Impact of resistance training sets performed until muscular failure with different loads on intraocular pressure and ocular perfusion pressure.
The aim of this article is to investigate the acute effects of bench press sets leading to muscular failure with different loads on intraocular pressure and ocular perfusion pressure. A randomized experimental study. Seventeen physically active young men performed four resistance training sets of bench press to muscular failure against different relative loads (65% one-repetition maximum vs 75% one-repetition maximum vs 85% one-repetition maximum vs 95% one-repetition maximum). Intraocular pressure was measured before and immediately after the execution of each of the four sets, and ocular perfusion pressure was also assessed before and after physical effort. We found that intraocular pressure increased after reaching muscular failure (p < 0.001, ƞ²= 0.52), being also dependent on the interaction load × point of measure (p < 0.001, ƞ²= 0.33). Our data demonstrated that higher intraocular pressure increases were found when participants performed the bench press exercise against heavier loads, showing statistical significance for the 75% one-repetition maximum (p = 0.020, d = -0.63, mean change = 0.9 mmHg), 85% one-repetition maximum (p = 0.035, d = -0.56, mean change = 1.4 mmHg), and 95% one-repetition maximum (p < 0.001, d = -1.36, mean change = 2.9 mmHg) relative loads. For its part, ocular perfusion pressure showed a reduction after exercise (p = 0.009, ƞ²= 0.35), being these changes independent on the load used. Bench press exercise leading to muscular failure provokes an acute intraocular pressure rise, with greater changes when heavier loads are used. Ocular perfusion pressure exhibited an acute reduction after exercise; however, its clinical relevance seems to be insignificant (lower to 4%). We argue that the use of heavy loads, when training to muscular failure, should be discouraged in order to avoid acute intraocular pressure fluctuations. Future studies should corroborate the generalizability of these findings in glaucoma patients.


[Heart failure in Italian intensive cardiac care units: data from the BLITZ-3 survey].
Only limited information about clinical characteristics, diagnostic procedures and therapeutic options is available in patients admitted to an intensive cardiac care unit (ICCU) for heart failure. The aim of this study was to evaluate causes of admission, clinical characteristics, diagnostic and therapeutic options, and outcome of patients admitted for heart failure in the ICCU network. The BLITZ-3 Registry prospectively included patients admitted by 332 Italian ICCUs. Data of the patients admitted with a principal diagnosis of heart failure are analyzed. From April 7 to 20, 2008, 6986 consecutive patients with acute cardiac conditions were admitted to ICCUs; 966 (14%) out of 6986 patients were admitted for acute heart failure. Heart failure was the second cause of admission after acute coronary syndromes (52%). Mean age of patients admitted for heart failure was 73 years, 42% were female, and diabetes accounted for 32% of heart failure patients. Most patients were admitted to the emergency department (62%), and were discharged by the cardiology ward (65%). Median length of stay in the ICCU was 4 days, and during the stay in ICCU 5% of the patients with heart failure died. Advanced age and elevated creatinine values were associated with a higher risk of death. Echocardiography was performed in 79% of heart failure patients, coronary angiography in 10%, assisted ventilation in 15%, ultrafiltration in 3%, and right catheterization in 1%. Diuretics were administered in 93% of patients admitted for acute heart failure, intravenous nitrates in 41%, inotropes in 22%, beta-blockers in 42%, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers in 66%. In a nationwide survey, acute heart failure accounted for 14% of hospital admissions in ICCUs. Patients admitted for heart failure are usually old, with frequent comorbidities. Diagnostic and therapeutic procedures are rarely used, with the exception of echocardiography.


CBLL1 is hypomethylated and correlates with cortical thickness in transgender men before gender affirming hormone treatment.
Gender identity refers to the consciousness of being a man, a woman or other condition. Although it is generally congruent with the sex assigned at birth, for some people it is not. If the incongruity is distressing, it is defined as gender dysphoria (GD). Here, we measured whole-genome DNA methylation by the Illumina © Infinium Human Methylation 850k array and reported its correlation with cortical thickness (CTh) in 22 transgender men (TM) experiencing GD versus 25 cisgender men (CM) and 28 cisgender women (CW). With respect to the methylation analysis, TM vs. CW showed significant differences in 35 CpGs, while 2155 CpGs were found when TM vs. CM were compared. With respect to correlation analysis, TM showed differences in methylation of CBLL1 and DLG1 genes that correlated with global and left hemisphere CTh. Both genes were hypomethylated in TM compared to the cisgender groups. Early onset TM showed a positive correlation between CBLL1 and several cortical regions in the frontal (left caudal middle frontal), temporal (right inferior temporal, left fusiform) and parietal cortices (left supramarginal and right paracentral). This is the first study relating CBLL1 methylation with CTh in transgender persons and supports a neurodevelopmental hypothesis of gender identity.


Area deprivation and trends in inequalities in self-rated health in Spain, 1987--2001.
This study examined the impact that individual social position and municipal area deprivation levels had on trends in inequalities in self-rated health in Spain, between 1987 and 2001. The study was based on cross-sectional data of the National Health Surveys of Spain for the years 1987, 1993, 1995, 1997, and 2001 (n=84,567). The indicators used were educational level and occupational class, and deprivation level as the indicator of municipal areas. Multilevel logistic regression models were made, with individuals nested into municipal areas. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated. To evaluate trends, the relative index of inequality was calculated. At the individual level, the likelihood of less-than-good health between those with no formal education as compared to those with graduate-level education increased from OR=2.66 (95% CI: 2.06-3.38) in 1987 to OR=3.62 (95% CI: 2.95-4.63) in 2001 among women. The values for men were OR=2.27 (95% CI: 1.89-2.72) and OR=2.94 (95% CI: 2.36-3.68) respectively. Living in areas with the highest deprivation levels as compared to the lowest systematically increased the likelihood of less-than-good health. The likelihood of reporting less-than-good health among women with no formal education as compared to women with graduate-level education in municipal areas with the highest deprivation levels increased from OR=3.61 (95% CI: 2.39-5.45) in 1987 to 4.85 (95% CI: 3.06-7.69) in 2001. Among men, the corresponding magnitudes were OR=2.07 (95% CI: 1.39-3.08) and OR=4.16 (95% CI: 2.52-6.89). Inequalities in self-rated health increased in Spain in this period. These inequalities may be explained by the social conditions existing throughout the period of reference, and the pattern varies according to gender, municipal area deprivation levels, and the individual indicator of social position used.


Thyme Extract Alleviates High-Fat Diet-Induced Obesity and Gut Dysfunction.
Prolonged intake of a high-fat diet (HFD) disturbs the composition of gut microbiota, contributing to the development of metabolic diseases, notably obesity and increased intestinal permeability. Thyme (Thymus vulgaris L.), an aromatic plant, is known for its several therapeutic properties. In this study, we explored the potential of thyme extract (TLE) to mitigate HFD-induced metabolic derangements and improve the gut environment. Eight-week-old C57BL/6 mice were administered 50 or 100 mg/kg TLE for eight weeks. Administration of 100 mg/kg TLE resulted in decreased weight gain and body fat percentage, alongside the regulation of serum biomarkers linked to obesity induced by a HFD. Moreover, TLE enhanced intestinal barrier function by increasing the expression of tight junction proteins and ameliorated colon shortening. TLE also altered the levels of various metabolites. Especially, when compared with a HFD, it was confirmed that 2-hydroxypalmitic acid and 3-indoleacrylic acid returned to normal levels after TLE treatment. Additionally, we investigated the correlation between fecal metabolites and metabolic parameters; deoxycholic acid displayed a positive correlation with most parameters, except for colon length. In contrast, hypoxanthine was negatively correlated with most parameters. These results suggest a promising role for thyme in ameliorating obesity and related gut conditions associated with a HFD.


[The blood serum lipid spectrum in diabetes mellitus].
Investigations of lipid metabolism in patients with disturbed GTT and manifest diabetes mellitus revealed in most cases dyslipidemia, decreasing significantly in the time course of therapy. The results of the investigations showed that the blood levels of cholesterol (CS), beta-lipoproteins, total lipids (TL) and free fatty acids (FFA) in patients with disturbed GTT were significantly increased as compared to healthy persons. In patients with DM of type I, CS, beta-lipoproteins, TL and FFA were significantly lowered after achieving compensation, and in patients with type II DM the above indices except CS and alpha-CS were also lowered. The detected lipid metabolic derangements were noted at the preclinical stage, i.e. in persons with disturbed GTT and were preserved at the stage of manifest forms of DM, necessitating adequate therapy.


Mammal-like striatal functions in Anolis. I. Distribution of serotonin receptor subtypes, and absence of striosome and matrix organization.
Serotonin (5-HT) 5-HT(2A) and 5-HT(2C) receptors are thought to play important roles in the mammalian striatum. As basal ganglia functions in general are thought highly conserved among amniotes, we decided to use in situ autoradiographic methods to determine the occurrence and distribution of pharmacologically mammal-like 5-HT(2A) and 5-HT(2C) receptors in the lizard, Anolis carolinensis, with particular attention to the striatum. We also determined the distributions of 5-HT(1A), 5-HT(1B/D), 5 HT(3), and 5-HT(uptake) receptors for comparison. All 5-HT receptors examined showed pharmacological binding specificity, and forebrain binding density distributions that resembled those reported for mammals. Anolis 5 HT(2A/C) and 5-HT(1A) site distributions were similar in both in vivo and ex vivo binding experiments. 5-HT(2A & C) receptors occur in both high and low affinity states, the former having preferential affinity for (125)I-(+/-)-2,5-dimethoxy-4-iodo-amphetamine hydrochloride ((125)I-DOI). In mammals (125)I-DOI binding shows a patchy density distribution in the striatum, being more dense in striosomes than in surrounding matrix. There was no evidence of any such patchy density of (125)I-DOI binding in the anole striatum, however. As a further indication that anoles do not possess a striosome and matrix striatal organization, neither (3)H-naloxone binding nor histochemical staining for acetylcholinesterase activity (AChE) were patchy. AChE did show a band-like striatal distribution, however, similar to that seen in birds.


Larynx and cervical trachea in humidification and heating of inhaled gases.
To evaluate the participation of the larynx and cervical trachea in conditioning inspired gases, we randomly allocated 16 mixed-breed dogs to two groups: group TT (tracheal tube; n = 8) and group LMA (laryngeal mask airway; n = 8). The dogs were anesthetized with pentobarbital sodium and mechanically ventilated for 3 hours. The parameters studied were temperature and absolute humidities of ambient, inhaled, and tracheal air. There was a small increase in tracheal air temperature compared to inhaled air temperature, but no significant difference between groups. The absolute humidity of tracheal air was greater in group LMA than in group TT (23 mg H2O * L(-1) and 14 mg H2O * L(-1), respectively; p < .0001). The difference in absolute humidity between the tracheal air and the inhaled air was higher in group LMA at all times (p < .0001). We conclude that the larynx and cervical trachea of the dog participate in humidification and heating of inhaled air by means of air contact with mucosa in this airway segment.


Multiscale semilocal interpolation with antialiasing.
Aliasing is a common artifact in low-resolution (LR) images generated by a downsampling process. Recovering the original high-resolution image from its LR counterpart while at the same time removing the aliasing artifacts is a challenging image interpolation problem. Since a natural image normally contains redundant similar patches, the values of missing pixels can be available at texture-relevant LR pixels. Based on this, we propose an iterative multiscale semilocal interpolation method that can effectively address the aliasing problem. The proposed method estimates each missing pixel from a set of texture-relevant semilocal LR pixels with the texture similarity iteratively measured from a sequence of patches of varying sizes. Specifically, in each iteration, top texture-relevant LR pixels are used to construct a data fidelity term in a maximum a posteriori estimation, and a bilateral total variation is used as the regularization term. Experimental results compared with existing interpolation methods demonstrate that our method can not only substantially alleviate the aliasing problem but also produce better results across a wide range of scenes both in terms of quantitative evaluation and subjective visual quality.


Prophylaxis of Mycobacterium tuberculosis H37Rv Infection in a Preclinical Mouse Model via Inhalation of Nebulized Bacteriophage D29.
Globally, more people die annually from tuberculosis than from any other single infectious agent. Unfortunately, there is no commercially-available vaccine that is sufficiently effective at preventing acquisition of pulmonary tuberculosis in adults. In this study, pre-exposure prophylactic pulmonary delivery of active aerosolized anti-tuberculosis bacteriophage D29 was evaluated as an option for protection against Mycobacterium tuberculosis infection. An average bacteriophage concentration of approximately 1 PFU/alveolus was achieved in the lungs of mice using a nose-only inhalation device optimized with a dose simulation technique and adapted for use with a vibrating mesh nebulizer. Within 30 minutes of bacteriophage delivery, the mice received either a low dose (∼50-100 CFU), or an ultra-low dose (∼5-10 CFU), of M. tuberculosis H37Rv aerosol to the lungs. A prophylactic effect was observed with bacteriophage aerosol pre-treatment significantly decreasing M. tuberculosis burden in mouse lungs 24 hours and 3 weeks post-challenge (p < 0.05). These novel results indicate that a sufficient dose of nebulized mycobacteriophage aerosol to the lungs may be a valuable intervention to provide extra protection to health care professionals and other individuals at risk of exposure to M. tuberculosis.


Treatment of Abnormalities of Potassium Homeostasis in CKD.
Hyperkalemia, defined as serum potassium concentrations greater than 5.0 to 5.5 mEq/L, is a frequent occurrence in clinical practice because of the increasing incidence and prevalence of CKD. Patients at risk for this disorder are commonly treated with drugs that interfere in the renin-angiotensin-aldosterone system, thereby enhancing the occurrence and prevalence of this disorder. Discontinuation of these drugs because of the development of hyperkalemia deprives these patients the renal-protective and cardiovascular benefits this class of pharmacology has been shown to provide. Here we provide the clinician strategies to both prevent and treat hyperkalemia in patients with CKD who are prescribed these drugs. We emphasize the importance of limiting dietary potassium intake and avoiding either prescribed or over-the-counter medications that may impair renal potassium excretion. We discuss the role of kaliuretic diuretics and correction of metabolic acidosis as a therapeutic strategy. Additionally, we discuss the role of new agents designed to bind potassium in the gastrointestinal tract that can be used to maintain normokalemia in patients who previously developed hyperkalemia on renin-angiotensin-aldosterone blockers. Finally, we provide a brief discussion on how best to treat hypokalemia in patients with CKD.


Systems biology approaches for advancing the discovery of effective drug combinations.
Complex diseases like cancer are regulated by large, interconnected networks with many pathways affecting cell proliferation, invasion, and drug resistance. However, current cancer therapy predominantly relies on the reductionist approach of one gene-one disease. Combinations of drugs may overcome drug resistance by limiting mutations and induction of escape pathways, but given the enormous number of possible drug combinations, strategies to reduce the search space and prioritize experiments are needed. In this review, we focus on the use of computational modeling, bioinformatics and high-throughput experimental methods for discovery of drug combinations. We highlight cutting-edge systems approaches, including large-scale modeling of cell signaling networks, network motif analysis, statistical association-based models, identifying correlations in gene signatures, functional genomics, and high-throughput combination screens. We also present a list of publicly available data and resources to aid in discovery of drug combinations. Integration of these systems approaches will enable faster discovery and translation of clinically relevant drug combinations. Graphical abstractSpectrum of Systems Biology Approaches for Drug Combinations.


Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.
The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.


Hyperconnected molecular glass network architectures with exceptional elastic properties.
Hyperconnected network architectures can endow nanomaterials with remarkable mechanical properties that are fundamentally controlled by designing connectivity into the intrinsic molecular structure. For hybrid organic-inorganic nanomaterials, here we show that by using 1,3,5 silyl benzene precursors, the connectivity of a silicon atom within the network extends beyond its chemical coordination number, resulting in a hyperconnected network with exceptional elastic stiffness, higher than that of fully dense silica. The exceptional intrinsic stiffness of these hyperconnected glass networks is demonstrated with molecular dynamics models and these model predictions are calibrated through the synthesis and characterization of an intrinsically porous hybrid glass processed from 1,3,5(triethoxysilyl)benzene. The proposed molecular design strategy applies to any materials system wherein the mechanical properties are controlled by the underlying network connectivity.


Statistical considerations for the interpretation of commonly utilized road traffic accident indicators: implications for developing countries.
The problem of increasing road traffic accidents and their consequences of death, injury and disability poses a growing concern, especially in developing countries. The objectives and nature of various indicators that countries with scarce economic resources can obtain are examined. The statistical and epidemiological considerations discussed should enable health care planners in developing countries to decide on the appropriate indicators required to properly assess the extent of the problem and aid in the amelioration and prevention of this "disease." Various indicators are discussed and a pragmatic guide examining their advantages and disadvantages is provided.


Glycemia management in critical care patients.
Over the last decade, the approach to clinical management of blood glucose concentration (BGC) in critical care patients has dramatically changed. In this editorial, the risks related to hypo, hyperglycemia and high BGC variability, optimal BGC target range and BGC monitoring devices for patients in the intensive care unit (ICU) will be discussed. Hypoglycemia has an increased risk of death, even after the occurrence of a single episode of mild hypoglycemia (BGC < 80 mg/dL), and it is also associated with an increase in the ICU length of stay, the major determinant of ICU costs. Hyperglycemia (with a threshold value of 180 mg/dL) is associated with an increased risk of death, longer length of stay and higher infective morbidity in ICU patients. In ICU patients, insulin infusion aimed at maintaining BGC within a 140-180 mg/dL target range (NICE-SUGAR protocol) is considered to be the state-of-the-art. Recent evidence suggests that a lower BGC target range (129-145 mg/dL) is safe and associated with lower mortality. In trauma patients without traumatic brain injury, tight BGC (target < 110 mg/dL) might be associated with lower mortality. Safe BGC targeting and estimation of optimal insulin dose titration should include an adequate nutrition protocol, the length of insulin infusion and the change in insulin sensitivity over time. Continuous glucose monitoring devices that provide accurate measurement can contribute to minimizing the risk of hypoglycemia and improve insulin titration. In conclusion, in ICU patients, safe and effective glycemia management is based on accurate glycemia monitoring and achievement of the optimal BGC target range by using insulin titration, along with an adequate nutritional protocol.


Laughter in popular games and in sport. The other health of human play.
Hurling in Cornwall, la soule in Britanny, Shrovetide football in England: Popular games have normally been treated as forerunners of modern sport, sport having regulated the space and the time of the game, the (non-) violence of behaviour, the control of results, the planning, strategy, tactics, techniques and evaluation of the competitive action. This is told as a story of social improvement and progress--and about turning unhealthy wildness into civilized 'healthy' sport activity. What sociological analysis of game-playing tended to ignore was the laughter of the participants. With the seriousness of modern sport, as it was established in the nineteenth century, a culture of laughter disappeared. This study tries to counter this mainstream by a phenomenology of laughter in popular games. A contrasting attention is turned towards the seriousness of sporting competition, the smile in modern sport and fitness, and the 'underground' dimension of laughter in modern sports. By comparative analysis, laughter reveals as a bodily discourse about the imperfect human being. It tells an oppositional story about the perfectionism in the order of Western thinking--in sports as well as in health. The bodily 'physiology' of laughter, the exploding psychical energy, and the inter-bodily social relations in laughter and play and game point towards the multi-dimensionality of health, as it was formulated by WHO: as "physical, mental, and social well-being".


The Profiling of Apoptosis-Associated MicroRNA Expressions of the Lung Adenocarcinoma.
The most common histopathological subtype of lung cancer is adenocarcinoma. MicroRNAs are a class of non-coding RNAs that play roles in the regulation of gene expression. MicroRNAs affecting apoptosis may have different roles in lung adenocarcinoma development, progression, and differentiation. The objective of this study is to profile all known microRNAs linked to apoptosis in normal and lung cancer tissue using real-time polymerase chain reaction. Tissues with adenocarcinoma and healthy tissues were taken from the same lung. The degree of differentiation of all tumors was determined. Expressions of 84 apoptosis-associated microRNAs in both tissues were analyzed by real-time polymerase chain reaction array. Eleven patients and 22 samples were included in the study. In the comparison of expression levels of apoptosis-associated microRNAs in normal and adenocarcinoma tissue, miR-134, miR-183-5p, miR-192-5p, miR-193b-3, miR-194-5p, miR-200c-3, miR212-3p, miR-25-3p, miR-449a, and miR-9-5p showed significant difference in downregulation. Receiver operating characteristic curve analysis of 10 identified microRNAs was performed, and cut-off values, sensitivity, and specificity were determined. No significant difference was found between microRNA expression levels in adenocarcinoma tissues classified as moderate-well to poorly differentiated. Differently, downregulated expressed apoptosis-associated microRNAs were detected in lung adenocarcinoma tissues. MicroRNAs can be used as biomarkers in the diagnosis in lung adenocarcinoma. The expression of microRNAs linked to apoptosis should be investigated in different lung cancer histological subtypes in order to identify potential biomarkers.


Neural response to catecholamine depletion in unmedicated subjects with major depressive disorder in remission and healthy subjects.
The pathophysiologic mechanism of major depressive disorder (MDD) has been consistently associated with altered catecholaminergic function, especially with decreased dopamine neurotransmission, by various sources of largely indirect evidence. An instructive paradigm for more directly investigating the relationship between catecholaminergic function and depression has involved the mood response to experimental catecholamine depletion (CD). To determine whether catecholaminergic dysfunction represents a trait abnormality in MDD and to identify brain circuitry abnormalities involved in the pathophysiologic mechanism of MDD. Randomized, double-blind, placebo-controlled, crossover, single-site experimental trial. Psychiatric outpatient clinic. Fifteen unmedicated subjects with MDD in full remission (hereinafter referred to as RMDD subjects) and 13 healthy controls. Induction of CD by oral administration of alpha-methylparatyrosine. Sham depletion used identical capsules containing hydrous lactose. Quantitative positron emission tomography of regional cerebral glucose utilization to study the neural effects of CD and sham depletion. Behavioral assessments included the Montgomery-Asberg Depression Rating Scale and the Snaith-Hamilton Pleasure Scale (anhedonia). Depressive and anhedonic symptoms increased during CD to a greater extent in RMDD subjects than in controls. In both groups, CD increased metabolism in the anteroventral striatum and decreased metabolism in the orbital gyri. In a limbic-cortical-striatal-pallidal-thalamic network previously implicated in MDD, composed of the ventromedial frontal polar cortex, midcingulate and subgenual anterior cingulate cortex, temporopolar cortex, ventral striatum, and thalamus, metabolism increased in RMDD subjects but decreased or remained unchanged in controls. Metabolic changes induced by CD in the left ventromedial frontal polar cortex correlated positively with depressive symptoms, whereas changes in the anteroventral striatum were correlated with anhedonic symptoms. This study provides direct evidence for catecholaminergic dysfunction as a trait abnormality in MDD. It demonstrates that depressive and anhedonic symptoms as a result of decreased catecholaminergic neurotransmission are related to elevated activity within the limbic-cortical-striatal-pallidal-thalamic circuitry.


Feature-enriched core percolation in multiplex networks.
Percolation models have long served as a paradigm for unraveling the structure and resilience of complex systems comprising interconnected nodes. In many real networks, nodes are identified by not only their connections but nontopological metadata such as age and gender in social systems, geographical location in infrastructure networks, and component contents in biochemical networks. However, there is little known regarding how the nontopological features influence network structures under percolation processes. In this paper we introduce a feature-enriched core percolation framework using a generic multiplex network approach. We thereby analytically determine the corona cluster, size, and number of edges of the feature-enriched cores. We find a hybrid percolation transition combining a jump and a square root singularity at the critical points in both the network connectivity and the feature space. Integrating the degree-feature distribution with the Farlie-Gumbel-Morgenstern copula, we show the existence of continuous and discrete percolation transitions for feature-enriched cores at critical correlation levels. The inner and outer cores are found to undergo distinct phase transitions under the feature-enriched percolation, all limited by a characteristic curve of the feature distribution.


Recent advances in alcoholic hepatitis.
Alcoholic hepatitis (AH) is an acute deterioration in liver function seen in the context of prolonged excessive alcohol consumption and is characterised by the rapid onset of jaundice. The diagnosis of AH has been controversial for many years: it is now accepted that there are clear clinical criteria which can be used to diagnose AH without the need for a liver biopsy. Corticosteroids remain the only treatment proven to be effective in reducing short-term mortality in severe AH; abstinence from alcohol is the most important factor in determining long-term survival. It is recommended a trial of corticosteroid therapy is considered only in those patients with high baseline 'static' scores (Glasgow Alcoholic Hepatitis score and model for end-stage liver disease). Response to corticosteroid therapy should be assessed using a 'dynamic' score such as the Lille score at day 7, with corticosteroids continuing only in patients with a favourable score. Infection and acute kidney injury are associated with poorer outcomes in AH. Early screening for and treatment of infection is recommended with antibiotic therapy overlapping with any subsequent corticosteroid treatment. A biomarker which predicts benefit from corticosteroids at baseline would avoid a trial of therapy to determine response. More efficacious therapeutic options for AH patients are required with N-acetylcysteine, granulocyte colony stimulating factor, faecal microbiota transplantation and routine antibiotics showing promise, but adequate controlled trials are needed to confirm efficacy. Liver transplant has an emerging role for some patients with severe AH not responding to corticosteroids and is likely to become more acceptable with improved methods of patient selection.


The PETfold and PETcofold web servers for intra- and intermolecular structures of multiple RNA sequences.
The function of non-coding RNA genes largely depends on their secondary structure and the interaction with other molecules. Thus, an accurate prediction of secondary structure and RNA-RNA interaction is essential for the understanding of biological roles and pathways associated with a specific RNA gene. We present web servers to analyze multiple RNA sequences for common RNA structure and for RNA interaction sites. The web servers are based on the recent PET (Probabilistic Evolutionary and Thermodynamic) models PETfold and PETcofold, but add user friendly features ranging from a graphical layer to interactive usage of the predictors. Additionally, the web servers provide direct access to annotated RNA alignments, such as the Rfam 10.0 database and multiple alignments of 16 vertebrate genomes with human. The web servers are freely available at: http://rth.dk/resources/petfold/


Supervised machine learning-based classification of oral malodor based on the microbiota in saliva samples.
This study presents an effective method of classifying oral malodor from oral microbiota in saliva by using a support vector machine (SVM), an artificial neural network (ANN), and a decision tree. This approach uses concentrations of methyl mercaptan in mouth air as an indicator of oral malodor, and peak areas of terminal restriction fragment (T-RF) length polymorphisms (T-RFLPs) of the 16S rRNA gene as data for supervised machine-learning methods, without identifying specific species producing oral malodorous compounds. 16S rRNA genes were amplified from saliva samples from 309 subjects, and T-RFLP analysis was carried out with the DNA fragments. T-RFLP analysis provides information on microbiota consisting of fragment lengths and peak areas corresponding to bacterial strains. The peak area is equivalent to the frequency of a specific fragment when one molecule is selected from terminal fragments. Another frequency is obtained by dividing the number of species-containing samples by the total number of samples. An SVM, an ANN, and a decision tree were trained based on these two frequencies in 308 samples and classified the presence or absence of methyl mercaptan in mouth air from the remaining subject. The proportion that trained SVM expressed as entropy achieved the highest classification accuracy, with a sensitivity of 51.1% and specificity of 95.0%. The ANN and decision tree provided lower classification accuracies, and only classification by the ANN was improved by weighting with entropy from the frequency of appearance in samples, which increased the accuracy to 81.9% with a sensitivity of 60.2% and a specificity of 90.5%. The decision tree showed low classification accuracy under all conditions. Using T-RF proportions and frequencies, models to classify the presence of methyl mercaptan, a volatile sulfur-containing compound that causes oral malodor, were developed. SVM classifiers successfully classified the presence of methyl mercaptan with high specificity, and this classification is expected to be useful for screening saliva for oral malodor before visits to specialist clinics. Classification by a SVM and an ANN does not require the identification of the oral microbiota species responsible for the malodor, and the ANN also does not require the proportions of T-RFs.


MAGI-1a functions as a scaffolding protein for the distal renal tubular basolateral K+ channels.
As the K(+) recycling pathway for renal Na(+) reabsorption, renal tubular K(+) channels participate in the fluid and electrolyte homeostasis. Previously, we showed that the Kir5.1/Kir4.1 heteromer, which is a heteromeric assembly of two inwardly rectifying K(+) channels, composes the principal basolateral K(+) channels in distal renal tubules and that two motifs in the carboxyl-terminal portion of the Kir4.1 subunit regulate its functional expression. In this study, by using yeast two-hybrid screening, we identified a new isoform of membrane-associated guanylate kinase with inverted domain structure 1 (MAGI-1a-long) as a scaffolding protein for the basolateral K(+) channels. MAGI-1a-long interacted with the PSD-95/Dlg/ZO-1 (PDZ)-binding motif of Kir4.1 by its fifth PDZ domain, and a high salt diet, which could suppress mineralocorticoid secretion, facilitated the interaction. The phosphorylation of serine 377 in the PDZ-binding motif disrupted the interaction, and the disruption of the interaction altered the intracellular localization of the channels from the basolateral side to perinuclear components. These results demonstrate that the phosphorylation-dependent scaffolding of the basolateral K(+) channels by MAGI-1a-long participates in the renal regulation of the fluid and electrolyte homeostasis.


Examining Cigar Pack Quantity Purchases by Cigar Type in the United States Between 2014 and 2017.
We examined whether the proportion of cigar pack quantity usually purchased differed between 2014 and 2017 in the United States. Data are from waves 1 and 4 of the Population Assessment of Tobacco and Health Study. The proportions of pack quantity purchases between waves for premium cigars, large cigars, cigarillos, and filtered cigars for 10-pack quantities (singles, 2-packs, 3-packs, 4-packs, 5-packs, 6-9-packs, 10-packs, 11--19-packs, 20-packs, ≥21 packs) were compared using Z-tests. We used multivariable linear regressions to examine factors associated with pack quantity. For premium cigars, most adults purchased singles, with 84.4% in 2014 and 88.7% in 2017. For large cigars, single purchases increased from 46.2% to 62.7% (p < .00001). Two-packs also increased, 3.1% to 10.2% (p < .00001), while 5-packs, 6-9 packs, 10-19-packs, and 20-packs decreased. For cigarillos, singles purchases increased from 51.9% to 57.5% (p = .0002). Two-packs also increased, 8.8% to 19.6% (p < .00001), while 3-packs, 4-packs, 5-packs, and 20-packs decreased. For filtered cigars, the most prevalent pack quantity shifted from 20-packs in 2014 (62.6%) to singles in 2017 (36.7%). Singles, 2-packs, 3-4-packs, and 5-packs increased while 11-19-packs, 20-packs, and ≥21 packs decreased. Days used were positively associated with pack quantity purchase for all cigar types in 2017, while the price was positively associated with premium cigar purchase. Between 2014 and 2017, the proportion of consumers who purchased singles remained at over 80% for premium cigars and increased for other cigars. Continued monitoring of use behaviors, including pack quantities by cigar type, is needed as the market and policy landscapes continue shifting. Single cigar purchases are prominent across cigar types. Five-packs and 20-packs were the second most common pack quantities across cigar types, with relatively low proportions for other pack quantities. These findings suggest adults consuming premium cigars consistently prefer singles, a pattern that was found across cigar types. However, when considering purchases made online, premium cigars are widely purchased in larger pack quantities. Age and race were associated with purchase for some cigar types at some years, days used were associated with the purchase quantity in 2017 for all cigar types, and price was positively associated across time periods for only premium cigar purchases. Thus, the factors associated with cigar pack quantity purchase remain unclear. As the cigar policy landscape shifts, continued monitoring of the purchase behaviors by cigar type will be important to better inform future cigar policies.


Susceptibility of Mycobacterium tuberculosis Cytochrome bd Oxidase Mutants to Compounds Targeting the Terminal Respiratory Oxidase, Cytochrome c.
We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.


[Binding of gamma-aminobutyric acid in the synaptic membranes of rat brain].
The Na+-dependent and Na+-independent (receptor) binding of [3H] GABA is measured in rat brain synaptic membranes. The receptor binding is presented by at least 3 types of binding sites differing in kinetic parameters (dissociation constants and maximal concentration of binding sites). Phenyl-GABA and pyracetam do not affect receptor binding while 4-hydroxybutyric acid, apamine and bicuculline inhibit it with inhibition constants K1=100, 2 and 1 micron, respectively. Solubilization of synaptic membranes with 2% potassium cholate in the presence of total brain phospholipids does not inactivate [3H] GABA receptor binding.


Sample site selection for tracer studies applying a unidirectional circulatory approach.
The optimal arterial or venous sites for infusion and sampling during isotopic tracer studies have not been established. This study determined the relationship of plasma and tissue enrichment (E) when isotopes were infused in an artery and sampled from a vein (av mode) or infused in a vein and sampled from an artery (va mode). Adult dogs were given primed constant infusions of [3-13C]lactate, [1-13C]leucine, and 14C-labeled bicarbonate. Simultaneous samples were drawn from the vena cava, aortic arch, and breath. Tissue samples were removed from skeletal muscle, liver, kidney, and gut. Breath samples were analyzed for 14CO2 by liquid scintillation counting and plasma isotopic enrichments of [13C]lactate, [13C]leucine, and alpha-[13C]ketoisocaproate (KIC) were determined by gas chromatography-mass spectrometry. By using the va mode, the plasma E for lactate and leucine were 30-40% above tissue E. The av mode provided an accurate reflection of tissue E for lactate, which equilibrates rapidly with tissues, and a reasonable estimate for leucine, which exchanges more slowly. The isotopic enrichment of plasma KIC more directly reflected tissue leucine E than did plasma leucine E, and KIC enrichment was insensitive to sampling site. We also evaluated theoretically a circulatory model that predicts venous isotopic enrichments when the va mode is used. We conclude that the av mode is optimal but that the problems arising from use of the va mode can be overcome by use of a metabolic product (i.e., KIC) or by calculation of venous specific activity with our circulatory mode.


Accelerated nonrigid intensity-based image registration using importance sampling.
Nonrigid image registration methods using intensity-based similarity metrics are becoming increasingly common tools to estimate many types of deformations. Nonrigid warps can be very flexible with a large number of parameters and gradient optimization schemes are widely used to estimate them. However, for large datasets, the computation of the gradient of the similarity metric with respect to these many parameters becomes very time consuming. Using a small random subset of image voxels to approximate the gradient can reduce computation time. This work focuses on the use of importance sampling to reduce the variance of this gradient approximation. The proposed importance sampling framework is based on an edge-dependent adaptive sampling distribution designed for use with intensity-based registration algorithms. We compare the performance of registration based on stochastic approximations with and without importance sampling to that using deterministic gradient descent. Empirical results, on simulated magnetic resonance brain data and real computed tomography inhale-exhale lung data from eight subjects, show that a combination of stochastic approximation methods and importance sampling accelerates the registration process while preserving accuracy.


Changing cluster composition in cluster randomised controlled trials: design and analysis considerations.
There are many methodological challenges in the conduct and analysis of cluster randomised controlled trials, but one that has received little attention is that of post-randomisation changes to cluster composition. To illustrate this, we focus on the issue of cluster merging, considering the impact on the design, analysis and interpretation of trial outcomes. We explored the effects of merging clusters on study power using standard methods of power calculation. We assessed the potential impacts on study findings of both homogeneous cluster merges (involving clusters randomised to the same arm of a trial) and heterogeneous merges (involving clusters randomised to different arms of a trial) by simulation. To determine the impact on bias and precision of treatment effect estimates, we applied standard methods of analysis to different populations under analysis. Cluster merging produced a systematic reduction in study power. This effect depended on the number of merges and was most pronounced when variability in cluster size was at its greatest. Simulations demonstrate that the impact on analysis was minimal when cluster merges were homogeneous, with impact on study power being balanced by a change in observed intracluster correlation coefficient (ICC). We found a decrease in study power when cluster merges were heterogeneous, and the estimate of treatment effect was attenuated. Examples of cluster merges found in previously published reports of cluster randomised trials were typically homogeneous rather than heterogeneous. Simulations demonstrated that trial findings in such cases would be unbiased. However, simulations also showed that any heterogeneous cluster merges would introduce bias that would be hard to quantify, as well as having negative impacts on the precision of estimates obtained. Further methodological development is warranted to better determine how to analyse such trials appropriately. Interim recommendations include avoidance of cluster merges where possible, discontinuation of clusters following heterogeneous merges, allowance for potential loss of clusters and additional variability in cluster size in the original sample size calculation, and use of appropriate ICC estimates that reflect cluster size.


Preparation-morphology-performance relationships in cobalt aerogels as supercapacitors.
The ability to direct the morphology of cobalt sol-gel materials by using the simple synthetic parameters in epoxide-driven polycondensations has been dramatically demonstrated, and the influence of such morphological differences upon the supercapacity of the materials has been explored. Precursor salt, epoxide, and solvent all influence the speed of the sol-gel transition and the size and shape of the features observed in the as-prepared materials, thereby leading to highly varied microstructures including spheres, sponge-like networks, and plate assemblies of varied size. These morphological features of the as-prepared cobalt aerogels were observed for the first time by high resolution scanning electron microscopy (HRSEM). The as-prepared aerogel materials were identified by powder X-ray diffraction and thermogravimetry as weakly crystalline or amorphous cobalt basic salts with the general formula Co(OH)(2-n)X(n) where X = Cl or NO3 according to the precursor salt used in the synthesis. For all samples, the morphology was preserved through mild calcining to afford spinel phase Co3O4 in a variety of microstructures. Wide-ranging specific surface areas were determined for the as-prepared and calcined phases by physisorption analysis in agreement with the morphologies observed by HRSEM. The Co3O4 aerogels were evaluated for their supercapacitive performance by cyclic voltammetry. The various specimens exhibit capacitances ranging from 110 to 550 F g(-1) depending upon the attributes of the particular aerogel material, and the best specimen was found to have good cycle stability. These results highlight the epoxide-driven sol-gel condensation as a versatile preparative route that provides wide scope in materials' properties and enables the analysis of structure-performance relationships in metal oxide materials.


Simulating a Community Mental Health Service During the COVID-19 Pandemic: Effects of Clinician-Clinician Encounters, Clinician-Patient-Family Encounters, Symptom-Triggered Protective Behaviour, and Household Clustering.
Objectives: Face-to-face healthcare, including psychiatric provision, must continue despite reduced interpersonal contact during the COVID-19 (SARS-CoV-2 coronavirus) pandemic. Community-based services might use domiciliary visits, consultations in healthcare settings, or remote consultations. Services might also alter direct contact between clinicians. We examined the effects of appointment types and clinician-clinician encounters upon infection rates. Design: Computer simulation. Methods: We modelled a COVID-19-like disease in a hypothetical community healthcare team, their patients, and patients' household contacts (family). In one condition, clinicians met patients and briefly met family (e.g., home visit or collateral history). In another, patients attended alone (e.g., clinic visit), segregated from each other. In another, face-to-face contact was eliminated (e.g., videoconferencing). We also varied clinician-clinician contact; baseline and ongoing "external" infection rates; whether overt symptoms reduced transmission risk behaviourally (e.g., via personal protective equipment, PPE); and household clustering. Results: Service organisation had minimal effects on whole-population infection under our assumptions but materially affected clinician infection. Appointment type and inter-clinician contact had greater effects at low external infection rates and without a behavioural symptom response. Clustering magnified the effect of appointment type. We discuss infection control and other factors affecting appointment choice and team organisation. Conclusions: Distancing between clinicians can have significant effects on team infection. Loss of clinicians to infection likely has an adverse impact on care, not modelled here. Appointments must account for clinical necessity as well as infection control. Interventions to reduce transmission risk can synergize, arguing for maximal distancing and behavioural measures (e.g., PPE) consistent with safe care.


Smoking is associated with worse outcomes of COVID-19 particularly among younger adults: a systematic review and meta-analysis.
Smoking impairs lung immune function and damages upper airways, increasing risks of contracting and severity of infectious diseases. This paper quantifies the association between smoking and COVID-19 disease progression. We searched PubMed and Embase for studies published from January 1-May 25, 2020. We included studies reporting smoking behavior of COVID-19 patients and progression of disease, including death. We used random effects meta-analysis, meta-regression and locally weighted regression and smoothing to examine relationships in the data. We identified 46 peer-reviewed papers with a total of 22,939 COVID-19 patients, 5421 (23.6%) experienced disease progression and 2914 (12.7%) with a history of smoking (current and former smokers). Among those with a history of smoking, 33.5% experienced disease progression, compared with 21.9% of non-smokers. The meta-analysis confirmed an association between ever smoking and COVID-19 progression (OR 1.59, 95% CI 1.33-1.89, p = 0.001). Ever smoking was associated with increased risk of death from COVID-19 (OR 1.19, 95% CI 1.02-1.39, p = 0.003). We found no significant difference (p = 0.864) between the effects of ever smoking on COVID-19 disease progression between adjusted and unadjusted analyses, suggesting that smoking is an independent risk factor for COVID-19 disease progression. We also found the risk of having COVID-19 progression higher among younger adults (p = 0.001), with the effect most pronounced among younger adults under about 45 years old. Smoking is an independent risk for having progression of COVID-19, including mortality. The effects seem to be higher among young people. Smoking prevention and cessation should remain a priority for the public, physicians, and public health professionals during the COVID-19 pandemic.


HE4 transcription- and splice variants-specific expression in endometrial cancer and correlation with patient survival.
We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC) tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.


Diaphorobacter oryzae sp. nov., isolated from a thiosulfate-oxidizing enrichment culture.
A taxonomic study was performed on two isolates, strains RF3(T) and RF21, recovered from a thiosulfate-oxidizing enrichment culture. Comparative 16S rRNA gene sequence analysis showed that these isolates were clearly affiliated with the class Betaproteobacteria. Strains RF3(T) and RF21 were most closely related to Diaphorobacter nitroreducens NA10B(T) (97.9 and 98.2 % 16S rRNA gene sequence similarity, respectively). The level of 16S rRNA gene sequence similarity between strains RF3(T) and RF21 was 99.8 %. The two isolates were Gram-negative, aerobic and denitrifying. Growth was observed at 7-35 degrees C. Physiological and biochemical properties of strains RF3(T) and RF21 were different from those of D. nitroreducens NA10B(T) in that strains RF3(T) and RF21 were able to utilize l-histidine, 2-ketogluconate and caprate, but unable to utilize suberate. The G+C contents of the genomic DNA of strains RF3(T) and RF21 were 62.9 and 62.8 mol%, respectively, and the predominant quinone was ubiquinone-8. Strains RF3(T) and RF21 possessed C(16 : 0), C(17 : 0) cyclo, and C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH as major fatty acids. DNA-DNA relatedness data indicated that strains RF3(T) and RF21 represent a genomic species separate from D. nitroreducens. On the basis of the evidence presented from this polyphasic analysis, it is suggested that strains RF3(T) and RF21 represent a novel species of the genus Diaphorobacter, for which the name Diaphorobacter oryzae sp. nov. is proposed. The type strain is RF3(T) (=KCTC 22225(T)=LMG 24467(T)).


Omega-3 fatty acid deficiency augments amphetamine-induced behavioral sensitization in adult mice: prevention by chronic lithium treatment.
Emerging data suggests that omega-3 fatty acid deficiency may be a risk factor for bipolar disorder. In the present study, we determined the effects of chronic dietary-induced omega-3 fatty acid deficiency and/or concomitant chronic lithium chloride (LiCl) treatment on amphetamine (AMPH)-induced behavioral sensitization, a phenomenon that may recruit neuroplastic mechanisms relevant to the pathophysiology of bipolar disorder. Adult male C57BL/6J mice were randomly assigned to one four diets: Control (alpha-linolenic-fortified), Control+LiCl (0.255%), alpha-linolenic-Deficient, or Deficient+LiCl (0.255%), and behavioral testing initiated 65 days later. Locomotor activity was determined following 3 intermittent (separated by 7d) injections of amphetamine (AMPH) (1mg/kg). After behavioral testing, red blood cell (RBC) and regional brain (prefrontal cortex, hippocampus, ventral striatum) fatty acid composition was determined by gas chromatography. Each diet group exhibited comparable locomotor activity following acute AMPH treatment. However, the development of sensitization following repeated AMPH treatment was significantly augmented in Deficient mice relative to controls, and this augmented response was prevented by chronic LiCl treatment. Relative to controls, Deficient mice exhibited deficits in RBC and regional brain docosahexaenoic acid (DHA, 22:6n-3) composition, reciprocal elevations in vaccenic acid (18:1n-7), arachidonic acid (AA, 20:4n-6), and docosapentaenoic acid (DPA, 22:5n-6) compositions, and elevations in AA:DHA, oleic acid:DHA, and DPA:DHA ratios. The fatty acid abnormalities in Deficient mice were not altered by concurrent chronic lithium treatment. Mice fed the Control+LiCl diet exhibited a significant increase in AA composition in RBC and all brain regions, and an elevated AA:DHA ratio in the prefrontal cortex and hippocampus, relative to Controls. Fatty acid composition in RBC and different brain regions were predominantly positively correlated. Within the ventral striatum, DHA composition was inversely correlated, and AA:DHA and oleic acid:DHA ratios positively correlated, with total distance traveled following the final AMPH treatment. These data indicate that alterations in fatty acid composition resulting from dietary-induced omega-3 fatty acid deficiency augment the development of AMPH-induced behavioral sensitization in a manner that is prevented by chronic lithium treatment. The implications of these findings for understanding the contribution of omega-3 fatty acid deficiency to the pathophysiology and progression of bipolar disorder are discussed.


Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines.
The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 A (where 1 A=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.


Awareness, treatment and control of cardiometabolic disorders in Chinese adults with diabetes: a national representative population study.
The diagnosis of diabetes has important clinic implications for the prevention and management of cardiometabolic disorders. We aimed to investigate the awareness, treatment and control of hypertension and dyslipidemia in previously-diagnosed and newly-diagnosed diabetes in Chinese adult population. We conducted a cross-sectional survey in a nationally representative sample of 98658 Chinese adults aged 18 years or older in 2010, using a complex, multistage, probability sampling design. Glycemic status were defined according to the 2010 American Diabetes Association criteria. Hypertension was diagnosed by the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Dyslipidemia was diagnosed by the 2004 National Cholesterol Education Program Adult Treatment Panel III. The weighted prevalence of hypertension and dyslipidemia gradually increased in adults with normal glucose regulation, prediabetes, newly-diagnosed diabetes and previously-diagnosed diabetes. Compared to newly-diagnosed diabetes patients, previously-diagnosed diabetes patients were more likely to be aware of hypertension (weighted percentage [95% confidence interval]: 55.2% [52.9%-57.5%] vs 37.6% [35.9%-39.3%]) and dyslipidemia (33.9% [31.8%-36.1%] vs 12.8% [11.7%-13.9%]), to receive blood pressure-lowing (43.7% [41.5%-46.0%] vs 27.5% [26.0%-29.0%]) and lipid-lowering (18.9% [17.2%-20.7%] vs 5.4% [4.6%-6.2%]) therapies, and to have controlled blood pressure (4.7% [3.5%-6.2%] vs 3.5% [2.6%-4.8%]) and lipid (15.9% [12.3%-20.3%] vs 9.5% [6.4%-13.8%]) levels. Detection and control of hypertension and dyslipidemia is far from optimal in Chinese adults, especially in newly-diagnosed diabetes. Improved screening for diabetes is required to promote a better prevention, treatment and control of hypertension and dyslipidemia in China.


A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS.
Development or regeneration of axons in several systems is accompanied by 20-100-fold increases in the synthesis of an acidic, axonally transported membrane protein with an apparent molecular weight of 43-50,000 (Benowitz and Lewis, 1983; Skene and Willard, 1981a, b), which we designate GAP-43. We have proposed that some step(s) in axon growth require production of GAP-43, and perhaps a small number of other "growth-associated proteins," at rates much higher than those typical of mature neurons. This hypothesis predicts that virtually all neurons synthesize GAP-43 at elevated levels during normal CNS development. Here we show that a protein similar to GAP-43 from regenerating toad nerves is prominent among the newly synthesized (35S-methionine-labeled) and total (Coomassie blue-stained) proteins in neonatal rat cerebral cortex and cerebellum, suggesting that synthesis of GAP-43 is indeed a common feature of many developing neurons. Synthesis and accumulation of the protein decline an order of magnitude as animals mature. Antibodies raised against the rat cortex GAP-43 also recognize electrophoretically similar proteins from regenerating toad optic nerves and from developing hamster sensorimotor cortex, indicating that structural features of GAP-43 are conserved in evolution. Cell-free translation of polyadenylated RNA from neonatal and adult cortex suggests that developmental regulation of GAP-43 synthesis is mediated largely through changes in mRNA abundance. These observations together suggest that developmental regulation of GAP-43 gene expression may be common to most vertebrate CNS neurons. GAP-43 remains detectable at a low level in adult rat cortex, and it co-migrates on two-dimensional gels with B-50, a synaptic membrane protein which is a preferred substrate for protein kinase C in adult brains. Phosphorylation of the protein by endogenous kinase(s) in vitro is 4-7-fold greater in growth cone membranes than in mature synaptic membranes, which raises the possibility that local modification of the protein in axon terminals may be synergistic with regulation of GAP-43 synthesis in the cell body.


New insights into the mechanism of notch signalling in fibrosis.
The Notch pathway is an evolutionary conserved signalling mechanism that regulates cellular fate and development in various types of cells. The full spectrum of Notch effects has been well studied over the last decade in the fields of development and embryogenesis. But only recently several studies emphasized the involvement of the Notch signalling pathway in fibrosis. This review summarizes the structure and activation of the Notch family members, and focuses on recent findings regarding the role of Notch in organ fibrogenesis, in humans and in animal models.


Direct freezing of whole blood enables analysis of leucocyte markers by flow cytometry: a proof-of-concept study.
Aim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry.


Retrospective investigation of the neutrophil-to-lymphocyte ratio in dogs with pneumonia: 49 cases (2011-2016).
To assess the utility of the neutrophil-to-lymphocyte ratio (NLR) in predicting outcome in canine pneumonia compared with routine hematological parameters and systemic inflammatory response syndrome (SIRS) scores. Retrospective study. University teaching hospital. Forty-nine client-owned dogs. None MEASUREMENTS AND MAIN RESULTS: Medical records were reviewed to identify dogs with a diagnosis of pneumonia from July 2011 to December 2016. Signalment, clinical findings, laboratory characteristics, and outcome were recorded. Inclusion criteria were a clinical and radiographic diagnosis of pneumonia, plus reference laboratory hematology at diagnosis. Cases that received steroids were excluded. Euthanized dogs were only included in statistical analysis if euthanized solely due to pneumonia severity. The NLR, total WBC count, neutrophil count, lymphocyte count, band neutrophil percent of total WBC count (%-bands), and percentage of cases diagnosed with SIRS were compared between survivors and nonsurvivors. Receiver operating characteristic curves were generated to identify optimal sensitivity and specificity cutoffs for nonsurvival to discharge. Two hundred records were retrieved; 49 cases fulfilled the inclusion criteria. Of these, 33 (67%) survived to discharge. The NLR did not differ significantly between the survivors and nonsurvivors, nor did total WBC count or neutrophil count. Survivors had a significantly lower %-bands than nonsurvivors (P < 0.001) and higher lymphocyte count (P = 0.004). The mortality rate did not differ significantly between dogs with and without SIRS. Receiver operating characteristic analysis identified a %-bands cutoff of 2.5% or higher had an 83% sensitivity and 79% specificity for nonsurvival. Unlike in human medicine, neither NLR nor SIRS scores predicted outcome in this cohort of dogs with pneumonia. However, survivors had a lower %-bands and higher lymphocyte count than nonsurvivors, which may be helpful prognostically in clinical cases.


[Antioxidant status of patients with thyrotoxicosis syndrome treated with the combined regimen containing epifamin].
The work purpose was research of degree of free-radical processes intensity and catalase activity in blood serum of patients with a thyreotoxicosis syndrome at traditional treatment and the combined therapy with epifamin. Patients (n=25) have been divided into 2 groups: the first group of patients--12 persons who are on traditional treatment (antithyreoid drugs, beta-adrenoblockers); the second group--13 persons, are padding to basic therapy received epifamin on 1 tablet (10 mg) 3 times a day 10-15 minutes prior to meal within 7 days. Patients with thyreotoxicosis syndrome had an intensification of free-radical oxidation that was traced at measurement of biochemiluminescence parameters in blood serum. At the combined therapy with epifamin less expressed intensity of free-radical processes, and also increasing of the general antioxidant activity of an organism that did not descend at traditional treatment was observed. At carrying out of standard therapy of thyreotoxicosis syndrome, including application of antithyreoid drugs and beta-adrenoblockers, catalase activity decreased, that unfavorable affected antioxidant potential of an organism. At the combined therapy with epifamin level of catalase activity, on the contrary, increased, that could influence positively on antioxidant protection of an organism.


Droplet Transportation on Liquid-Infused Asymmetrically Structured Surfaces by Mechanical Oscillation and Viscosity Control.
The transportation of droplets on solid surfaces has received significant attention owing to its importance in biochemical analysis and microfluidics. In this study, we propose a novel strategy for controlling droplet motion by combining an asymmetric structure and infused lubricating oil on a vibrating substrate. The transportation of droplets with volumes ranging from 10 to 90 μL was realized, and the movement speed could be adjusted from 1.45 to 10.87 mm/s. Typical droplet manipulations, including droplet transportation along a long trajectory and selective movement of multiple droplets, were successfully demonstrated. Through experimental exploration and theoretical analysis, we showed that the adjustment of droplet transport velocity involves an intricate interaction among the Ohnesorge number, droplet volume, and input amplitude. It can potentially be used for the more complex manipulation of liquid droplets in microfluidic and biochemical analysis systems.


Health Beliefs Model as a template for measurement of intern satisfaction in an ambulatory care clinical training program.
The Health Beliefs Model was originally developed in the 1950s to assess patient behavior toward preventative health strategies, and has evolved into a tool for measuring a variety of health related topics. Interns in an ambulatory care clinical training program were asked to complete a 62 item self-administered questionnaire based on concepts central to a modified Health Beliefs Model for assessment of their satisfaction with their internship program. A logistic regression was undertaken to identify relationships between interns' perceptions toward susceptibility and seriousness of illness, delivery of health care through outpatient clinics, supervisory professional personnel and their overall internship satisfaction rating. The data implies that the modified Health Beliefs Model is of value in predicting intern satisfaction.


Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion.
Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.


Endometrial expression and in vitro modulation of the iron transporter divalent metal transporter-1: implications for endometriosis.
To evaluate divalent metal transporter-1 (DMT1) expression in healthy women's and endometriosis patients' endometrium and to analyze DMT1 and ferritin light chain (Fn-L) expression modulation by iron overload and IL-1β in endometrial stromal cells (ESCs). Observational and experimental study. University hospital research laboratory. Thirty-one healthy women and 24 endometriosis patients. Menstrual, proliferative, and secretory endometrial biopsies. Isolated ESCs from seven endometrial biopsies incubated with IL-1β or FeSO4 overload for 24 hours. Divalent metal transporter-1 endometrial protein expression assessed by immunohistochemistry and Western blot. Divalent metal transporter-1 and Fn-L proteins expression in stimulated ESCs evaluated by Western blot. Divalent metal transporter-1 is expressed throughout the menstrual cycle in human endometrium. Four endometrial DMT1 variants were identified accordingly to their molecular weight: DMT-80, -65, -55, and -50. Endometrial expression of DMT-80 and -55 is higher in endometriosis patients than in healthy women. In ESCs, iron overload induces an overexpression of DMT-80, DMT-50, and Fn-L, whereas IL-1β increases DMT-80 and -50 expressions and decreases Fn-L expression. Divalent metal transporter-1 overexpression in endometriosis patients' endometrium can increase iron influx to endometrial cells, inducing oxidative stress-mediated proinflammatory signaling. In turn, endometriosis-related conditions, as iron overload and inflammation (IL-1β), enhance endometriosis patients endometrial DMT1 expression, creating a vicious circle on DMT-1-modulated pathways.


Use of Proximal Suboccipital Segment of Occipital Artery as Donor for Posterior Circulation Revascularization Surgery: Technical Note.
The distal suboccipital segment of the occipital artery is commonly used as a donor vessel for aneurysms of the posterior circulation requiring revascularization techniques. Isolating this segment of vessel can be challenging due to its course in and out of the various layers of the suboccipital musculature. We describe the use of the proximal suboccipital segment of the occipital artery within the occipital groove at the lateral skull base as a donor segment for an interposition graft in revascularization surgery. We present a case detailing the use of this technique for treatment of a dissecting PICA aneurysm and photographs from cadaveric dissections demonstrating relevant anatomy. Our patient is a 62-year-old man who presented with a high-grade subarachnoid hemorrhage from a proximal dissecting-type aneurysm of the posterior inferior cerebellar artery (PICA). Endovascular options were limited to coil sacrifice of the parent vessel, and thus the patient was taken for a far lateral craniotomy, trapping of the aneurysm, and revascularization of the PICA territory via a saphenous vein interposition graft sewn proximally and distally to the occipital artery within the occipital groove and a cortical segment of the affected PICA, respectively. Postoperative imaging demonstrated filling of the PICA territory via the graft, and the patient ultimately recovered from his subarachnoid hemorrhage. The use of the proximal suboccipital segment of the occipital artery is a viable option for a donor in posterior fossa revascularization surgery.


The health behaviour and wellbeing of older seafarers on Merseyside - indicated changes through brief interventions.
There is significant evidence of the poor health of seafarers that arises both from the rigours of their trade and, for many, the associated lifestyles. Such poor health can continue in later life. The objective of the research is to report on a specific project that provided brief interventions to assist older (ex-) seafarers and to establish the effect of those interventions on their knowledge, behaviours, health and wellbeing. Older seafarers were recruited to the project. Brief interventions were provided by which the knowledge of a number of older seafarers with health needs was raised about the options available to them; and the implications for their lifestyles and behaviours were addressed. Initial and final interviews were undertaken to determine any changes in self-reported health and wellbeing using both EQ5D and the Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) measures. Post project interviews took place with a sample of the older seafarers. A good level of understanding was found among the older seafarers regarding their own health. This meant that a precondition was in place, for many, by which changes in behaviours and lifestyles could take place. An important outcome was the indicated benefits of the brief interventions for self-reported wellbeing, though not statistically significant at the 95% level of confidence. Endeavours within the project to reach some of those who could benefit from the brief interventions were successful. Just over half changed their behaviours or were thinking of so doing. Wellbeing gains arising were indicated.


Seven-coordinate homoleptic metal carbonyls in the gas phase.
Gas-phase metal carbonyl cations of the vanadium-group metals (V(+), Nb(+), Ta(+)) were produced in a molecular beam by laser vaporization and then mass-analyzed and size-selected in a time-of-flight spectrometer and studied with IR laser photodissociation spectroscopy in the carbonyl-stretching region. The abundances in the mass spectra, the fragmentation patterns, and the IR spectra provided a combined approach that revealed the coordination numbers in these systems. Although seven-coordinate structures would have 18 electrons in each case, V(CO)(6)(+) was found to be formed rather than V(CO)(7)(+). Nb(+) formed both six- and seven-coordinate species, while Ta(+) formed only the Ta(CO)(7)(+) complex. Density functional theory computations were used to predict the IR spectra for these systems, which are dramatically different for the six- and seven-coordinate structures and in excellent agreement with the measurements. V(CO)(6)(+) and Nb(CO)(6)(+) have structures slightly distorted from octahedral, while Nb(CO)(7)(+) and Ta(CO)(7)(+) have C(3v) capped octahedral structures.


The measure of precarious employment and its impact on the mental health of workers: A systematic review 2007-2020.
Precarious employment conditions can influence the worker's mental health; however, there is no consensus regarding the definition of precarious employment or the way it is measured. The objective is to identify existing research of the conceptual framework, the ways to measure precarious employment and its impact on the mental health of workers. A systematic review with the strategic search for observational-empirical and qualitative studies published between 2007 and 2020 in Embase, Scopus and PubMed, full text, in English and Spanish. Studies were selected and excluded, according to eligibility criteria. Two independent reviewers and one arbitrator evaluated the quality of selected papers with the STROBE guidelines for observational studies and SRQR for qualitative ones. 408 studies were obtained, 21 met inclusion criteria, in 14 studies precarious employment was measured one-dimensionally. Of these, 11 with the dimension of temporality and three with insecurity. Four studies it was measured in a multidimensional way, with the Employment Precariousness Scale (EPRES) and in three qualitative designs, with different categories of analysis. Mental health was measured with SF-36 (n = 4), GHQ-12 (n = 3) and CES-D (n = 3). The concept of precarious employment requires a multidimensional construct, although the tendency to measure precarious employment is one-dimensional. Regardless of how is measured, this has a negative impact on the mental health of workers. The outcomes related to this social determinant included depression, depressive symptoms, psychological distress, stress, and suicidal thoughts. Youths, women, people with low levels of education and immigrants are the groups that show the major precariousness.


Cholesterol supports headgroup superlattice domain formation in fluid phospholipid/cholesterol bilayers.
Fluorescence and Fourier transform infrared (FTIR) spectroscopic techniques were used to explore the effect of added cholesterol on the composition-dependent formation of putative phospholipid headgroup superlattices in fluid 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers. Steady-state fluorescence anisotropy measurements of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) revealed significant dips at several POPE-to-phospholipid mole fractions (X(PE)'s) when the cholesterol-to-lipid mole fraction (X(CHOL)) was fixed at 0.00, 0.35, 0.40, and 0.50. Most of the observed dips occur at or close to critical X(PE)'s predicted by the Headgroup Superlattice (SL) model, suggesting that phospholipid headgroups of different structures tend to adopt regular distributions even in the presence of cholesterol. Time-resolved fluorescence anisotropy measurements revealed that DPH-PC senses a disordered and highly mobile microenvironment in the POPE/POPC/CHOL bilayers at those critical X(PE)'s, indicating that this probe may partition to defect regions in the bilayers. The presence of coexisting packing defect regions and regularly distributed SL domains is a key feature predicted by the Headgroup SL model. Importantly, probe-free FTIR measurements of acyl chain C-H, interfacial carbonyl, and headgroup phosphate stretching peak frequencies revealed the presence of abrupt changes at X(PE)'s close to those observed in the fluorescence data. When X(PE) was varied from 0.60 to 0.72 and X(CHOL) from 0.34 to 0.46, a clear dip at the lipid composition coordinates (X(PE), X(CHOL)) approximately (0.68, 0.40) was observed in the three-dimensional surface plots of DPH-PC anisotropy as well as the carbonyl and phosphate stretching frequencies. The critical X(CHOL) at 0.40 agrees with the Cholesterol SL model, which assumes that cholesterol and phospholipid form SL domains at the lipid acyl chain level. In conclusion, this study provides evidence that cholesterol supports formation of phospholipid headgroup SLs in fluid state ternary lipid bilayers. The feasibility of the parallel existence of SLs at the lipid headgroup and acyl chain levels supports the relevance of the lipid SL model for the membranes of eukaryotic cells that typically contain significant amounts of cholesterol. We speculate that lipid SL formation may play a central role in the regulation of membrane lipid compositions, maintenance of organelle boundaries, and other crucial phenomena in those cells.


The walking dead: macrophage inflammation and death in atherosclerosis.
To highlight recent studies that describe novel inflammatory and signaling mechanisms that regulate macrophage death in atherosclerosis. Macrophages contribute to all stages of atherosclerosis. The traditional dogma states that in homeostatic conditions, macrophages undergo apoptosis and are efficiently phagocytosed to be cleared by a process called efferocytosis. In advanced atherosclerosis, however, defective efferocytosis results in secondary necrosis of these uncleared apoptotic cells, which ultimately contributes to the formation of the characteristic necrotic core and the vulnerable plaque. Here, we outline the different types of lesional macrophage death: apoptosis, autophagic and the newly defined necroptosis (i.e. a type of programmed necrosis). Recent discoveries demonstrate that macrophage necroptosis directly contributes to necrotic core formation and plaque instability. Further, promoting the resolution of inflammation using preresolving mediators has been shown to enhance efferocytosis and decrease plaque vulnerability. Finally, the canonical 'don't eat me' signal CD47 has recently been described as playing an important role in atherosclerotic lesion progression by impairing efficient efferocytosis. Although we have made significant strides in improving our understanding of cell death and clearance mechanisms in atherosclerosis, there still remains unanswered questions as to how these pathways can be harnessed using therapeutics to promote lesion regression and disease stability. Improving our understanding of the mechanisms that regulate macrophage death in atherosclerosis, in particular apoptosis, necroptosis and efferocytosis, will provide novel therapeutic opportunities to resolve atherosclerosis and promote plaque stability.


Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation.
We reported previously that both subtypes of estrogen receptors, ERalpha and ERbeta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERalpha and ERbeta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERbeta, but immortalized and cancer cells express much higher amounts of ERalpha than primary cells. Higher levels of ERalpha mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERalpha-selective agonist PPT, the ERbeta-selective agonist DPN, and specific small interfering RNA against ERalpha or ERbeta, we found that ERbeta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERalpha predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17beta-estradiol (E(2)) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E(2)-induced urothelial cell proliferation. E(2) up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERalpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.


Flow-generating capability of the isolated skeletal muscle pump.
We sought to test directly whether the mechanical forces produced during rhythmic muscle contraction and relaxation act on the muscle vasculature in a manner sufficient to initiate and sustain blood flow. To accomplish this goal, we evaluated the mechanical performance of the isolated skeletal muscle pump. The hindlimb skeletal muscle pump was isolated by reversibly connecting the inferior vena cava and terminal aorta with extracorporeal tubing in 15- to 20-kg anesthetized pigs (n = 5). During electrically evoked contractions (1/s), hindlimb muscles were made to perfuse themselves by diverting the venous blood propelled out of the muscles into the shunt tubing, which had been prefilled with fresh arterial blood. This caused arterial blood to be pushed into the distal aorta and then through the muscles (shunt open, proximal aorta and vena cava clamped). In essence, the muscles perfused themselves for brief periods by driving blood around a "short-circuit" that isolates muscle from the remainder of the circulation, analogous to isolated heart-lung preparations. Because the large, short shunt offers a negligible resistance to flow, the arterial-venous pressure difference across the limbs was continuously zero, and thus the energy to drive flow through muscle could come only from the muscle pump. The increase in blood flow during normal heart-perfused contractions (with only the shunt tubing clamped) was compared with shunt-perfused contractions in which the large veins were preloaded with extra blood volume. Muscle blood flow increased by 87 +/- 11 and 110 +/- 21 (SE) ml/min in the first few seconds after the onset of shunt-perfused and heart-perfused contractions, respectively (P > 0.4). We conclude that the mechanical forces produced by muscle contraction and relaxation act on the muscle vasculature in a manner sufficient to generate a significant flow of blood.


Transition from Initial Hypoactivity to Hyperactivity in Cortical Layer V Pyramidal Neurons after Traumatic Brain Injury In Vivo.
Traumatic brain injury (TBI) often results in structural damage and a loss of neurons that is commonly accompanied by early changes in neuronal electrical activity. Loss of neuronal activity has been hypothesized to contribute to post-traumatic epileptogenesis through the regulation of homeostatic plasticity. The existence of activity loss in cortical neurons after TBI and its subsequent transition into hyperactivity over time is not well characterized, however, particularly in models of TBI in vivo. In the current study, changes in neuronal activity in the primary motor cortex after moderate controlled cortical impact (CCI) in mice were studied using a single-unit recording technique in vivo. Recordings were made at different time points after CCI from cortical layer V pyramidal neurons that were within 1-2 mm from the anterior edge of the injured foci. Within 1-4 h after CCI, the frequency of spontaneous single-unit activity depressed significantly, with the mean firing frequency decreasing from 2.59 ± 0.18 Hz in the sham group to 1.05 ± 0.20 Hz of the injured group. The firing frequencies recovered to the normal level at 1 day and 7 days post-CCI, but became significantly higher at 3 days and 14 days post-CCI. The results suggest that TBI caused initial loss of activity in neurons of the perilesional cortical region, which was followed by compensatory recovery and enhancement of activity. These time-dependent changes in neuronal activity may contribute to the development of hyperexcitability through homeostatic activity regulation.


Lyophilized Drug Product Cake Appearance: What Is Acceptable?
Cake appearance is an important attribute of freeze-dried products, which may or may not be critical with respect to product quality (i.e., safety and efficacy). Striving for "uniform and elegant" cake appearance may continue to remain an important goal during the design and development of a lyophilized drug product. However, "sometimes" a non-ideal cake appearance has no impact on product quality and is an inherent characteristic of the product (due to formulation, drug product presentation, and freeze-drying process). This commentary provides a summary of challenges related to visual appearance testing of freeze-dried products, particularly on how to judge the criticality of cake appearance. Furthermore, a harmonized nomenclature and description for variations in cake appearance from the ideal expectation of uniform and elegant is provided, including representative images. Finally, a science and risk-based approach is discussed on establishing acceptance criteria for cake appearance.


A critical review of the epidemiology of Agent Orange/TCDD and prostate cancer.
To inform risk assessment and regulatory decision-making, the relationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and prostate cancer requires clarification. This article systematically and critically reviews the epidemiologic evidence on the association between exposure to TCDD or Agent Orange, a TCDD-contaminated herbicide used during the Vietnam War, and prostate cancer risk. Articles evaluated include 11 studies of three cohorts, four case-control or cross-sectional studies, and three case-only studies of military veterans with information on estimated Agent Orange or TCDD exposure; 13 studies of seven cohorts, one case-control study, and eight proportionate morbidity or mortality studies of Vietnam veterans without information on Agent Orange exposure; 11 cohort studies of workers with occupational exposure to TCDD; and two studies of one community cohort with environmental exposure to TCDD. The most informative studies, including those of Vietnam veterans involved in Agent Orange spraying or other handling, herbicide manufacturing or spraying workers with occupational TCDD exposure, and community members exposed to TCDD through an industrial accident, consistently reported no significant increase in prostate cancer incidence or mortality. Only some potentially confounded studies of Vietnam veterans compared with the general population, studies with unreliable estimates of Agent Orange exposure, and analyses of selected subgroups of Vietnam veterans reported positive associations. Overall, epidemiologic research offers no consistent or convincing evidence of a causal relationship between exposure to Agent Orange or TCDD and prostate cancer. More accurate exposure assessment is needed in large epidemiologic studies to rule out a causal association more conclusively.


The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.
Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.