive (fertility, contraception, menstrual pattern) characteristics. DBS samples were collected timed to the early follicular phase (cycle Days 3-7) for menstruating individuals and on a random day for amenorrheic individuals; saliva samples were collected three time points within 1 day. FSH and LH were measured by DBS ELISAs, cortisol was measured by ELISA and estradiol was measured by liquid chromatography tandem mass spectrometry. The median age of participants was 34.0 years (range 19-41) at a median of 6.0 years since cancer diagnosis. The most common cancer was breast (32.1%). Median PSS-10 score was 15 (range 0-36), with 5.3% scoring ≥26, the cut point suggestive of severe stress. Cortisol levels followed a diurnal pattern and cortisol AUC was negatively correlated with PSS-10 scores (P = 0.03). Neither PSS-10 scores nor cortisol AUC were associated with FSH, LH, estradiol levels or menstrual pattern. Waking and evening cortisol and the cortisol awakening response also were not related to ovarian function measures. Our analysis is limited by its cross-sectional nature, heterogeneity of cancer diagnosis and treatments and low prevalence of severe stress. The lack of association between psychosocial stress and a variety of ovarian function measures in female AYA cancer survivors suggests that psychosocial stress does not have a significant impact on the reproductive axis of AYA survivors. This finding is important in counseling this population on their menstrual pattern and family building plans. NIH HD080952, South Korea Health Industry Development Institute HI18C1837 (JK). Dr A.D. works for Bluebird Bio, Inc., Dr D.Z. works for ZRT Labs and Dr P.M.S. works for Ansh Labs, which did not sponsor, support or have oversight of this research. Other authors report no competing interests. N/A.


Rabies in Iraq: trends in human cases 2001-2010 and characterisation of animal rabies strains from Baghdad.
Control of rabies requires a consistent supply of dependable resources, constructive cooperation between veterinary and public health authorities, and systematic surveillance. These are challenging in any circumstances, but particularly during conflict. Here we describe available human rabies surveillance data from Iraq, results of renewed sampling for rabies in animals, and the first genetic characterisation of circulating rabies strains from Iraq. Human rabies is notifiable, with reported cases increasing since 2003, and a marked increase in Baghdad between 2009 and 2010. These changes coincide with increasing numbers of reported dog bites. There is no laboratory confirmation of disease or virus characterisation and no systematic surveillance for rabies in animals. To address these issues, brain samples were collected from domestic animals in the greater Baghdad region and tested for rabies. Three of 40 brain samples were positive using the fluorescent antibody test and hemi-nested RT-PCR for rabies virus (RABV). Bayesian phylogenetic analysis using partial nucleoprotein gene sequences derived from the samples demonstrated the viruses belong to a single virus variant and share a common ancestor with viruses from neighbouring countries, 22 (95% HPD 14-32) years ago. These include countries lying to the west, north and east of Iraq, some of which also have other virus variants circulating concurrently. These results suggest possible multiple introductions of rabies into the Middle East, and regular trans-boundary movement of disease. Although 4000 years have passed since the original description of disease consistent with rabies, animals and humans are still dying of this preventable and neglected zoonosis.


Extending the In Vivo Residence Time of Macrophage Membrane-Coated Nanoparticles through Genetic Modification.
Nanoparticles coated with natural cell membranes have emerged as a promising class of biomimetic nanomedicine with significant clinical potential. Among them, macrophage membrane-coated nanoparticles hold particular appeal due to their versatility in drug delivery and biological neutralization applications. This study employs a genetic engineering approach to enhance their in vivo residence times, aiming to further improve their performance. Specifically, macrophages are engineered to express proline-alanine-serine (PAS) peptide chains, which provide additional protection against opsonization and phagocytosis. The resulting modified nanoparticles demonstrate prolonged residence times when administered intravenously or introduced intratracheally, surpassing those coated with the wild-type membrane. The longer residence times also contribute to enhanced nanoparticle efficacy in inhibiting inflammatory cytokines in mouse models of lipopolysaccharide-induced lung injury and sublethal endotoxemia, respectively. This study underscores the effectiveness of genetic modification in extending the in vivo residence times of macrophage membrane-coated nanoparticles. This approach can be readily extended to modify other cell membrane-coated nanoparticles toward more favorable biomedical applications.


Inotropic and chronotropic effects of propranolol in isolated atrium of rats with fructose-induced insulin-resistance
Insulin resistance (IR) is a metabolic deficiency associated with type 2 diabetes and commonly related to the pathogenesis of cardiovascular diseases, being the determining factor of the metabolic syndrome. This research aims to understand the chronotropic and inotropic effects of Propranolol in isolated atrium of rats with fructose-induced insulin-resistance. For this reason, 16 male Sprague-Dawley rats were assigned to two groups and given ad libitum access to one of the following diets: Perrarina® dog chow or Perrarina® dog chow supplemented with vegetable shortening and with fructose (20%) and sucrose (20%) added to the water supply. Both groups were maintained on their respective dietary regimens for eight months. At the end of this period insulin resistance was verified by routine blood test. The rat hearts were rapidly removed, and the atria were dissected and kept in Krebs solutions (37ºC, pH 7.4; 95% O2 - 5% CO2) in an isolated organ bath Letica®, connected to a polygraph Grass®, registering atria frequency. The Student´s t-test was used to evaluate statistical differences between the two groups (p<0.05). Cumulative dose-response curves with isoproterenol were established in basal condition, and after fifteen minutes of pre-incubation with propranolol (1x10-6 M). A significant positive chronotropic effect was observed in IR rats (68.92±4.16 vs 44.92±4.08 beats/sec of control; p<0.001). The maximum force of atrial contraction after pre-incubation with propranolol was significantly higher in the IR group (91.67±2.10 vs 42.6 ± 3.5; p<0.001). These findings suggest that a blunted response of atrial β-adrenoceptor to propranolol exists in rats with fructose- induced insulin-resistance.


PLGA nanoparticle formulations of risperidone: preparation and neuropharmacological evaluation.
The aim of this work was to develop extended-release poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles of risperidone and thermal-responsive in situ gel containing risperidone nanoparticles for parenteral (subcutaneous) delivery and to reduce the dose-dependent extrapyramidal side effects of risperidone. PLGA nanoparticles of risperidone were designed by nanoprecipitation method using polymeric stabilizer (Poloxamer 407). The prepared nanoparticles were characterized for particle size by photon correlation spectroscopy and atomic force microscopy. Poloxamer 407-based in situ gel containing PLGA nanoparticles of risperidone was prepared by modified cold method to control the initial rapid release from the nanoparticles. The in vivo efficacy (antipsychotic effect) of prepared formulations (nanoparticles and in situ gel containing nanoparticles) was studied by administering them subcutaneously to mice. Extrapyramidal side effects of the formulations were also studied. The particle size of the prepared nanoparticles ranged between 85 and 219 nm. About 89% to 95% drug encapsulation efficiency was achieved when risperidone was loaded at 1.7% to 8.3% by weight of the polymer. During in vivo studies prepared risperidone formulations showed an antipsychotic effect that was significantly prolonged over that of risperidone solution for up to 72 hours with fewer extrapyramidal side effects. The prolonged effect of risperidone was obtained from the risperidone formulations administered subcutaneously, and this may improve the treatment of psychotic disorders by dose reduction. The development of extended-release poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles of risperidone is reported in this paper, along with the development of thermal-responsive in situ gel containing risperidone nanoparticles for parenteral (subcutaneous) delivery and to reduce the dose-dependent extrapyramidal side effects. In vivo studies showed a significantly prolonged antipsychotic effect with fewer extrapyramidal side effects.


Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.
Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze co-occurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. We compared results obtained with the middle-down and the bottom-up strategy in calculating PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, we performed an external correction using a synthetic peptide library with known peptide relative abundance. Corrected bottom-up data were used as reference. Calculated abundances of single PTMs showed similar deviations from the reference when comparing middle-down and uncorrected bottom-up results. Moreover, we show that the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, we evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs.


HIV-1 virologic failure and acquired drug resistance among first-line antiretroviral experienced adults at a rural HIV clinic in coastal Kenya: a cross-sectional study.
An increasing number of people on antiretroviral therapy (ART) in sub-Saharan Africa has led to declines in HIV related morbidity and mortality. However, virologic failure (VF) and acquired drug resistance (ADR) may negatively affect these gains. This study describes the prevalence and correlates of HIV-1 VF and ADR among first-line ART experienced adults at a rural HIV clinic in Coastal Kenya. HIV-infected adults on first-line ART for ≥6 months were cross-sectionally recruited between November 2008 and March 2011. The primary outcome was VF, defined as a one-off plasma viral load of ≥400 copies/ml. The secondary outcome was ADR, defined as the presence of resistance associated mutations. Logistic regression and Fishers exact test were used to describe correlates of VF and ADR respectively. Of the 232 eligible participants on ART over a median duration of 13.9 months, 57 (24.6% [95% CI: 19.2 - 30.6]) had VF. Fifty-five viraemic samples were successfully amplified and sequenced. Of these, 29 (52.7% [95% CI: 38.8 - 66.3]) had at least one ADR, with 25 samples having dual-class resistance mutations. The most prevalent ADR mutations were the M184V (n = 24), K103N/S (n = 14) and Y181C/Y/I/V (n = 8). Twenty-six of the 55 successfully amplified viraemic samples (47.3%) did not have any detectable resistance mutation. Younger age (15-34 vs. ≥35 years: adjusted odd ratios [95% CI], p-value: 0.3 [0.1-0.6], p = 0.002) and unsatisfactory adherence (<95% vs. ≥95%: 3.0 [1.5-6.5], p = 0.003) were strong correlates of VF. Younger age, unsatisfactory adherence and high viral load were also strong correlates of ADR. High levels of VF and ADR were observed in younger patients and those with unsatisfactory adherence. Youth-friendly ART initiatives and strengthened adherence support should be prioritized in this Coastal Kenyan setting. To prevent unnecessary/premature switches, targeted HIV drug resistance testing for patients with confirmed VF should be considered.


Rationing decisions: from diversity to consensus.
As rationing decisions become more of an immediate reality for healthcare practitioners it is important to design mechanisms that facilitate carefully deliberated outcomes. No individual can be expected to be able to cover wide debate on their own, so an exercise has been designed that helps generate consensus decisions from diverse opinions. The exercise was piloted with two groups, an undergraduate medical class and the members of a general practice. Though the aims were different for each group, the tool was useful to both for producing the desired outcomes. Expert and non-expert knowledge were drawn upon and rationing prioritisation lists regarding funding of infertility treatment were generated. A description of the exercise and the results produced by the two groups are provided, as well as the theoretical placement for the significance of forming consensus from diversity.


Re-expression of SynGAP protein in adulthood improves translatable measures of brain function and behavior.
It remains unclear to what extent neurodevelopmental disorder (NDD) risk genes retain functions into adulthood and how they may influence disease phenotypes. SYNGAP1 haploinsufficiency causes a severe NDD defined by autistic traits, cognitive impairment, and epilepsy. To determine if this gene retains therapeutically-relevant biological functions into adulthood, we performed a gene restoration technique in a mouse model for SYNGAP1 haploinsufficiency. Adult restoration of SynGAP protein improved behavioral and electrophysiological measures of memory and seizure. This included the elimination of interictal events that worsened during sleep. These events may be a biomarker for generalized cortical dysfunction in SYNGAP1 disorders because they also worsened during sleep in the human patient population. We conclude that SynGAP protein retains biological functions throughout adulthood and that non-developmental functions may contribute to disease phenotypes. Thus, treatments that target debilitating aspects of severe NDDs, such as medically-refractory seizures and cognitive impairment, may be effective in adult patients.


Ubiquitylation as a quality control system for intracellular proteins.
Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins.


Novel therapies for pediatric acute lymphoblastic leukemia.
This review summarizes the current novel therapy landscape in pediatric acute lymphoblastic leukemia (ALL), with a focus on key clinical trials which will shape the future direction of care for these children. Recent landmark immunotherapy trials in B-ALL have demonstrated significant benefit for children, adolescents, and young adults with relapsed/refractory high-risk leukemia. Due to these successes, current trials are asking the question as to whether immunotherapy can be successfully incorporated upfront. Additionally, therapies targeting novel antigens or molecular pathways are being developed, providing new options for children previously thought to have incurable leukemia. As survival for ALL has relatively plateaued with maximizing intensity through conventional chemotherapy, continued preclinical and clinical study of novel immunotherapeutic and targeted agents is crucial to further improve outcomes in childhood leukemia.


Chemical hyperthyroidism: serum triiodothyronine levels in clinically euthyroid individuals treated with levothyroxine.
We have observed many patients treated with levothyroxine sodium who have elevated serum thyroxine (T4) levels but appear clinically euthyroid. Such patients generally have normal serum triiodothyronine (T3) values. A retrospective review at The Johns Hopkins Hospital, Baltimore, comparing the correlation of T3 and T4 values in levothyroxine-treated patients with that in patients not so treated was carried out from 1977 to 1979. Mean free thyroxine index (FTI) value in 104 levothyroxine-treated patients was 4.70 +/- 0.2 and mean T3 value was 177 +/- 9 ng/dL. In a group of 50 hyperthyroid patients, mean FTI value was 7.26. +/- 0.5, with a mean T3 value of 389 +/- 26 ng/dL. In 71 euthyroid patients, mean FTI value was 2.36 +/- 0.1, with a T3 value of 137 +/- 3 ng/dL. Computed ratios of T3 to FTI and T3 to T4 were significantly lower in the group treated with levothyroxine than in either the hyperthyroid or euthyroid nontreated groups. Levothyroxine-treated patients with high T4 levels but normal T3 levels were clinically euthyroid. Patients not treated with levothyroxine with similarly elevated T4 levels had elevated T3 levels and were clinically hyperthyroid. It is concluded that lower relative T3 levels in levothyroxine-treated patients may explain why these patients appear clinically euthyroid despite elevated T4 values. Serum T3 determination is the procedure of choice for evaluation of levothyroxine-treated individuals. Furthermore, an elevated FTI value in such an individual does not, in itself, dictate need to reduce dosage.


Transition from non-monotonic to monotonic electrical diffuse layers: impact of confinement on ionic liquids.
Intense investigations of room temperature ionic liquids have revealed not only their advantages in a wide range of technological applications but also triggered scientific debates about charge distribution properties within the bulk and near the solid-liquid interfaces. While many observations report on an alternating charge layering (i.e., spatially extended decaying charge density oscillations), there are recent conjectures that ionic liquids bear similarity to dilute electrolytes. Using a modified Poisson-Nernst-Planck model for ionic liquids (after Bazant et al., Phys. Rev. Lett. 2011, 106, 046102), we show that both behaviors are fundamental properties of ionic liquids. The transition from the non-monotonic (oscillatory) to the monotonic structure of electrical diffuse layers appears to non-trivially depend on ionic density in the bulk, electrostatic correlation length, confinement and surface properties. Consequently, the results not only reconcile the empirical results but also provide a powerful methodology to gain insights into the nonlinear aspects of concentrated electrolytes.


Volar Dislocation of Second, Third and Fourth Carpometacarpal Joints in Association with a Bennet's Fracture of the Thumb Carpo-Metacarpal Dislocation: A Case Report.
Injuries of the carpometacarpal joints of the long fingers are infrequent and go often unnoticed if a thorough clinical exploration and complete radiological assessment is not carried out. The overall frequency of carpometacarpal joints injuries is of 1-2% among trauma of the wrist and the carpus. These are normally secondary to high energy trauma (such as a car or a motorbike accidents). Among these kinds of injuries, the volar dislocation of the metacarpals is much less frequent than the dorsal dislocation, being even rarer the cases in which the three middle metacarpals are involved. In this case report, we present a case of volar dislocation of the middle three carpometacarpal joints in association with a Bennet's fracture of the thumb in a 30-year-old male. He was treated in the operating room with reduction and stabilization using Kirschner wires, which allowed a satisfactory recovery of the mobility of the fingers eight weeks after the intervention. It is important to produce an admission diagnosis of this kind of injuries to be able to treat them immediately. Treatment must be performed quickly to reduce and stabilize the dislocation, since this will avoid the vasculo-nervous compression or the edema which increases cutaneous suffering and the risk of complications.


Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.
Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.


Breath-hold BOLD fMRI without CO2 sampling enables estimation of venous cerebral blood volume: potential use in normalization of stimulus-evoked BOLD fMRI data.
BOLD fMRI signal has been used in conjunction with vasodilatory stimulation as a marker of cerebrovascular reactivity (CVR): the relative change in cerebral blood flow (CBF) arising from a unit change in the vasodilatory stimulus. Using numerical simulations, we demonstrate that the variability in the relative BOLD signal change induced by vasodilation is strongly influenced by the variability in deoxyhemoglobin-containing cerebral blood volume (CBV), as this source of variability is likely to be more prominent than that of CVR. It may, therefore, be more appropriate to describe the relative BOLD signal change induced by an isometabolic vasodilation as a proxy of deoxygenated CBV (CBVdHb) rather than CVR. With this in mind, a new method was implemented to map a marker of CBVdHb, termed BOLD-CBV, based on the normalization of voxel-wise BOLD signal variation by an estimate of the intravascular venous BOLD signal from voxels filled with venous blood. The intravascular venous BOLD signal variation, recorded during repeated breath-holding, was extracted from the superior sagittal sinus in a cohort of 27 healthy volunteers and used as a regressor across the whole brain, yielding maps of BOLD-CBV. In the same cohort, we demonstrated the potential use of BOLD-CBV for the normalization of stimulus-evoked BOLD fMRI by comparing group-level BOLD fMRI responses to a visuomotor learning task with and without the inclusion of voxel-wise vascular covariates of BOLD-CBV and the BOLD signal change per mmHg variation in end-tidal carbon dioxide (BOLD-CVR). The empirical measure of BOLD-CBV accounted for more between-subject variability in the motor task-induced BOLD responses than BOLD-CVR estimated from end-tidal carbon dioxide recordings. The new method can potentially increase the power of group fMRI studies by including a measure of vascular characteristics and has the strong practical advantage of not requiring experimental measurement of end-tidal carbon dioxide, unlike traditional methods to estimate BOLD-CVR. It also more closely represents a specific physiological characteristic of brain vasculature than BOLD-CVR, namely blood volume.


Monooctanoin-associated pulmonary edema.
Monooctanoin is a cholesterol solvent indicated for dissolution of retained biliary stones. We summarize four reports--one from the U.S. Food and Drug Administration's Spontaneous Reporting System for Adverse Drug Reactions and three from published medical literature--of noncardiogenic pulmonary edema during intrabiliary monooctanoin in the United States. Based on these data, we show that pulmonary edema during intrabiliary monooctanoin infusion may occur in approximately one per 1000 patients treated.


Physiologically realistic formation of autoassociative memory in networks with theta/gamma oscillations: role of fast NMDA channels.
Recordings from brain regions involved in memory function show dual oscillations in which each cycle of a low-frequency theta oscillation (5-8 Hz) is subdivided into about seven subcycles by high frequency gamma oscillations (20-60 Hz). It has been proposed (Lisman and Idiart 1995) that such networks are a multiplexed short-term memory (STM) buffer that can actively maintain about seven memories, a capability of human STM. A memory is encoded by a subset of principal neurons that fire synchronously in a particular gamma subcycle. Firing is maintained by a membrane process intrinsic to each cell. We now extend this model by incorporating recurrent connections with modifiable synapses to store long-term memory (LTM). The repetition provided by STM gradually modifies synapses in a physiologically realistic way. Because different memories are active in different gamma subcycles, the formation of autoassociative LTM requires that synaptic modification depend on N-methyl-D-aspartate (NMDA) channels having a time constant of deactivation that is of the same order as the duration of a gamma subcycle (15-50 msec). Many types of NMDA channels have longer time constants (150 msec), as for instance those found in the hippocampus, but both fast and slow NMDA channels are present in cortex. This is the first proposal for the special role of these fast NMDA channels. The STM for novel items must depend on activity-dependent changes intrinsic to neurons rather than recurrent connections, which have not developed the required selectivity. Because these intrinsic mechanisms are not error-correcting, STM will become slowly corrupted by noise. This limits the accuracy with which LTM can become encoded after a single presentation. Accurate encoding of items in LTM can be achieved by multiple presentations, provided different memory items are presented in a varied interleaved order. Our results indicate that a limited memory-capacity STM model can be integrated in the same network with a high-capacity LTM model.


Borna disease virus phosphoprotein represses p53-mediated transcriptional activity by interference with HMGB1.
Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that has a broad host range in warm-blooded animals, probably including humans. Recently, it was demonstrated that a 24-kDa phosphoprotein (P) of BDV directly binds to a multifunctional protein, amphoterin-HMGB1, and inhibits its function in cultured neural cells (W. Kamitani, Y. Shoya, T. Kobayashi, M. Watanabe, B. J. Lee, G. Zhang, K. Tomonaga, and K. Ikuta, J. Virol. 75:8742-8751, 2001). This observation suggested that expression of BDV P may cause deleterious effects in cellular functions by interference with HMGB1. In this study, we further investigated the significance of the binding between P and HMGB1. We demonstrated that P directly binds to the A-box domain on HMGB1, which is also responsible for interaction with a tumor suppression factor, p53. Recent works have demonstrated that binding between HMGB1 and p53 enhances p53-mediated transcriptional activity. Thus, we examined whether BDV P affects the transcriptional activity of p53 by interference with HMGB1. Mammalian two-hybrid analysis revealed that p53 and P competitively interfere with the binding of each protein to HMGB1 in a p53-deficient cell line, NCI-H1299. In addition, P was able to significantly decrease p53-mediated transcriptional activation of the cyclin G promoter. Furthermore, we showed that activation of p21(waf1) expression was repressed in cyclosporine-treated BDV-infected cells, as well as p53-transduced NCI-H1299 cells. These results suggested that BDV P may be a unique inhibitor of p53 activity via binding to HMGB1.


Classification of Mycobacterium farcinogenes and Mycobacterium senegalense by immunodiffusion and thin-layer chromatography of long-chain components.
Comparative immunodiffusion studies and thin-layer chromatographic analyses of whole-organism acid methanolysates were performed on 37 strains of Mycobacterium farcinogenes, Mycobacterium senegalense and Nocardia farcinica. The latter were clearly distinguished from the mycobacteria in containing a single mycolic acid methyl ester and showing more precipitinogens with nocardial than with mycobacterial and rhodococcal reference systems. The distribution of precipitinogens showed that M. farcinogenes and M. senegalense were very closely related and that both showed a greater affinity to Mycobacterium fortuitum than to any of the other established species of Mycobacterium tested. The complex pattern of alpha-mycolates and characteristic polar mycolates found in both M. farcinogenes and M. senegalense has only previously been found in M. fortuitum and Mycobacterium smegmatis.


Ultra-low loss Si3N4 waveguides with low nonlinearity and high power handling capability.
We investigate the nonlinearity of ultra-low loss Si3N4-core and SiO2-cladding rectangular waveguides. The nonlinearity is modeled using Maxwell's wave equation with a small amount of refractive index perturbation. Effective n2 is used to describe the third-order nonlinearity, which is linearly proportional to the optical intensity. The effective n2 measured using continuous-wave self-phase modulation shows agreement with the theoretical calculation. The waveguide with 2.8-μm wide and 80-nm thick Si3N4 core has low loss and high power handling capability, with an effective n2 of about 9×10(-16) cm2/W.


Bioconcentration and biotransformation of selenite versus selenate exposed periphyton and subsequent toxicity to the Mayfly Centroptilum triangulifer.
Little is known about the bioaccumulation dynamics, biotransformation processes, or subsequent toxicity to consumers of dissolved selenite (SeO3) versus selenate (SeO4) uptake into aquatic primary producer communities. To address these data gaps, we examined SeO3 and SeO4 bioconcentration into complex freshwater periphyton communities under static and static-renewal conditions. Further, we explored periphyton biotransformation of Se species using X-ray absorption near edge structure (XANES) spectroscopy analysis and changes in the periphyton associated microbial consortium using denaturing gradient gel electrophoresis (DGGE). Last, we fed differentially treated periphyton to the mayfly Centroptilum triangulifer in full life cycle exposures to assess toxicity. Selenite exposed periphyton readily bioconcentrated Se while, in contrast, initial periphyton uptake of SeO4 was negligible, but over time periphyton [Se] increased steadily in conjunction with the formation of dissolved SeO3. XANES analyses revealed that both SeO3 and SeO4 treated periphyton biotransformed Se similarly with speciation dominated by organo-selenide (∼61%). Mayfly survival, secondary production, and time to emergence were similar in both SeO3 and SeO4 treated periphyton exposures with significant adverse effects at 12.8 μg g(-1) ((d.w.) secondary production) and 36 μg g(-1) ((d.w.) survival and development time). Overall, dissolved selenium speciation, residence time, and organisms at the base of aquatic food webs appear to be the principal determinants of Se bioaccumulation and toxicity.


Tikhonov regularized solutions for improvement of signal-to-noise ratio in case of auditory-evoked potentials.
In the present study, standard Tikhonov regularization (STR) Technique and the subspace regularization (SR) method have been applied to remove the additive EEG noise on average auditory-evoked potential (EP) signals. In methodological manner, the difference between these methods is the formation of regularization matrices which are used to solve the weighted problem of EP estimation. Those methods are compared to ensemble averaging (EA) with respect to signal-to-noise-ratio (SNR) improvement in experimental studies, simulations and pseudo-simulations. The results of tests no superiority of the SR in comparison to STR has been observed. In addition, the STR is found to be less computational complex. Moreover, results support the theoretical fact that the STR was introduced to be optimum for smooth solutions whereas the SR allows sharp variations in solutions. Thus, the STR is found to be more useful in removing the noise with the average signal remaining.


Versatility of azide in serendipitous assembly of copper(II) magnetic polyclusters.
Engineering at the molecular level is one of the most exciting new developments for the generation of functional materials. However, the concept of designing polynuclear extended structures from bottom up is still not mature. Although progress has been made with secondary building units (SBUs) in metal organic frameworks (MOFs), the control seems to be just an illusion when it comes to bridging ligands such as the azide ion. When we say that the azido ligand is versatile in its bridging capabilities, what we mean is that it would be difficult to predict or control its bridging properties. However, this kind of serendipity is not always bad news. For example, scientists have shown that the azido ligand can mediate magnetic exchanges between paramagnetic metals in a predictable fashion (usually depending upon the bonding geometries). Therefore, it is a well-respected ligand in polynuclear assemblies. Serendipitous assemblies offer new magnetic structures that we may not otherwise even think about synthesizing. The azido ligand forms a variety of complexes with copper(II) using different blocking amines or pyridine based ligands. Its structural nature changes upon changing the substitution on amine, as well as the amount of blocking ligand. In principle, if we take any of these complexes and provide more coordination sites to the bridging azido ligands by removing a fraction of the blocking ligands, we can get new complexes with intricate structural networks and therefore different magnetic properties with the same components as used for the parent complex. In this Account, we mainly discuss the development of a number of new topological and magnetic exchange systems synthesized using this concept. Not all of these new complexes can be grouped according to their basic building structures or even by the ratio of the metal to blocking ligand. Therefore, we divided the discussion by the nuclearity of the basic building structures. Some of the complexes with the same nuclearities have very similar or even almost identical basic structures. However, the way these building units are joined together (by the azido bridges) to form the overall extended structures differ almost in every case. The complexes having the Cu6 core are particularly interesting from a structural point of view. Although they have almost identical basic structures, some of them are extended in three dimensions, but two of them are extended in two dimensions by two different bridging networks. In the complexes having linear Cu4 basic units, we find that using similar ligands does not always give the same bridging networks even within the basic building structures. These complexes have also enriched the field of molecular magnetism. One of the complexes with a Cu3 building unit has provided us with the opportunity to study the competing behavior of two different kinds of magnetic exchange mechanism (ferromagnetic and antiferromagnetic) acting simultaneously between two metal ions. Through density functional theory calculations, we showed how they work independently and their additive nature to produce the overall effect. The exciting methodology for the generation of copper(II) polyclusters presented in this Account will provide the opportunity to explore analogous serendipitous assembly of diverse structures with interesting magnetic behavior using other transition metal ions having more than one unpaired electrons.


Rimonabant: a novel selective cannabinoid-1 receptor antagonist for treatment of obesity.
The pharmacology, pharmacokinetics, clinical efficacy, safety, drug interactions, and dosage and administration of rimonabant in the treatment of obesity and related metabolic factors are reviewed. Discovery of the cannabinoid receptors has led to the development of rimonabant, a cannabinoid-1 (CB(1)) antagonist. Selective blockade of this receptor has been shown to lead to decreased appetite and food intake in animal models. Clinical studies have shown that rimonabant 20 mg once daily produces significant decreases in weight and waist circumference in obese human subjects and improves the lipid profile and glucose control. The frequency of metabolic syndrome also decreased significantly with rimonabant 20 mg daily. Limited data are available regarding the pharmacokinetics and pharmacodynamics of rimonabant. Preclinical data have demonstrated a long duration of action. As of yet, no drug-drug, drug-food, or drug-disease interactions have been identified with rimonabant. Adverse reactions occurred rarely, with nausea, dizziness, diarrhea, arthralgia, and back pain being the most common. Psychiatric disorders, including depression and anxiety, were the most common reasons for subjects to withdraw from rimonabant studies. Rimonabant has been shown to be safe for up to two years of treatment. Further research will clarify currently unknown areas, including pharmacokinetics, drug interactions, and the drug's role in standard therapy. Rimonabant, a selective CB(1) antagonist, is a novel treatment option for obese and overweight individuals. Significant weight loss, decrease in waist circumference, and improvements in lipid profile and glucose control have been shown in clinical trials of rimonabant.


Nucleoside-2',3'/3',5'-bis(thio)phosphate antioxidants are also capable of disassembly of amyloid beta42-Zn(ii)/Cu(ii) aggregates via Zn(ii)/Cu(ii)-chelation.
Currently, there is an urgent need for biocompatible metal-ion chelators capable of antioxidant activity and disassembly of amyloid beta (Aβ)-aggregates as potential therapeutics for Alzheimer's disease (AD). We recently demonstrated the promising antioxidant activity of adenine/guanine 2',3' or 3',5'-bis(thio)phosphate analogues, 2'-dA/G3'5'PO/S and A2'3'PO/S, and their affinity to Zn(ii)-ions. These findings encouraged us to evaluate them as agents for the dissolution of Aβ42-Zn(ii)/Cu(ii) aggregates. Specifically, we explored their ability to bind Cu(ii)/Zn(ii)-ions, the geometry and stoichiometry of these complexes, Cu(ii)/Zn(ii)-binding-sites and binding mode, and the ability of these analogues to dissolve Aβ42-Zn(ii)/Cu(ii) aggregates, as well as their effect on the secondary structure of those aggregates. Finally, we identified the most promising agents for dissolution of Aβ42-Zn(ii)/Cu(ii) aggregates. Specifically, we observed the formation of a 1 : 1 complex between 2'-dG3'5'PO and Cu(ii), involving O4 ligands. Zn(ii) was coordinated by both thiophosphate groups of 2'-dA3'5'PS and A2'3'PS involving O2S2 ligands in a 1 : 1 stoichiometry. A2'3'PS dissolves Aβ42-Zn(ii) and Aβ42-Cu(ii) aggregates as effectively as, and 2.5-fold more effectively than EDTA, respectively. Furthermore, 2'-dG3'5'PS and A2'3'PS reverted the Aβ42-M(ii) structure, back to that of the free Aβ42. Finally, cryo-TEM and TEM images confirmed the disassembly of Aβ42 and Aβ42-M(ii) aggregates by A2'3'PS. Hence, 2'-dG3'5'PS and A2'3'PS may serve as promising scaffolds for new AD therapeutics, acting as both effective antioxidants and agents for solubilization of Aβ42-Cu(ii)/Zn(ii) aggregates.


The Northwest Forest Plan as a model for broad-scale ecosystem management: a social perspective.
I evaluated the Northwest Forest Plan as a model for ecosystem management to achieve social and economic goals in communities located around federal forests in the US. Pacific Northwest. My assessment is based on the results of socioeconomic monitoring conducted to evaluate progress in achieving the plan's goals during its first 10 years. The assessment criteria I used related to economic development and social justice. The Northwest Forest Plan incorporated economic development and social justice goals in its design. Socioeconomic monitoring results indicate that plan implementation to achieve those goals met with mixed success, however I hypothesize there are two important reasons the plan's socioeconomic goals were not fully met: some of the key assumptions underlying the implementation strategies were flawed and agency institutional capacity to achieve the goals was limited. To improve broad-scale ecosystem management in the future, decision makers should ensure that natural-resource management policies are socially acceptable; land-management agencies have the institutional capacity to achieve their management goals; and social and economic management goals (and the strategies for implementing them) are based on accurate assumptions about the relations between the resources being managed and well-being in local communities. One of the difficulties of incorporating economic development and social justice goals in conservation initiatives is finding ways to link conservation behavior and development activities. From a social perspective, the Northwest Forest Plan as a model for ecosystem management is perhaps most valuable in its attempt to link the biophysical and socioeconomic goals of forest management by creating high-quality jobs for residents of forest communities in forest stewardship and ecosystem management work, thereby contributing to conservation.


Intestinal absorption mechanism of amphoteric beta-lactam antibiotics II: Michaelis-Menten kinetics of cyclacillin absorption and its pharmacokinetic analysis in rats.
The absorption of cyclacillin at pH 7.0 by the rat small intestine was investigated using in situ perfusion. At the lowest dose of 95 microgram/ml, the antibiotic disappearance was rapid and followed first-order kinetics, with the disappearance being 85% at 100 min. At the intermediate concentrations of 770 and 1200 microgram/ml, the disappearance after 100 min was 69 and 54%, respectively, and semilogarithmic plots clearly showed convex curvatures. At the highest concentration of 30 mg/ml, cyclacillin disappeared slowly from the perfusate, in an apparent first-order fashion. The disappearance was 26% after 100 min of perfusion and was similar in extent at 5.2 mg/ml. This concentration-time profile was satisfactorily fitted to the simultaneous Michaelis-Menten and first-order kinetic equations. The area under the blood concentration versus time curve (AUC) after a single intraduodenal dose of cyclacillin was almost consistent with the AUC after the equivalent intravenous dose (10 mg/kg). Additional evidence from a pharmacokinetic analysis of steady-state blood concentrations after constant infusion of cyclacillin through the portal vein and the small intestinal lumen indicated that cyclacillin absorption by the rat intestinal tissue at relatively low concentrations (less than 1 mg/ml) followed solely Michaelis-Menten kinetics. Cyclacillin may be transported by certain types of carrier-mediated mechanisms.


Deletion mapping of DNA regions required for SV40 early region promoter function in vivo.
The SV40 early region promoter, previously localized to the DNA segment bounded by the HpaII and HindIII restriction sites (nucleotides 346 and 5171), was further defined by construction of an extensive set of deletions within this region and measurement of their effects on (a) viral DNA replication, (b) virus multiplication and the ability to complement early and late mutations, (c) transformation of rat cells, (d) large T antigen formation, and (e) the location of the 5' ends of early mRNAs. One set of mutations is represented by deletions that begin at the HpaII site and extend unidirectionally for varying lengths toward the BglI site at ori. A second set of mutants contains deletions that start at ori and extend unidirectionally for varying lengths towards the HpaII site. A third set of mutants, with deletions or duplications of various lengths and boundaries, lie between the HpaII and BglI sites. Our studies indicate the following. (a) Ori, the sequence needed for initiating SV40 DNA replication, extends from the sequences needed for initiating SV40 DNA replication, extends from the sequences needed to bind T antigen to the palindrome in site II to nucleotide 34, the late region edge of the AT block. Flanking sequences adjacent to the AT block facilitate DNA replication. (b) The SV40 early region promoter comprises two functionally distinct nucleotide sequence elements. One is flanked by nucleotides 5231 and 107, and contains the RNA initiation sites at nucleotides 5231-5237, a positioning element resembling the TATAAATA consensus sequence about 20-25 nucleotides upstream, and an RNA polymerase II recognition sequence contributed by short GC-rich sequences clustered between nucleotides 35 and 107; we refer to this as the RNA polymerase II interaction site. The second distinct sequence element is contained within each of two 72-bp segments located between nucleotides 107 and 250; the behavior of this element suggests that it may influence the accessibility of RNA polymerase II for the interaction site or the efficiency of RNA chain initiation. Large T antigen binding sites I, II, and III overlap with the putative RNA polymerase II interaction site; since large T antigen does not prevent elongation of RNA transcripts initiated upstream, T antigen probably represses early region expression by preventing RNA polymerase II binding to the promoter.


Life history, code of honor, and emotional responses to inequality in an economic game.
The code of honor, which is characterized by a preoccupation with reputation and willingness to take retaliatory action, has been used extensively to explain individual and cultural differences in peoples' tendencies to behave aggressively. However, research on the relationship between the code of honor and emotional responses to social interactions has been limited in scope, focusing primarily on anger in response to insults and reputational threats. Here we broaden this scope by examining the relationship between code of honor and emotional reactions in response to an unfair economic exchange that resulted in unequal monetary earnings among 3 laboratory participants. We found that endorsement of the code of honor was related to anger and envy in response to unfair monetary distributions. Interestingly, code of honor predicted envy above and beyond what could be accounted for by anger, but the converse was not the case. This suggests that the code of honor influenced perceptions of how subjects viewed their own earnings relative to those of others, which consequently was responsible for their apparent anger as a result of the economic transaction. Furthermore, the unique relationship between code of honor and envy was present only for subjects who received unfair treatment and not for subjects who merely witnessed unfair treatment. Additionally, we replicated previous findings that harsh childhood environmental conditions are associated with endorsement of the code of honor, highlighting the potential value of incorporating a life history theoretical approach to investigating individual differences in endorsement of the code of honor.


Role of charcoal tattooing in localization of recurred papillary thyroid carcinoma: initial experiences.
Surgical excision is the definitive treatment for localized recurrence of papillary thyroid carcinoma. Reoperation for recurrence, however, is challenging and associated with increased operative times and complication rates. For safe and effective reoperation, ultrasound-guided charcoal tattooing localization can be used. The aim of this study was to investigate the feasibility and safety of the ultrasound-guided charcoal tattooing localization. Between November 2012 and August 2013, ten patients underwent preoperative charcoal tattooing localization for twelve recurrent lesions. Patient demographics, pathologic features, and operation results were reviewed. The technical success rate of charcoal tattooing was 100%. Eight patients had one recurrent lesion, and two patients had double lesions. Among these 12 recurrent lesions, three (25%) were found in level II, four (33%) in level IV, four (33%) in level VI, and one (8%) was found in the thyroidectomy bed site. The mean size of lesions was 0.87 ± 0.35 cm. Of these 10 patients, eight patients underwent selective lymph node dissection, one patient underwent modified radical neck dissection, and one patient underwent recurrent mass excision. Transient hypocalcemia developed in one patient, and no recurrent laryngeal nerve palsy occurred. There were no major complications related to the injection of the charcoal. The mean follow-up period after reoperation was 8.6 ± 2.7 months; in the follow-up ultrasound, there were no remnant lesions in all patients. Preoperative ultrasound-guided charcoal tattooing localization for recurrent thyroid cancer appears to be a feasible and safe procedure for reoperation. Further evaluation is warranted in larger patients' cohorts.


Evaluation of the Electroporation Efficiency of a Grid Electrode for Electrochemotherapy: From Numerical Model to In Vitro Tests.
Electrochemotherapy (ECT) is a local anticancer treatment based on the combination of chemotherapy and short, tumor-permeabilizing, voltage pulses delivered using needle electrodes or plate electrodes. The application of ECT to large skin surface tumors is time consuming due to technical limitations of currently available voltage applicators. The availability of large pulse applicators with few and more spaced needle electrodes could be useful in the clinic, since they could allow managing large and spread tumors while limiting the duration and the invasiveness of the procedure. In this article, a grid electrode with 2-cm spaced needles has been studied by means of numerical models. The electroporation efficiency has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. The computational results show the distribution of the electric field in a model of the treated tissue. These results are helpful to evaluate the effect of the needle distance on the electric field distribution. Furthermore, the in vitro tests showed that the grid electrode proposed is suitable to electropore, by a single application, a cell culture covering an area of 55 cm(2). In conclusion, our data might represent substantial improvement in ECT in order to achieve a more homogeneous and time-saving treatment, with benefits for patients with cancer.


Gelatinase activity in synovial fluid and synovium obtained from healthy and osteoarthritic joints of dogs.
To determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions. 35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII). MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints. Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue. Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD.


Noise robust one-dimensional blind deconvolution of medical ultrasound images.
Recently, several blind cepstral deconvolution methods for medical ultrasound images were compared experimentally. The results indicated that the generalized cepstrum or the complex cepstrum with phase unwrapping give the blind homomorphic deconvolution algorithms with the best performance. However, the frequency domain phase unwrapping for pulse estimation, which is an essential part of both methods, is sensitive to the sensor noise when the values of the spectrum are small due to the randomness of the tissue response. The noise introduces abrupt changes in the phase. The phase degradation due to the noise causes variable spatial and gray scale resolution in image sequences following deconvolution. This paper introduces a noise robust Bayesian phase unwrapping method using a noncausal Markov random chain model. The prior regularizing term accounts for the noise and smoothes the phase. The phase unwrapping is formulated as a least mean square optimization problem. The optimization is done noniteratively by solving a difference equation using the cosine transform. The resulting improvement in radial and lateral blind deconvolution is demonstrated on six short ultrasound image sequences recorded in vitro or in vivo.


Decline in survival after liver transplantation.
Twenty-three recent cases of orthotopic liver transplantation were individually reviewed in an effort to determine why survival had declined from the 50% one-year survival rate of an immediately precedent series. In the series of 23, only six (26%) achieved one-year survival. Faulty case selection, technical complications, the use of damaged organs, and complications of immunosuppression were the main causes of death. Attention was directed to the possible use of preoperative lymphoid depletion to improve the effectiveness and safety of immunosuppression.


Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows.
The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108 v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104 v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108 v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.


The Gut Microbiota in Multiple Sclerosis: An Overview of Clinical Trials.
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, and degenerative disease that affects the central nervous system. A recent study showed that interaction between the immune system and the gut microbiota plays a crucial role in the development of MS. This review reports the clinical studies carried out in recent years that aimed to evaluate the composition of the microbiota in patients with relapsing-remitting MS (RR-MS). We also report what is available in the literature regarding the effectiveness of fecal microbiota transplantation and the role of the diet in restoring the intestinal bacterial population. Studies report that patients with RR-MS have a microbiota that, compared with healthy controls, has higher amounts of Pedobacteria, Flavobacterium, Pseudomonas, Mycoplana, Acinetobacter, Eggerthella, Dorea, Blautia, Streptococcus and Akkermansia. In contrast, MS patients have a microbiota with impoverished microbial populations of Prevotella, Bacteroides, Parabacteroides, Haemophilus, Sutterella, Adlercreutzia, Coprobacillus, Lactobacillus, Clostridium, Anaerostipes and Faecalibacterium. In conclusion, the restoration of the microbial population in patients with RR-MS appears to reduce inflammatory events and the reactivation of the immune system.


The effect of colloid and crystalloid preloading on thromboelastography prior to Cesarean delivery.
Fluid preloading with colloids reduces hypotension after spinal anesthesia for Cesarean delivery more effectively than crystalloids. However, the effects of fluid preloading regimens on coagulation in pregnant patients remain unresolved. The aim of this study was to compare the effects on coagulation of fluid preloading with 6% hydroxyethyl starch (HES) and lactated Ringer's (LR) solution using thromboelastography (TEG) with kaolin-activated whole blood in healthy pregnant patients prior to spinal anesthesia for Cesarean delivery. After obtaining Ethics committee approval, 30 parturients were prospectively randomized prior to spinal anesthesia for elective Cesarean delivery to receive fluid preloading with either 1500 mL LR or 500 mL 6% HES over 30 min. Thromboelastography was performed immediately prior to and after fluid preloading. Standard TEG parameters were analyzed in terms of r time (min), k time (min), alpha angle (degrees) and maximum amplitude (mm). Group HES had statistically significant longer reaction times (r) and clot formation times (k) after fluid loading compared to baseline values (P < 0.05 respectively), although these post-fluid loading TEG parameters remained within a normal reference range. No significant differences in TEG values were seen after preloading within the LR group. Fluid preloading with 500 mL 6% HES in healthy parturients produced mild coagulation effects, as measured with TEG, prior to spinal anesthesia for Cesarean delivery. No significant effects on coagulation with TEG were observed following preloading with 1500 mL LR.


Altered glycoconjugates in cultures of retinitis pigmentosa retinal pigment epithelium.
These studies have shown that these are some alterations in the complex carbohydrates associated with the cell surface of RPE cells from donors with dominant retinitis pigmentosa. Differences are found both in proteoglycans and in glycoproteins. Proteoglycans of different sizes and glycosaminoglycan composition are associated with distinct cellular compartments of normal RPE cells. In the case of dominant retinitis pigmentosa studied here, there are some alterations in RPE proteoglycans. Within a given cellular compartment, there may be differences in the size distribution and/or the ratios of the proteoglycan size classes. In addition, there may be altered glycosaminoglycan content found between peaks of comparable size. There also appears to be more microheterogenity in oligosaccharides derived from surface glycoproteins of retinitis pigmentosa RPE cells. This may be due to differences in the total number of carbohydrate units or in the organization of the branching structure as suggested by altered size distribution and lectin-reactivity, respectively. While the reason for these differences is not clear, they could be due to abnormal processing and/or increased degradation. Considering their importance, alterations in cell surface glycoconjugates could affect the ability of RPE cells to interact with their environment and to maintain a healthy status.


Cognitive Normal Older Adults with APOE-2 Allele Show a Distinctive Functional Connectivity Pattern in Response to Cerebral Aβ Deposition.
The ε2 allele of apolipoprotein E (ε2) has neuroprotective effects against beta-amyloid (Aβ) pathology in Alzheimer's disease (AD). However, its impact on the functional connectivity and hub efficiency in cognitively normal older adults (CN) with ε2 is unclear. We investigated the functional connectivity differences in the default mode network (DMN), salience network, and central executive network (CEN) between A-PET-negative (N = 29) and A-PET-positive (N = 15) CNs with ε2/ε2 or ε2/ε3 genotypes. The A-PET-positive CNs exhibited a lower anterior DMN functional connectivity, higher posterior DMN functional connectivity, and increased CEN functional connectivity compared to the A-PET-negative CNs. Cerebral Aβ retention was negatively correlated with anterior DMN functional connectivity and positively correlated with posterior DMN and anterior CEN functional connectivity. A graph theory analysis showed that the A-PET-positive CNs displayed a higher betweenness centrality in the middle frontal gyrus (left) and medial fronto-parietal regions (left). The betweenness centrality in the middle frontal gyrus (left) was positively correlated with Aβ retention. Our findings reveal a reversed anterior-posterior dissociation in the DMN functional connectivity and heightened CEN functional connectivity in A-PET-positive CNs with ε2. Hub efficiencies, measured by betweenness centrality, were increased in the DMN and CEN of the A-PET-positive CNs with ε2. These results suggest unique functional connectivity responses to Aβ pathology in CN individuals with ε2.


A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.
In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.


Computed tomographic evaluation of the canine intercondylar notch in normal and cruciate deficient stifles.
In the human and veterinary orthopaedic literature it has been implied that intercondylar notch stenosis is a mechanical factor in cranial cruciate ligament rupture and intraarticular graft failure. The patients in this study were classified as normal (32), unilateral cruciate rupture (23), or bilateral cruciate rupture (17). The dogs were placed under general anaesthesia and both stifles were scanned via computed tomography (CT) as previously described. Three CT slices at predetermined levels were evaluated within the notch. Measurements included opening notch angle, notch width and height, condyle width, and notch width index (notch width/condyle width) at two different heights within the notch. Intercondylar notch measurements at the most cranial extent were significantly more narrow in unilateral and bilaterally affected stifles when compared to the normal population. Significant differences were noted in the opening notch angle (ONA), notch width index (NWI), NWI at two thirds notch height (NWI2/3), and tibial slope index (TSI). No significant differences were noted between unilateral and bilateral affected stifles. Increased mechanical contact of the cranial cruciate ligament with a stenotic intercondylar notch may predispose the ligament to mechanical wear and structural weakening. Intercondylar notch measurements have been used as a tool to predict the risk of anterior cruciate ligament injury in young human athletes, and to assess the risk factors for intra-articular graft replacements. Our findings may be useful in developing similar predictive models using stifle CT scans.


Spatial bistability generates hunchback expression sharpness in the Drosophila embryo.
During embryonic development, the positional information provided by concentration gradients of maternal factors directs pattern formation by providing spatially dependent cues for gene expression. In the fruit fly, Drosophila melanogaster, a classic example of this is the sharp on-off activation of the hunchback (hb) gene at midembryo, in response to local concentrations of the smooth anterior-posterior Bicoid (Bcd) gradient. The regulatory region for hb contains multiple binding sites for the Bcd protein as well as multiple binding sites for the Hb protein. Some previous studies have suggested that Bcd is sufficient for properly sharpened Hb expression, yet other evidence suggests a need for additional regulation. We experimentally quantified the dynamics of hb gene expression in flies that were wild-type, were mutant for hb self-regulation or Bcd binding, or contained an artificial promoter construct consisting of six Bcd and two Hb sites. In addition to these experiments, we developed a reaction-diffusion model of hb transcription, with Bcd cooperative binding and hb self-regulation, and used Zero Eigenvalue Analysis to look for multiple stationary states in the reaction network. Our model reproduces the hb developmental dynamics and correctly predicts the mutant patterns. Analysis of our model indicates that the Hb sharpness can be produced by spatial bistability, in which hb self-regulation produces two stable levels of expression. In the absence of self-regulation, the bistable behavior vanishes and Hb sharpness is disrupted. Bcd cooperative binding affects the position where bistability occurs but is not itself sufficient for a sharp Hb pattern. Our results show that the control of Hb sharpness and positioning, by hb self-regulation and Bcd cooperativity, respectively, are separate processes that can be altered independently. Our model, which matches the changes in Hb position and sharpness observed in different experiments, provides a theoretical framework for understanding the data and in particular indicates that spatial bistability can play a central role in threshold-dependent reading mechanisms of positional information.


Superior canal dehiscence effect on hearing thresholds: animal model.
Superior semicircular dehiscence syndrome is associated with vestibular symptoms and an air-bone gap component in the audiogram, apparently caused by the creation of a pathological bony "third window" in the superior semicircular canal. The aim of this study was to evaluate changes in auditory air- and bone-conduction thresholds to low- and high-frequency stimuli in an animal model of a bony fenestration facing the aerated mastoid cavity. Anatomic, audiological. Tertiary university-affiliated medical center. A small hole was drilled in the bony apical portion of the superior semicircular canal facing the mastoid bulla/cavity, with preservation of the membranous labyrinth, in 5 adult-size fat sand rats. Auditory brain stem responses to clicks and 1-kHz tone bursts delivered by air and bone conduction before surgery, after opening the bulla, and after fenestration. After fenestration, a significant air-bone gap was measured in response to clicks (mean ± standard deviation, 37 ± 5.8 dB) and bursts (mean ± standard deviation, 34 ± 14.5 dB). The gap was attributable solely to the significant deterioration in air-conduction thresholds, in the absence of a significant change in bone conduction thresholds. The pattern of auditory brain response changes closely resembled that reported for middle ear dysfunction, namely, an increase in absolute latency of waves I, III, and V without significant alterations in interpeak latency differences. Bony fenestration of the superior semicircular canal toward an aerated cavity in a rodent model mimics the auditory loss pattern of patients with superior semicircular dehiscence syndrome. The dehiscent membrane accounts for the auditory changes.


Physiological limits for "subliminal" perception.
Cortical, subcortical, and peripheral sensory nerve potentials were studied in cats to determine comparative thresholds of response. Stimuli capable of eliciting responses in peripheral nerve also invariably elicited responses from thalamus and cortex. Together with relevant data from human studies, the results indicate that stimuli subthreshold for perception do not affect the nervous system. They also have implications for studies of "subliminal" perception.


A critical role for beta-endorphin in cocaine-seeking behavior.
Endogenous beta-endorphin levels in the brain are elevated in response to cocaine and are downstream of the mesolimbic dopaminergic system. However, beta-endorphin's direct involvement in cocaine reinforcement has not been demonstrated. In the present study, a single bilateral microinjection of anti-beta-endorphin antibodies (4 microg) to the nucleus accumbens during the maintenance phase of cocaine self-administration (1 mg/kg/infusion) significantly increased the number of active and inactive lever responses. The increase in lever responses is reminiscent of rat behavior during extinction of cocaine self-administration. Further, a cocaine dose-response demonstrates that the increased lever presses in anti-beta-endorphin antibody-injected rats was still present after substitution with a lower dose of cocaine. These findings support a critical role for beta-endorphin in the cocaine brain reward system.


High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.
Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.