A prospective epidemiological study of injuries in four English professional football clubs.
To define the causes of injuries to players in English professional football during competition and training. Lost time injuries to professional and youth players were prospectively recorded by physiotherapists at four English League clubs over the period 1994 to 1997. Data recorded included information related to the injury, date and place of occurrence, type of activity, and extrinsic Playing factors. In all, 67% of all injuries occurred during competition. The overall injury frequency rate (IFR) was 8.5 injuries/1000 hours, with the IFR during competitions (27.7) being significantly (p < 0.01) higher than that during training (3.5). The IFRs for youth players were found to increase over the second half of the season, whereas they decreased for professional players. There were no significant differences in IFRs for professional and youth players during training. There were significantly (p < 0.01) injuries in competition in the 15 minute periods at the end of each half. Strains (41%), sprains (20%), and contusions (20%) represented the major types of injury. The thigh (23%), the ankle (17%), knee (14%), and lower leg (13%) represented the major locations of injury, with significantly (p < 0.01) more injuries to the dominant body side. Reinjury counted for 22% of all injuries. Only 12% of all injuries were caused by a breach of the rules of football, although player to player contact was involved in 41% of all injuries. The overall level of injury to professional footballers has been showed to be around 1000 times higher times higher than for industrial occupations generally regarded as high risk. The high level of muscle strains, in particular, indicates possible weakness in fitness training programmes and use of warming up and cooling down procedures by clubs and the need for benchmarking players' levels of fitness and performance. Increasing levels of injury to youth players as a season progresses emphasizes the importance of controlling the exposure of young players to high levels of competition.


OnabotulinumtoxinA vs Sacral Neuromodulation on Refractory Urgency Urinary Incontinence in Women: A Randomized Clinical Trial.
Women with refractory urgency urinary incontinence are treated with sacral neuromodulation and onabotulinumtoxinA with limited comparative information. To assess whether onabotulinumtoxinA is superior to sacral neuromodulation in controlling refractory episodes of urgency urinary incontinence. Multicenter open-label randomized trial (February 2012-January 2015) at 9 US medical centers involving 381 women with refractory urgency urinary incontinence. Cystoscopic intradetrusor injection of 200 U of onabotulinumtoxinA (n = 192) or sacral neuromodulation (n = 189). Primary outcome, change from baseline mean number of daily urgency urinary incontinence episodes over 6 months, was measured with monthly 3-day diaries. Secondary outcomes included change from baseline in urinary symptom scores in the Overactive Bladder Questionnaire Short Form (SF); range, 0-100, higher scores indicating worse symptoms; Overactive Bladder Satisfaction questionnaire; range, 0-100; includes 5 subscales, higher scores indicating better satisfaction; and adverse events. Of the 364 women (mean [SD] age, 63.0 [11.6] years) in the intention-to-treat population, 190 women in the onabotulinumtoxinA group had a greater reduction in 6-month mean number of episodes of urgency incontinence per day than did the 174 in the sacral neuromodulation group (-3.9 vs -3.3 episodes per day; mean difference, 0.63; 95% CI, 0.13 to 1.14; P = .01). Participants treated with onabotulinumtoxinA showed greater improvement in the Overactive Bladder Questionnaire SF for symptom bother (-46.7 vs -38.6; mean difference, 8.1; 95% CI, 3.0 to 13.3; P = .002); treatment satisfaction (67.7 vs 59.8; mean difference, 7.8; 95% CI, 1.6 to 14.1; P = .01) and treatment endorsement (78.1 vs 67.6; mean difference; 10.4, 95% CI, 4.3 to 16.5; P < .001) than treatment with sacral neuromodulation. There were no differences in convenience (67.6 vs 70.2; mean difference, -2.5; 95% CI, -8.1 to 3.0; P = .36), adverse effects (88.4 vs 85.1; mean difference, 3.3; 95% CI, -1.9 to 8.5; P = .22), and treatment preference (92.% vs 89%; risk difference, -3%; 95% CI, -16% to 10%; P = .49). Urinary tract infections were more frequent in the onabotulinumtoxinA group (35% vs 11%; risk difference, -23%; 95% CI, -33% to -13%; P < .001). The need for self-catheterization was 8% and 2% at 1 and 6 months in the onabotulinumtoxinA group. Neuromodulation device revisions and removals occurred in 3%. Among women with refractory urgency urinary incontinence, treatment with onabotulinumtoxinA compared with sacral neuromodulation resulted in a small daily improvement in episodes that although statistically significant is of uncertain clinical importance. In addition, it resulted in a higher risk of urinary tract infections and need for transient self-catheterizations.


Equilibria and Speciation of Chloramines, Bromamines, and Bromochloramines in Water.
The stabilities and speciation of the halamines in water are difficult to characterize experimentally. We provide theoretical estimates of aqueous standard free energies of formation for inorganic chloramines, bromamines, and bromochloramines, based on high-accuracy theoretical standard free energies of formation in gas phase combined with quantum chemical estimates of Henry's law constant. Based on comparisons between several theoretical and experimental datasets, we assign an error of 1.1-1.2 log unit for equilibrium constants of several reactions leading to halamines in water. The reactions of ammonia with HOCl or HOBr that lead to dichloramine, trichloramine, and tribromamine are found to be thermodynamically more favorable than was previously believed. The newly reported equilibrium data also allow us to propose rate constant values for some hydrolysis and disproportionation reactions of dichloramine, monobromamine, and bromochloramine. Finally, theoretical results indicate aqueous acid dissociation constant (pKa) values of 1.5 ± 1 for NH3Cl+, 0.8 ± 1 for NH3Br+, 11.8 ± 1 for NHCl2, and 12.5 ± 1 for NHBrCl. The present report provides a comprehensive data set describing the free energies of the neutral inorganic halamines, the anionic conjugate base species, and the cationic conjugate acid species, with approximately uniform uncertainty bounds assigned throughout.


Willingness to pay and size of health benefit: an integrated model to test for 'sensitivity to scale'.
A key theoretical prediction concerning willingness to pay is that it is positively correlated with benefit size and is assessed by testing the 'sensitivity to scale (scope)'. 'External' (between-sample) sensitivity tests are usually regarded as less powerful than 'internal' (within-subject) tests. However, the latter may suffer from 'anchoring' effects. This paper studies the statistical power of these tests by questioning the distributional assumption of empirical data. We present an integrated model to capture both internal and external variations, while controlling for sample heterogeneity, applied to data from a survey estimating the value of reducing symptom-days. Results indicate that once data is properly transformed, WTP becomes 'scale sensitive' and consistent with diminishing marginal utility theory.


Extracorporeal membrane oxygenation-assisted primary percutaneous coronary intervention may improve survival of patients with acute myocardial infarction complicated by profound cardiogenic shock.
The aim of this study was to evaluate the impact of extracorporeal membrane oxygenation (ECMO) assistance on the clinical outcome of patients with acute myocardial infarction (AMI) that is complicated by profound cardiogenic shock (CS) who received primary percutaneous coronary intervention (PCI). We collected patients from January 2004 through December 2006 (stage 1); 25 patients who presented with AMI and received primary PCI and had profound CS were enrolled in the study. Intraaortic balloon counterpulsation (IABP) was the only modality for extracorporeal support in our hospital. From January 2007 through December 2009 (stage 2), 33 patients who presented with AMI and received primary PCI and had profound CS were enrolled; for this stage; both intra-aortic balloon counter-pulsation and ECMO support were available in our facility. A Kaplan-Meier survival analysis displayed significantly improved survival for patients in stage 2 (P = .001; 1-year survival in stage 1 vs 2; 24% vs 63.64%). Patients presenting with either STEMI (ST segment elevation myocardial infarction) or NSTEMI (Non-ST segment elevation myocardial infarction) benefited from ECMO-assisted PCI (P < .05). In stage 1, patients with refractory ventricular tachycardia/ventricular fibrillation had a very low survival rate; however, in stage 2, the survival rate of patients with and without refractory ventricular tachycardia/ventricular fibrillation was similar (P = .316). Extracorporeal membrane oxygenation-assisted PCI for patients with AMI that is complicated by profound CS may improve the 30-day and 1-year survival rates.


A conceptual framework for a clinical nurse leader program.
Developing a map to guide the design of a novel educational program for nursing students can assist educators in piecing together identified conceptual variables in a logical and reasonable manner. We developed a conceptual model for a clinical nurse leader (CNL) program to present to a school of nursing faculty and a state board of nursing for approval and adoption. The concept map was created following a literature review and analysis of the American Association of Colleges of Nursing description of the CNL. Following the identification of certain variables, such as theories underpinning nursing practice, clinical processes, and education, each team member researched identified variables of the conceptual diagram, wrote a thoughtful explanation of the components, and frequently met with the others to discuss each element of the model and the relationships among the variables. Over time, an emerging model developed and was adopted by the school of nursing and approved by the state board of nursing. An explanation of the components of the conceptual framework and their relationship to the CNL role may assist educators in developing and implementing other emerging CNL programs.


Welfare assessment of novel on-farm killing methods for poultry.
There is a need for novel mechanical devices for dispatching poultry on farm following the introduction of EU Regulation (EC) no. 1099/2009 On the Protection of Animals at the Time of Killing. We examined three novel mechanical killing devices: Modified Armadillo, Modified Rabbit Zinger, a novel mechanical cervical dislocation device; and traditional manual cervical dislocation. The four killing methods were tested on 230 chickens across four batches. We measured behavioural, electroencephalogram and post-mortem outcomes in anesthetized laying hens and broilers at two life stages (juveniles and adults/slaughter age). Graeco Latin-Square designs systematically randomized killing treatment, bird type, age and kill order. All birds were lightly anaesthetized immediately prior to the killing treatment with inhalation of Sevoflurane. The novel mechanical cervical dislocation method had the highest kill success rate (single application attempt only, with no signs of recovery) of a mechanical method (96%). The Modified Armadillo was the least reliable with 49% kill success. Spectral analysis of electroencephalogram signals at 2 s intervals for successfully killed birds only revealed progressive decreases in median frequency alongside increases in total power. Later, total power decreased as the birds exhibited isoelectric electroencephalogram signal. Latencies to pre-defined spectral ranges associated with unconsciousness showed that birds subjected to manual and novel mechanical cervical dislocation achieved these states sooner than birds subjected to the modified Armadillo. Nevertheless all methods exhibited short latencies (<4 s). The Modified Rabbit Zinger had the shortest duration of reflex persistence for nictitating membrane, pupillary and rhythmic breathing post method application. Of the methods tested, the novel mechanical cervical dislocation device is the most promising mechanical method for killing poultry on-farm based on a range of behavioural, electroencephalogram and anatomical parameters. This device has the potential to fulfil the current need for a mechanical alternative to manual cervical dislocation.


The fate of E- and P-cadherin during the early stages of apoptosis.
Caspases are responsible for the proteolysis of many cytoskeletal proteins in apoptotic cells. It has been demonstrated here that during cisplatin-induced apoptosis of human embryo retinoblasts both E- and P-cadherin were degraded by caspases, giving initially major polypeptide products of apparent molecular weights 48 K and 104 K respectively. This proteolysis occurred over a similar time-scale to the observed degradation of PARP and to the onset of DNA fragmentation but appreciably later than p53 induction and cleavage of Mdm2 and p21. Addition of caspase inhibitors such as Z-VAD-FMK inhibited apoptosis and cadherin degradation. Co-immunoprecipitation studies carried out on viable cells confirmed previously observed complexes between cadherins and alpha and beta catenin and between the catenins themselves. These interactions were sustained in apoptotic cells as long as the protein components remained intact. Using confocal microscopy it has been shown that cytoskeletal changes associated with apoptosis precede degradation of catenins and cadherins by several hours. In particular, after addition of cisplatin relatively rapid (within 3 h) re-localization of adherens junction proteins from the cell periphery to the cytoplasm was observed whereas little cadherin or catenin degradation occurred until 10 h. We conclude that neither caspase-mediated degradation of cytoskeletal components nor disruption of adherens junction protein-protein interactions is required for morphological change.


Inactivation of the interoceptive insula disrupts drug craving and malaise induced by lithium.
Addiction profoundly alters motivational circuits so that drugs become powerful reinforcers of behavior. The interoceptive system continuously updates homeostatic and emotional information that are important elements in motivational decisions. We tested the idea that interoceptive information is essential in drug craving and in the behavioral signs of malaise. We inactivated the primary interoceptive cortex in amphetamine-experienced rats, which prevented the urge to seek amphetamine in a place preference task. Interoceptive insula inactivation also blunted the signs of malaise induced by acute lithium administration. Drug-seeking and malaise both induced Fos expression, a marker of neuronal activation, in the insula. We conclude that the insular cortex is a key structure in the perception of bodily needs that provides direction to motivated behaviors.


Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.
Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.


A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia.
Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 x 10(9)/L) due to these diseases were enrolled in this multicenter phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony-stimulating factor (filgrastim) (3.45 to 11.50 micrograms/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of > or = 1.5 x 10(9)/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently; adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.


Masseter muscle rigidity associated with glycine1306-to-alanine mutation in the adult muscle sodium channel alpha-subunit gene.
Succinylcholine-induced masseter muscle rigidity (MMR) is a potentially life-threatening complication of anesthesia and is closely correlated with the heterogeneous disorder malignant hyperthermia (MH) susceptibility. MMR also is identified with a variety of neuromuscular disorders, including the myotonias, that are associated with abnormal in vitro contracture test (IVCT) results. Recently, mutations in the adult skeletal muscle sodium channel alpha-subunit gene (SCN4A) have been shown to cause generalized nondystrophic myotonias, some of which are associated with mild nonspecific symptoms. The purpose of the current investigation was to begin to evaluate the molecular genetic relationship between known mutations in the SCN4A gene, MMR, and the results of the IVCT used to diagnose MH-susceptibility. A single extended pedigree of 16 individuals was ascertained through a proband who experienced MMR and whole-body rigidity after succinylcholine administration. Subsequently, four individuals were shown to have a mild form of myotonia on clinical and laboratory examination. IVCT was carried out according to standardized protocols. Mutations in the SCN4A gene were sought in exons 22 and 24 using single-strand conformational analyses. Variability in the SCN4A gene sequence was confirmed by direct DNA sequence analyses. Four individuals with myotonia were shown to carry a guanine-to-cytosine mutation at nucleotide position 3917 of the reported SCN4A sequence. This DNA mutation was coinherited with MMR and an abnormal IVCT result in this family. Previous studies have demonstrated that the glycine1306-to-alanine substitution is associated with a mild clinical syndrome referred to as myotonia fluctuans. The current report provides direct evidence that succinylcholine-induced MMR, whole-body rigidity, and an abnormal IVCT result are associated with a mutation in the SCN4A gene.


'Reaching the hard to reach'--lessons learned from the VCS (voluntary and community Sector). A qualitative study.
The notion 'hard to reach' is a contested and ambiguous term that is commonly used within the spheres of social care and health, especially in discourse around health and social inequalities. There is a need to address health inequalities and to engage in services the marginalized and socially excluded sectors of society. This paper describes a pilot study involving interviews with representatives from eight Voluntary and Community Sector (VCS) organisations. The purpose of the study was to explore the notion of 'hard to reach' and perceptions of the barriers and facilitators to accessing services for 'hard to reach' groups from a voluntary and community sector perspective. The 'hard to reach' may include drug users, people living with HIV, people from sexual minority communities, asylum seekers, refugees, people from black and ethnic minority communities, and homeless people although defining the notion of the 'hard to reach' is not straight forward. It may be that certain groups resist engaging in treatment services and are deemed hard to reach by a particular service or from a societal stance. There are a number of potential barriers for people who may try and access services, including people having bad experiences in the past; location and opening times of services and how services are funded and managed. A number of areas of commonality are found in terms of how access to services for 'hard to reach' individuals and groups could be improved including: respectful treatment of service users, establishing trust with service users, offering service flexibility, partnership working with other organisations and harnessing service user involvement. If health services are to engage with groups that are deemed 'hard to reach' and marginalised from mainstream health services, the experiences and practices for engagement from within the VCS may serve as useful lessons for service improvement for statutory health services.


Investigation of a national outbreak of STEC Escherichia coli O157 using online consumer panel control methods: Great Britain, October 2014.
In October 2014, Public Health England (PHE) identified cases of Shiga toxin-producing Escherichia coli (STEC) serogroup O157 sharing a multiple locus variable-number tandem repeat analysis (MLVA) profile. We conducted a case-control study using multivariable logistic regression to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) testing a range of exposures. Cases were defined as laboratory-confirmed STEC O157 with the implicated MLVA profile, were UK residents aged ⩾18 years with symptom onset between 25 September and 30 October 2014, and had no history of travel abroad within 5 days of symptom onset. One hundred and two cases were identified. Cases were mostly female (65%; median age 49, range 2-92 years). It was the second largest outbreak seen in England, to date, and a case-control study was conducted using market research panel controls and online survey methods. These methods were instrumental in the rapid data collection and analysis necessary to allow traceback investigations for short shelf-life products. This is a new method of control recruitment and this is the first in which it was a standalone recruitment method. The case-control study suggested a strong association between consumption of a ready-to-eat food and disease (aOR 28, 95% CI 5·0-157) from one retailer. No reactive microbiological testing of food items during the outbreak was possible due to the short shelf-life of the product. Collaboration with industrial bodies is needed to ensure timely traceback exercises to identify contamination events and initiate appropriate and focused microbiological testing and implement control measures.


Temporal variation selects for diet-microbe co-metabolic traits in the gut of Gorilla spp.
Although the critical role that our gastrointestinal microbes play in host physiology is now well established, we know little about the factors that influenced the evolution of primate gut microbiomes. To further understand current gut microbiome configurations and diet-microbe co-metabolic fingerprints in primates, from an evolutionary perspective, we characterized fecal bacterial communities and metabolomic profiles in 228 fecal samples of lowland and mountain gorillas (G. g. gorilla and G. b. beringei, respectively), our closest evolutionary relatives after chimpanzees. Our results demonstrate that the gut microbiomes and metabolomes of these two species exhibit significantly different patterns. This is supported by increased abundance of metabolites and bacterial taxa associated with fiber metabolism in mountain gorillas, and enrichment of markers associated with simple sugar, lipid and sterol turnover in the lowland species. However, longitudinal sampling shows that both species' microbiomes and metabolomes converge when hosts face similar dietary constraints, associated with low fruit availability in their habitats. By showing differences and convergence of diet-microbe co-metabolic fingerprints in two geographically isolated primate species, under specific dietary stimuli, we suggest that dietary constraints triggered during their adaptive radiation were potential factors behind the species-specific microbiome patterns observed in primates today.


Teaching Missing Data Methodology to Undergraduates Using a Group-Based Project Within a Six-Week Summer Program.
Missing data mechanisms, methods of handling missing data, and the potential impact of missing data on study results are usually not taught until graduate school. However, the appropriate handling of missing data is fundamental to biomedical research and should be introduced earlier on in a student's education. The Summer Institute for Training in Biostatistics (SIBS) provides practical experience to motivate trainees to pursue graduate training and biomedical research. Since 2010, SIBS Pittsburgh has demonstrated the feasibility of introducing missing data concepts to trainees in a small-group project-based setting that involves both simulation and data analysis. After learning about missing data mechanisms and statistical techniques, trainees apply what they have learned to a NIDDK/NIH-funded Hepatitis C treatment study, to examine how various hypothesized missing data patterns can affect results. A simulation is also used to examine the bias and precision of these methods under each missing data pattern. Our experience shows that under such project-based training, advanced topics, such as missing data, can be presented to trainees with limited statistical preparation, and ultimately, can further their statistical literacy and reasoning. The tools presented here are provided in the Appendix.


Identification of Risk Factors for Postoperative Cerebrospinal Fluid Leakage and Comparison of Two Alternative Dural Augmentation Techniques in Posterior Fossa and Spinal Surgeries.
To investigate comparative efficacy of a novel absorbable adhesive membrane (TissuePatchDuralTM "TPD") and a fibrin glue (Tisseel "T") in reducing cerebrospinal fluid (CSF) leaks after posterior fossa and spinal procedures, and also to identify potential risk factors for CSF leakage. This is a single-center, retrospective cohort study of 123 consecutive posterior fossa (n=77) and spinal (n=46) surgeries. Patients were grouped based on dural sealants used 2-group comparison: TPD (n=56) vs. no-TPD (n=67) and 3-group comparison: T only (n=43), TPD only (n=32) vs TPD+T (n=35). Mean age was 38.9 ± 22.2 years (62 males, 61 females). Baseline characteristics were similar between groups. Neither 2-group (TPD: 10.4% vs no-TPD: 8.9%; p=0.778) nor 3-group (T: 9.3% vs TPD: 6.3% vs TPD+T: 14.3%; p=0.539) comparisons revealed a significant difference in postoperative CSF leakage rates. Multivariate analysis showed that diagnosis (non-tumoral vs. tumor) (OR: 5.487; 95% CI: 1.118-26.937; p=0.036); previous surgery (OR: 9.268; 95% CI: 1.911-44.958; p=0.006), postoperative hydrocephalus (OR: 5.456; 95% CI: 1.250-23.821; p=0.024) were independent predictors of postoperative CSF leakage. TissuePatchDural < sup > TM < /sup > is a novel dural sealant patch which can be safely used to reinforce dural closure in posterior fossa and spinal surgeries, and its efficacy is comparable to widely used fibrin glue (Tisseel). Non-tumoral pathologies, previous surgery, and postoperative hydrocephalus appear to be independent risk factors for postoperative CSF leakage.


RNA-Seq reveals dynamic changes of gene expression in key stages of intestine regeneration in the sea cucumber Apostichopus japonicus. [corrected].
Sea cucumbers (Holothuroidea; Echinodermata) have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs) in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ~30000 ESTs. We used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe). This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M) reads were sequenced in every library. Over 2400 up-regulated genes (>10%) and over 1000 down-regulated genes (~5%) were observed at 3 and 7dpe (log2Ratio ≥ 1, FDR ≤ 0.001). Specific "Go terms" revealed that the DEGs (Differentially Expressed Genes) performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term), the "Notch signaling pathway," the "ECM-receptor interaction" and the "Cytokine-cytokine receptor interaction" were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs) were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques. Our studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched pathways that contribute to intestine regeneration in sea cucumbers. This provides a foundation for future studies on the genetics/molecular mechanisms associated with intestine regeneration.


Benchtop NMR for Online Reaction Monitoring of the Biocatalytic Synthesis of Aromatic Amino Alcohols.
Online analytics provides insights into the progress of an ongoing reaction without the need for extensive sampling and offline analysis. In this study, we investigated benchtop NMR as an online reaction monitoring tool for complex enzyme cascade reactions. Online NMR was used to monitor a two-step cascade beginning with an aromatic aldehyde and leading to an aromatic amino alcohol as the final product, applying two different enzymes and a variety of co-substrates and intermediates. Benchtop NMR enabled the concentration of the reaction components to be detected in buffered systems in the single-digit mM range without using deuterated solvent. The concentrations determined via NMR were correlated with offline samples analyzed via uHPLC and displayed a good correlation between the two methods. In summary, benchtop NMR proved to be a sensitive, selective and reliable method for online reaction monitoring in (multi-step) biosynthesis. In future, online analytic systems such as the benchtop NMR devices described might not only enable direct monitoring of the reaction, but may also form the basis for self-regulation in biocatalytic reactions.


The protective effect of familial longevity persists after age 100: Findings from the Danish national registers.
A recent study suggested that the protective effect of familial longevity becomes negligible for centenarians. However, the authors assessed the dependence on familial longevity in centenarians by comparing centenarians with one parent surviving to age 80+ to centenarians whose same-sexed parent did not survive to age 80. Here we test whether the protective effect of familial longevity persists after age 100 using more restrictive definitions of long-lived families. Long-lived sibships were identified through three nationwide, consecutive studies in Denmark, including families with either at least two siblings aged 90+ or a Family Longevity Selection Score (FLoSS) above 7. Long-lived siblings enrolled in these studies and who reached age 100 were included. For each sibling, five controls matched on sex and year of birth were randomly selected among centenarians in the Danish population. Survival time from age 100 was described with Kaplan-Meier curves for siblings and controls separately. Survival analyses were performed using stratified Cox proportional hazards models. A total of 340 individuals from long-lived sibships who survived to age 100 and 1,700 controls were included. Among the long-lived siblings and controls, 1,650 (81%) were women. The results showed that long-lived siblings presented better overall survival after age 100 than sporadic long-livers (HR=0.80 95%CI=0.71-0.91), with even lower estimate (HR=0.65 95%CI=0.50-0.85) if familial longevity was defined by FLoSS. The present study, with virtually no loss to follow-up, demonstrated a persistence of protective effect of familial longevity after age 100.


Signals transduced via insulin-like growth factor I receptor (IGF(R)) mediate resistance to retinoic acid-induced cell growth arrest in a human neuroblastoma cell line.
Retinoic Acid (RA) has been shown to control growth and induce differentiation in a number of human neuroblastoma (NB) cell lines. However, a number of NB cell lines may be termed resistant to RA as they fail to growth arrest and differentiate. In studying the mechanism mediating RA-resistance, we noted that invariably RA-resistant NB cell lines constitutively express Insulin-like Growth Factor 2 (IGF2) (Gaetano, 1991b). The NB cell line LAN-1-15N (15N) represented an interesting model in which to study the development of RA-resistance as initially 15N cells are growth arrested by RA, however with prolonged culture (8-10 days) cells begin to proliferate. Coincidentally, RA induces IGF2 mRNA and protein secretion in 15N NB cells (Matsumoto, 1992). In this study we isolated RA-resistant 15N cell lines and analyzed their growth properties and changes in cell cycle related (cdc2, cdk2, cyclins A, B, D and E) and early response (fos and jun) gene expression to evaluate the role IGF2 may play in mediating RA resistance. We found that exogenous IGF2 stimulates growth in 15N and is capable of altering RA induced inhibition of NB cell growth. Finally we show that by blocking the Insulin-like Growth Factor Receptor (IGF1(R)) with a monoclonal antibody (alpha-IR3) in the presence of RA the growth of RAR cell lines could be completely blocked. These data are consistent with the concept that signals by IGF2 and transduced via the IGF1(R) can mediate resistance to the growth inhibiting properties of RA.


Instructed knowledge shapes feedback-driven aversive learning in striatum and orbitofrontal cortex, but not the amygdala.
Socially-conveyed rules and instructions strongly shape expectations and emotions. Yet most neuroscientific studies of learning consider reinforcement history alone, irrespective of knowledge acquired through other means. We examined fear conditioning and reversal in humans to test whether instructed knowledge modulates the neural mechanisms of feedback-driven learning. One group was informed about contingencies and reversals. A second group learned only from reinforcement. We combined quantitative models with functional magnetic resonance imaging and found that instructions induced dissociations in the neural systems of aversive learning. Responses in striatum and orbitofrontal cortex updated with instructions and correlated with prefrontal responses to instructions. Amygdala responses were influenced by reinforcement similarly in both groups and did not update with instructions. Results extend work on instructed reward learning and reveal novel dissociations that have not been observed with punishments or rewards. Findings support theories of specialized threat-detection and may have implications for fear maintenance in anxiety.


Diversity and complexity in DNA recognition by transcription factors.
Sequence preferences of DNA binding proteins are a primary mechanism by which cells interpret the genome. Despite the central importance of these proteins in physiology, development, and evolution, comprehensive DNA binding specificities have been determined experimentally for only a few proteins. Here, we used microarrays containing all 10-base pair sequences to examine the binding specificities of 104 distinct mouse DNA binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in the evolution of transcriptional regulatory networks.


Nanoconnectomic upper bound on the variability of synaptic plasticity.
Information in a computer is quantified by the number of bits that can be stored and recovered. An important question about the brain is how much information can be stored at a synapse through synaptic plasticity, which depends on the history of probabilistic synaptic activity. The strong correlation between size and efficacy of a synapse allowed us to estimate the variability of synaptic plasticity. In an EM reconstruction of hippocampal neuropil we found single axons making two or more synaptic contacts onto the same dendrites, having shared histories of presynaptic and postsynaptic activity. The spine heads and neck diameters, but not neck lengths, of these pairs were nearly identical in size. We found that there is a minimum of 26 distinguishable synaptic strengths, corresponding to storing 4.7 bits of information at each synapse. Because of stochastic variability of synaptic activation the observed precision requires averaging activity over several minutes.


GPs' use of gut feelings when assessing cancer risk: a qualitative study in UK primary care.
The use of gut feelings to guide clinical decision making in primary care has been frequently described but is not considered a legitimate reason for cancer referral. To explore the role that gut feeling plays in clinical decision making in primary care. Qualitative interview study with 19 GPs in Oxfordshire, UK. GPs who had referred patients to a cancer pathway based on a gut feeling as a referral criterion were invited to participate. Interviews were conducted between November 2019 and January 2020, and transcripts were analysed using the one sheet of paper method. Gut feeling was seen as an essential part of decision making that facilitated appropriate and timely care. GPs distanced their gut feelings from descriptions that could be seen as unscientific, describing successful use as reliant on experience and clinical knowledge. This was especially true for patients who fell within a 'grey area' where clinical guidelines did not match the GP's assessment of cancer risk, either because the guidance inadequately represented or did not include the patient's presentation. GPs sought to legitimise their gut feelings by gathering objective clinical evidence, careful examination of referral procedures, and consultation with colleagues. GPs described their gut feelings as important to decision making in primary care and a necessary addition to clinical guidance. The steps taken to legitimise their gut feelings matched that expected in good clinical practice.


Collective phases of strongly interacting cavity photons.
We study a coupled array of coherently driven photonic cavities, which maps onto a driven-dissipative XY spin- 1 2 model with ferromagnetic couplings in the limit of strong optical nonlinearities. Using a site-decoupled mean-field approximation, we identify steady-state phases with canted antiferromagnetic order, in addition to limit cycle phases, where oscillatory dynamics persist indefinitely. We also identify collective bistable phases, where the system supports two steady states among spatially uniform, antiferromagnetic, and limit cycle phases. We compare these mean-field results to exact quantum trajectory simulations for finite one-dimensional arrays. The exact results exhibit short-range antiferromagnetic order for parameters that have significant overlap with the mean-field phase diagram. In the mean-field bistable regime, the exact quantum dynamics exhibits real-time collective switching between macroscopically distinguishable states. We present a clear physical picture for this dynamics and establish a simple relationship between the switching times and properties of the quantum Liouvillian.


Performance of the Pulmonary Embolism Rule-out Criteria (the PERC rule) combined with low clinical probability in high prevalence population.
PERC rule was created to rule out pulmonary embolism (PE) without further exams, with residual PE risk<2%. Its safety is currently not confirmed in high PE prevalence populations even when combined with low clinical probability assessed by revised Geneva score (RGS). As PERC rule and RGS are 2 similar explicit rules with many redundant criteria, we hypothesized that the combination of PERC rule with gestalt clinical probability could resolve this limitation. We collected prospectively documented clinical gestalt assessments and retrospectively calculated PERC rules and RGS from a prospective study of PE suspected patients. We analyzed performance of combinations of negative PERC with low clinical probability assessed by both methods in high overall PE prevalence population. Among the final study population (n = 959), the overall PE prevalence was 29.8%. Seventy-four patients (7.7%) were classified as PERC negative and among them, 4 patients (5.4%) had final diagnosis of PE. When negative PERC was combined with low pretest probability assessed by RGS or gestalt assessment, PE prevalence was respectively 6.2% and 0%. This last combination reaches threshold target of 2% and unnecessary exams could easily have been avoided in this subgroup (6%). However, it confidence interval was still wide (0%; CI 0-5). PERC rule combined with low gestalt probability seems to identify a group of patients for whom PE could easily be ruled out without additional test. A larger study is needed to confirm this result and to ensure safety.


The Clonal Invariant NKT Cell Repertoire in People with Type 1 Diabetes Is Characterized by a Loss of Clones Expressing High-Affinity TCRs.
Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.


Lipid peroxidation and platelet membrane fluidity--implications for Alzheimer's disease?
In humans, the fluidity of cell membranes generally decreases with age. Unexpectedly, several laboratories have found increased fluidity of platelet membranes (mainly endoplasmic reticulum) in patients with Alzheimer's disease (AD) compared with controls. In the present study, free radical induced lipid peroxidation was found to increase the fluidity of platelet membranes. Hydroxyl radicals were generated in the presence of Fe2+ and EDTA at low concentrations of ascorbate. It is hypothesised that platelet membranes are unable to restore their microviscosity by incorporating cholesterol. There may be a link between the result obtained in this study, the recently discovered decreased cholesterol content of affected AD neuronal membranes, and the increased frequency of epsilon 4 apolipoprotein E (a cholesterol carrier) found in AD patients.


Mechanical behavior of shark vertebral centra at biologically relevant strains.
Cartilaginous shark skeletons experience axial deformation at the intervertebral joints, but also within the mineralized cartilaginous centrum, which can compress to between 3% and 8% of its original length in a free-swimming shark. Previous studies have focused on shark centra mechanical properties when loaded to failure; our goal was to determine properties when compressed to a biologically relevant strain. We selected vertebrae from six shark species and from the anterior and posterior regions of the vertebral column. Centra were X-radiographed to measure double cone proportion and apex angles, and were mechanically tested at three displacement rates to 4% strain. We determined the variation in toughness and stiffness of vertebral centra among shark species and ontogenetic stages, testing strain rates, and compared anterior and posterior regions of the vertebral column. Our results suggest that toughness and stiffness, which are positively correlated, may be operating in concert to support lateral body undulations, while providing efficient energy transmission and return in these swift-swimming apex predators. We analyzed the contribution of double cone proportion and apex angle to centra mechanical behavior. We found that the greatest stiffness and toughness were in the youngest sharks and from the posterior body, and there was significant interspecific variation. Significant inverse correlations were found between mechanical properties and double cone apex angle suggesting that properties can be partially attributed to the angle forming the double cone apex. These comparative data highlight the importance of understanding cartilaginous skeleton mechanics under a wide variety of loading conditions representative of swimming behaviors seen in the wild.


Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media.
To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses.


NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties.
Recent evidence suggests that sulfhydryl species can react with oxides of nitrogen under physiologic conditions and thereby stabilize endothelium-derived relaxing factor (EDRF) activity, but the presence of a specific in vivo thiol carrier for nitric oxide (NO) remains controversial. The single free sulfhydryl of serum albumin is the most abundant thiol species in plasma (approximately 0.5 mM) and is particularly reactive towards NO. To examine the potential role of serum albumin in endogenous nitric oxide metabolism, we synthesized S-nitroso-BSA (S-NO-BSA), a model S-nitroso-protein, and examined its effects on platelet function and coronary and systemic vascular tone in 16 mongrel dogs. Intravenous bolus S-NO-BSA markedly reduced mean arterial pressure in a dose-dependent manner and proved seven and a half-fold less potent than intravenous nitroglycerin and 10-fold less potent than intravenous S-nitroso-cysteine (half-maximal response of 75 nmol/kg compared to 10 and 7.5 nmol/kg, respectively; P < 0.05); when given by intravenous infusion (half-maximal response = 10 nmol/kg per min), however, S-NO-BSA and nitroglycerin were equipotent. Intravenous bolus S-NO-BSA had a greater duration of action than either nitroglycerin or S-nitroso-cysteine and produced marked prolongation of the template bleeding time associated with dose-dependent inhibition of ex vivo platelet aggregation (half-maximal response approximately 70 nmol/kg). Intracoronary S-NO-BSA increased coronary blood flow (mean +/- SEM) less effectively than nitroprusside, acetylcholine, or S-nitroso-cysteine (165% +/- 24% vs. 315% +/- 82%, 483% +/- 55%, or 475% +/- 66%, respectively; P < 0.05) although with much longer duration of action. On a molar basis, S-nitroso-cysteine proved more effective than S-nitroso-BSA, nitroprusside, or acetylcholine as an epicardial coronary vasodilator. Thus, serum albumin reacts with oxides of nitrogen to form a stable S-nitroso-thiol with properties reminiscent of authentic EDRF supporting the view that protein associated thiol may participate in the action and metabolism of EDRF.


Evaluation of higher plant virus resistance genes in the green alga, Chlorella variabilis NC64A, during the early phase of infection with Paramecium bursaria chlorella virus-1.
With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host-virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, like higher plants, may employ certain RNA silencing pathways to defend itself against virus infection. To our knowledge this is the first examination of RNA silencing genes in algae beyond core proteins, and the first analysis of their transcription during virus infection.


Physical performance during high-intensity resistance exercise in normoxic and hypoxic conditions.
This study aimed to determine whether different levels of hypoxia affect physical performance during high-intensity resistance exercise or subsequent cardiovascular and perceptual responses. Twelve resistance-trained young men (age, 25.3 ± 4.3 years; height, 179.0 ± 4.5 cm; body mass, 83.4 ± 9.1 kg) were tested for 1 repetition maximum (1RM) in the back squat and deadlift. Following this, participants completed 3 separate randomized trials of 5 × 5 repetitions at 80% 1RM, with 3 minutes rest between sets, in normoxia (NORM; fraction of inspired oxygen [FIO2] = 0.21), moderate-level hypoxia (FIO2 = 0.16), or high-level hypoxia (FIO2 = 0.13) by a portable hypoxic unit. Peak and mean force and power variables were monitored during exercise. Arterial oxygen saturation (SpO2), heart rate (HR), and rating of perceived exertion (RPE) were assessed immediately following each set. No differences in force or power variables were evident between conditions. Similar trends were evident in these variables across each set and across the exercise session in each condition. SpO2 was lower in hypoxic conditions than in NORM, whereas HR was higher following sets performed in hypoxia. There were no differences between conditions in RPE. These results indicate that a hypoxic stimulus during high-intensity resistance exercise does not alter physical performance during repetitions and sets or affect how strenuous exercise is perceived to be. This novel training strategy can be used without adversely affecting the physical training dose experienced and may provide benefits over the equivalent training in NORM.


Plasmon-less surface enhanced Raman spectra induced by self-organized networks of silica nanoparticles produced by femtosecond lasers.
Raman spectroscopy is the workhorse for label-free analysis of molecules. It relies on the inelastic scattering of incoming monochromatic light impinging molecules of interest. This effect leads to a very weak emission of light spectrum that provides a signature of the molecules being observed. Considerable efforts have been made over the last decades, in particular with the development of Surface Enhanced Raman Spectroscopy (SERS), to enhance the intensity of the emitted signal so that ultimately, traces of molecules can be detected. Here, we show that dense self-organized networks of quasi-monodisperse nanoparticles redepositing during femtosecond laser ablation of trenches in fused silica can lead to a significant field enhancement effect, enabling the Raman detection of a single-molecule layer deposited on the surface (so called monolayer). Unlike previously reported for SERS experiments, here, there is no metal layer promoting plasmonics effects causing localized field enhancement. The method for producing SERS substrates is therefore quite straightforward and low cost.


Asthma as a risk factor for COPD in a longitudinal study.
For several years, asthma and COPD have been regarded as distinct entities, with distinct clinical courses. However, despite distinctive physiologic features at the time of diagnosis, and different risk factors, the two diseases over time may develop features that are quite similar. To evaluate the association between physician-diagnosed asthma and the subsequent development of COPD in a cohort of 3,099 adult subjects from Tucson, AZ. A prospective observational study. Participants completed up to 12 standard respiratory questionnaires and 11 spirometry lung function measurements over a period of 20 years. Survival curves (with time to development of COPD as the dependent variable) were compared between subjects with asthma and subjects without asthma at the initial survey. Subjects with active asthma (n = 192) had significantly higher hazard ratios than inactive (n = 156) or nonasthmatic subjects (n = 2751) for acquiring COPD. As compared with nonasthmatics, active asthmatics had a 10-times-higher risk for acquiring symptoms of chronic bronchitis (95% confidence interval [CI], 4.94 to 20.25), 17-times-higher risk of receiving a diagnosis of emphysema (95% CI, 8.31 to 34.83), and 12.5-times-higher risk of fulfilling COPD criteria (95% CI, 6.84 to 22.84), even after adjusting for smoking history and other potential confounders. Physician-diagnosed asthma is significantly associated with an increased risk for CB, emphysema, and COPD.


F-Actin-Driven CD28-CD80 Localization in the Immune Synapse.
During immunological synapse (IS) formation, T cell receptor (TCR) signaling complexes, integrins, and costimulatory molecules exhibit a particular spatial localization. Here, we develop an agent-based model for the IS formation based on TCR peptide-bound major histocompatibility complex (pMHC) and leukocyte-function-associated antigen 1 (LFA-1) intracellular activation molecule 1 (ICAM-1) dynamics, including CD28 binding to a costimulatory ligand, coupling of molecules to the centripetal actin flow, and size-based segregation (SBS). A radial gradient of LFA-1 in the peripheral supramolecular activation cluster (pSMAC) toward the central supramolecular activation cluster (cSMAC) emerged as a combined consequence of actin binding and diffusion and modified the positioning of other molecules. The simulations predict a mechanism of CD28 movement, according to which CD28-CD80 complexes passively follow TCR-pMHC microclusters. However, the characteristic CD28-CD80 localization in a ring pattern around the cSMAC only emerges with a particular CD28-actin coupling strength that induces a centripetal motion. These results have implications for the understanding of T cell activation and fate decisions.


Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.
Hepatitis E virus (HEV) causes 14 million infections and 60,000 deaths per year globally, with immunocompromised persons and pregnant women experiencing severe symptoms. Although ribavirin can be used to treat chronic hepatitis E, toxicity in pregnant patients and the emergence of resistant strains are major concerns. Therefore there is an imminent need for effective HEV antiviral agents. The aims of this study were to develop a drug screening platform and to discover novel approaches to targeting steps within the viral life cycle. We developed a screening platform for molecules inhibiting HEV replication and selected a candidate, isocotoin. Isocotoin inhibits HEV replication through interference with heat shock protein 90 (HSP90), a host factor not previously known to be involved in HEV replication. Additional work is required to understand the compound's translational potential, however this suggests that HSP90-modulating molecules, which are in clinical development as anti-cancer agents, may be promising therapies against HEV.


Tracheal intubation in traumatic brain injury: a multicentre prospective observational study.
We aimed to study the associations between pre- and in-hospital tracheal intubation and outcomes in traumatic brain injury (TBI), and whether the association varied according to injury severity. Data from the international prospective pan-European cohort study, Collaborative European NeuroTrauma Effectiveness Research for TBI (CENTER-TBI), were used (n=4509). For prehospital intubation, we excluded self-presenters. For in-hospital intubation, patients whose tracheas were intubated on-scene were excluded. The association between intubation and outcome was analysed with ordinal regression with adjustment for the International Mission for Prognosis and Analysis of Clinical Trials in TBI variables and extracranial injury. We assessed whether the effect of intubation varied by injury severity by testing the added value of an interaction term with likelihood ratio tests. In the prehospital analysis, 890/3736 (24%) patients had their tracheas intubated at scene. In the in-hospital analysis, 460/2930 (16%) patients had their tracheas intubated in the emergency department. There was no adjusted overall effect on functional outcome of prehospital intubation (odds ratio=1.01; 95% confidence interval, 0.79-1.28; P=0.96), and the adjusted overall effect of in-hospital intubation was not significant (odds ratio=0.86; 95% confidence interval, 0.65-1.13; P=0.28). However, prehospital intubation was associated with better functional outcome in patients with higher thorax and abdominal Abbreviated Injury Scale scores (P=0.009 and P=0.02, respectively), whereas in-hospital intubation was associated with better outcome in patients with lower Glasgow Coma Scale scores (P=0.01): in-hospital intubation was associated with better functional outcome in patients with Glasgow Coma Scale scores of 10 or lower. The benefits and harms of tracheal intubation should be carefully evaluated in patients with TBI to optimise benefit. This study suggests that extracranial injury should influence the decision in the prehospital setting, and level of consciousness in the in-hospital setting. NCT02210221.


F statistics in Drosophila buzzatii: selection, population size and inbreeding.
Drosophila buzzatii is confined to reproducing in a well defined patchy environment consisting of rotting cactus cladodes which are ephemeral, permitting at most three generations. Flies emerging from such rots were used to estimate the additive genetic variance within rots and the genetic variance between rots for body size and also were electrophoresed to determine their genotypes at six polymorphic loci. F statistics were estimated from body size and allozyme data. The FST derived from body size was significantly larger than the allozyme FST. It is proposed this is due to selective differentiation of body size. The allozyme FST is used to estimate effective population size: 10 < N < 50. It is suggested that the regularly observed positive FIS's could be due to partial sib mating, S. If so, the estimated lower bound is S = 0.258. Experiments are identified which could support or contradict these interpretations.


Perfectionism and sense of coherence among patients with eating disorders.
There is a substantial body of research on eating disorders and perfectionism. Also there are several studies on eating disorders and sense of coherence (SOC), but studies regarding all three subjects are sparse. Perfectionism and the degree of SOC are considered central and aggravating aspects of psychiatric conditions, not least in relation to eating disorders. The present study aimed to describe the relationship between perfectionism as operationalized by Garner in the Eating Disorder Inventory-2 and SOC as defined by Antonovsky in the SOC-29 scale. The hypothesis was that SOC should be negatively associated with perfectionism. Data from the two self-measuring instruments collected from 95 consecutively recruited eating disorder outpatients were analysed with descriptive and inferential statistics. The patients in the present study scored consistently with other Swedish eating disorder samples on the Perfectionism subscale in the Eating Disorder Inventory-2 (EDI-P) and on the SOC-29, indicating a higher degree of perfectionism and weaker SOC than normal population groups. Perfectionism was significantly correlated to SOC. The correlation was negative, confirming the study hypothesis. The hypothesis was further confirmed in a subgroup analysis comparing patients with different degrees of SOC related to their EDI-P scores. Perfectionism is associated with SOC in patients with eating disorders. The clinical implications derived from the study could be a recommendation to focus on the SOC in patients with an eating disorder with the hope of lowering the patients' perfectionism as well.


High risk of HIV in non-brothel based female sex workers in India.
Heterosexual contact is the most common mode of HIV transmission in India that is largely linked to sex work. We assessed the non-use of condoms in sex work and with regular sex partners by female sex workers (FSWs), and identified its associations that could assist in planning HIV prevention programmes. Detailed documentation of various aspects of sex work, and sexual behaviour with regular sex partners, was done through confidential interviews for 6,648 FSWs in 13 districts in the Indian state of Andhra Pradesh. Multivariate analysis was done to understand condom non-use with clients. 5,010 (75.4%), 1,499 (22.5%), and 139 (2.1%) FSWs were street-, home-, and brothel-based, respectively. Of the total 6,648 FSWs, 6,165 (92.7%) had penetrative vaginal/anal sex with at least one client in the last 15 days, and of these 2,907 (47.2%; 95% CI 41.2-53.2%) reported non-use of condom with at least one of her last three clients. Lack of knowledge that HIV could be prevented (odds ratio 5.01; 95% CI 4.38-5.73), no access to free condoms (odds ratio 3.45; 95% CI 2.99-3.98), being street-based as compared with brothel-based (odds ratio 3.36; 95% CI 1.87-6.04), and no participation in FSW support groups (odds ratio 2.02; 95% CI 1.50-2.70) were the most significant predictors of condom non-use with clients. Other associations included lower social support, lower income, age >24 years, illiteracy, and living in medium-size urban or rural areas. Of the 2582 who had penetrative sex with regular sex partner within the last 7 days, 2428 (94%; 95% CI 92.1-95.9%) had not used condom at last sex, and 1032 (41.8%) had neither used condom consistently with clients nor with regular sex partner. About half the FSWs do not use condom consistently with their clients in this Indian state putting them at high risk of HIV infection. Non-brothel-based FSWs, who form the majority of sex workers in India, were at a significantly higher risk of HIV infection as compared with brothel-based FSWs. With their high vulnerability, the success of expansion of HIV prevention efforts will depend on achieving and sustaining an environment that enables HIV prevention with the non-brothel based FSWs.


A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling.
The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.


Specialty supplements and breast cancer risk in the VITamins And Lifestyle (VITAL) Cohort.
Use of nonvitamin, nonmineral "specialty" supplements has increased substantially over recent decades. Several supplements may have anti-inflammatory or anticancer properties. Additionally, supplements taken for symptoms of menopause have been associated with reduced risk of breast cancer in two case-control studies. However, there have been no prospective studies of the association between the long-term use of these supplements and breast cancer risk. Participants were female members of the VITamins And Lifestyle (VITAL) Cohort. Postmenopausal women, ages 50 to 76 years, who were residents of western Washington State, completed a 24-page baseline questionnaire in 2000 to 2002 (n = 35,016). Participants were queried on their recency (current versus past), frequency (days/week), and duration (years) of specialty supplement use. Incident invasive breast cancers (n = 880) from 2000 to 2007 were obtained from the Surveillance, Epidemiology, and End Results registry. Multivariable-adjusted hazards ratios (HR) and 95% confidence intervals (95% CI) were estimated by Cox proportional hazards models. Current use of fish oil was associated with reduced risk of breast cancer (HR, 0.68; 95% CI, 0.50-0.92). Ten-year average use was suggestive of reduced risk (P trend = 0.09). These results held for ductal but not lobular cancers. The remaining specialty supplements were not associated with breast cancer risk: Specifically, use of supplements sometimes taken for menopausal symptoms (black cohosh, dong quai, soy, or St. John's wort) was not associated with risk. Fish oil may be inversely associated with breast cancer risk. Fish oil is a potential candidate for chemoprevention studies. Until that time, it is not recommended for individual use for breast cancer prevention.


Make Better Choices (MBC): study design of a randomized controlled trial testing optimal technology-supported change in multiple diet and physical activity risk behaviors.
Suboptimal diet and physical inactivity are prevalent, co-occurring chronic disease risk factors, yet little is known about how to maximize multiple risk behavior change. Make Better Choices, a randomized controlled trial, tests competing hypotheses about the optimal way to promote healthy change in four bundled risk behaviors: high saturated fat intake, low fruit and vegetable intake, low physical activity, and high sedentary leisure screen time. The study aim is to determine which combination of two behavior change goals--one dietary, one activity--yields greatest overall healthy lifestyle change. Adults (n = 200) with poor quality diet and sedentary lifestyle will be recruited and screened for study eligibility. Participants will be trained to record their diet and activities onto a personal data assistant, and use it to complete two weeks of baseline. Those who continue to show all four risk behaviors after baseline recording will be randomized to one of four behavior change prescriptions: 1) increase fruits and vegetables and increase physical activity, 2) decrease saturated fat and increase physical activity, 3) increase fruits and vegetable and decrease saturated fat, or 4) decrease saturated fat and decrease sedentary activity. They will use decision support feedback on the personal digital assistant and receive counseling from a coach to alter their diet and activity during a 3-week prescription period when payment is contingent upon meeting behavior change goals. They will continue recording on an intermittent schedule during a 4.5-month maintenance period when payment is not contingent upon goal attainment. The primary outcome is overall healthy lifestyle change, aggregated across all four risk behaviors. The Make Better Choices trial tests a disseminable lifestyle intervention supported by handheld technology. Findings will fill a gap in knowledge about optimal goal prescription to facilitate simultaneous diet and activity change. Results will shed light on which goal prescription maximizes healthful lifestyle change. Clinical Trials Gov. Identifier NCT00113672.


A Comparison of Three Different Warm-Ups on 800-Meter Running Performance in Elite Division I Track Athletes - A Pilot Study.
Track and Field athletes perform different types of warm-ups at varying levels of volume and/or intensity prior to competition. Theoretically, this prepares sport specific muscles for activity by increasing muscle temperature, thus mitigating the chance for injury. There is a paucity of information regarding the optimum level for warm-ups regarding maximizing performance in middle distance events. The aim of this study was to examine the effects of three different warm-ups on 800-meter performance. Thirteen Division I student-athletes (seven males and six females) from the Southeastern Conference (SEC) who were middle distance runners participated in this study. We utilized a randomized, cross-over study design to test low, medium, and high-volume warm-up protocols on 800-meter performance. Trials were conducted over a span of three weeks on a SEC University outdoor track. We used a 2 (Sex) x 3 (Warm-Up Protocol) mixed-factor ANOVA, and our results show a main effect of warm-up volume that is not moderated by sex. Post-hoc tests reveal a high-volume warm-up yields superior results for the 800-meter run in comparison to a medium volume warm-up, which provides better results than a low-volume warm-up. These findings may be of value in providing information in program design for coaches on the most effective warm-up protocols for 800-meter runners. Identifying the best warm-up protocol to prime an 800-meter runner for peak performance may not only assist in preventing injury, but enhance performance thus leading to an increased of achievement, and confidence in personal ability.


A potential link between transgene silencing and poly(A) tails.
Argonaute proteins function in gene silencing induced by double-stranded RNA (dsRNA) in various organisms. In Drosophila, the Argonaute proteins AGO1 and AGO2 have been implicated in post-transcriptional gene-silencing (PTGS)/RNA interference (RNAi). In this study, we found that AGO1 and AGO2 depletion caused the accumulation of multicopied enhanced green fluorescence protein (EGFP) transgene transcripts in Drosophila S2 cells. Depletion of AGO1, the essential factor for miRNA biogenesis, led to an increased transcriptional rate of the transgenes. In contrast, depletion of AGO2, the essential factor for siRNA-directed RNAi, resulted in EGFP mRNA stabilization with concomitant shortening of the EGFP mRNA poly(A) tail. Our findings suggest that AGO1 and AGO2 mediate multicopied transgene silencing by different mechanisms. Intriguingly, Dicer2 depletion phenocopies AGO2 depletion, with an increase in EGFP protein levels and shortening of the EGFP mRNA poly(A) tail. The possibility that AGO2 and Dicer2 involve, at least in part, poly(A) length maintenance of transgene mRNA suggests a potentially important link between transgene silencing and poly(A) tails.


Immunofluorescence Tomography of Mouse Ocular Surface Epithelial Stem Cells and Their Niche Microenvironment.
Currently, there are no definitive immunomarkers for epithelial stem cells (corneal and conjunctival) or their poorly understood niche microenvironment. The H2B-GFP/K5tTA mouse enables visualization of label-retaining cells (LRCs), which exhibit the functional marker of stem cell quiescence. We used immunofluorescence tomography to evaluate putative stem cell markers and LRCs of the mouse ocular surface. H2B-GFP/K5tTA mice were pulsed for 56 days and then chased with doxycycline to label LRCs. Limbus and eyelid tissue was 3-dimensionally (3-D) reconstructed using immunofluorescence tomography to identify and characterize LRCs using the putative stem cell markers sox9, keratin 19, lrig1, blimp1, and abcb5. After 28 days of chase, LRCs were localized to the entire limbus epithelium and, infrequently, the anterior limbal stroma. Label-retaining cells comprised 3% of limbal epithelial cells after 56 days of chase. Conjunctival LRCs were localized to the fornix and comprised 4% of the total fornix epithelial cells. No stem cell immunomarker was specific for ocular surface LRCs; however, blimp1 enriched for limbal basal epithelial cells and 100% of green fluorescent protein-positive (GFP+) cells at the limbus and fornix were found to be lrig1-positive. Label-retaining cells represent a larger population of the mouse limbus than previously thought. They decrease in number with increased doxycycline chase, suggesting that LRC populations with different cell cycle lengths exist at the limbus. We conclude that current immunomarkers are unable to colocalize with the functional marker of epithelial stem cell quiescence; however, blimp1 may enrich for limbal epithelial basal cells.


Skew t Mixture Latent State-Trait Analysis: A Monte Carlo Simulation Study on Statistical Performance.
This simulation study assessed the statistical performance of a skew t mixture latent state-trait (LST) model for the analysis of longitudinal data. The model aims to identify interpretable latent classes with class-specific LST model parameters. A skew t-distribution within classes is allowed to account for non-normal outcomes. This flexible function covers heavy tails and may reduce the risk of identifying spurious classes, e.g., in case of outliers. Sample size, number of occasions and skewness of the trait variable were varied. Generally, parameter estimation accuracy increases with increasing numbers of observations and occasions. Larger bias compared to other parameters occurs for parameters referring to the skew t-distribution and variances of the latent trait variables. Standard error estimation accuracy shows diffuse patterns across conditions and parameters. Overall model performance is acceptable for large conditions, even though none of the models is free from bias. The application of the skew t mixture model in case of large numbers of occasions and observations may be possible, but results should be treated with caution. Moreover, the skew t approach may be useful for other mixture models.


Depth sensitivity analysis of functional near-infrared spectroscopy measurement using three-dimensional Monte Carlo modelling-based magnetic resonance imaging.
Theoretical analysis of spatial distribution of near-infrared light propagation in head tissues is very important in brain function measurement, since it is impossible to measure the effective optical path length of the detected signal or the effect of optical fibre arrangement on the regions of measurement or its sensitivity. In this study a realistic head model generated from structure data from magnetic resonance imaging (MRI) was introduced into a three-dimensional Monte Carlo code and the sensitivity of functional near-infrared measurement was analysed. The effects of the distance between source and detector, and of the optical properties of the probed tissues, on the sensitivity of the optical measurement to deep layers of the adult head were investigated. The spatial sensitivity profiles of photons in the head, the so-called banana shape, and the partial mean optical path lengths in the skin-scalp and brain tissues were calculated, so that the contribution of different parts of the head to near-infrared spectroscopy signals could be examined. It was shown that the signal detected in brain function measurements was greatly affected by the heterogeneity of the head tissue and its scattering properties, particularly for the shorter interfibre distances.


Programming of cells for death under experimental conditions: relevance to the tumor problem.
An attempt was made in this study to determine more precisely the nature of the factors that are involved in the programming of cells for a form of terminal cellular differentiation that results in death. These studies demonstrated that both the cytokinesins, which are potent inhibitors of plant and animal adenosine 3':5'-cyclic monophosphate phosphodiesterases, and 8-bromoadenosine 3':5'-cyclic monophosphate, which is a stable, biologically active form of adenosine 3':5'-cyclic monophosphate, are highly effective in encouraging differentiation of parenchyma cells into tracheary elements with accompanying death. Since adenosine 3':5'-cyclic monophosphate and theophylline when used together were also effective, the results reported here suggest that adenosine 3':5'-cyclic monophosphate is somehow importantly involved in the conversion of parenchyma cells into tracheary elements in this system. The possible significance to the tumor problem generally of the programming of cells for terminal differentiation, with or without resulting death, is discussed.


Genetic diversity within and among aecia of the wheat rust fungus Puccinia striiformis on the alternate host Berberis vulgaris.
An isolate of the fungus Puccinia striiformis, causing yellow (stripe) rust on cereals and grasses, was selfed on the alternate (sexual) host, Berberis vulgaris. This enabled us to investigate genetic variability of progeny isolates within and among aecia. Nine aecial clusters each consisting of an aecium (single aecial cup) and nine clusters containing multiple aecial cups were selected from 18 B. vulgaris leaves. Aeciospores from each cluster were inoculated on susceptible wheat seedlings and 64 progeny isolates were recovered. Molecular genotyping using 37 simple sequence repeat markers confirmed the parental origin of all progeny isolates. Thirteen molecular markers, which were heterozygous in the parental isolate, were used to analyse genetic diversity within and among aecial cups. The 64 progeny isolates resulted in 22 unique recombinant multilocus genotypes and none of them were resampled in different aecial clusters. Isolates derived from a single cup were always of the same genotype whereas isolates originating from clusters containing up to nine aecial cups revealed one to three genotypes per cluster. These results implied that each aecium was the result of a successful fertilization in a corresponding pycnium and that an aecium consisted of genetically identical aeciospores probably multiplied via repetitive mitotic divisions. Furthermore, the results suggested that aecia within a cluster were the result of independent fertilization events often involving genetically different pycniospores. The application of molecular markers represented a major advance in comparison to previous studies depending on phenotypic responses on host plants. The study allowed significant conclusions about fundamental aspects of the biology and genetics of an important cereal rust fungus.


Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function.
In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.


Surgical Treatment of Cervical Spondylotic Myelopathy in the Elderly: Outcomes in Patients Aged 80 Years or Older.
Retrospective multicenter study. We aimed to assess the outcomes following posterior cervical decompression for cervical spondylotic myelopathy (CSM) in a large sample of patients older than 80 years. Although age is a predictor of surgical outcomes for CSM, most previous studies have only investigated the effect of age for patients aged 65 to 75 years, and surgical outcomes remain unclear for older patients. Patients older than 50 years who underwent posterior cervical decompression for CSM were enrolled from 17 institutions. The patients were grouped into a young-old group (<80 yrs old) and an old-old group (≥80 yrs). Clinical information, comorbidities, perioperative complications, Japanese Orthopaedic Association (JOA) scores, Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), visual analog scale (VAS) scores, and radiographic parameters were compared between the two groups. All patients were followed for at least 1 year after surgery. The young-old and old-old groups included 491 and 77 patients, respectively. The JOA score and quality of life measured by JOACMEQ improved significantly after surgery in both groups. Compared with the young-old group, the old-old group had lower preoperative JOA scores (9.6 vs. 11.0), lower final JOA scores (12.6 vs. 14.0), and lower recovery rates (42% vs. 50%). However, there were no significant differences in perioperative complications or in the VAS scores at the final assessments. The old-old group had a higher mean preoperative sagittal Cobb angle at C2-C5, though this was not statistically significant postoperatively. Posterior decompression surgery is safe and beneficial for patients older than 80 years with CSM, despite having a more limited neurological recovery compared with younger patients. 3.


Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.
One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.


Repetition of suicide attempts: data from emergency care settings in five culturally different low- and middle-income countries participating in the WHO SUPRE-MISS Study.
Attempted suicide is a strong risk factor for subsequent suicidal behaviors. Innovative strategies to deal with people who have attempted suicide are needed, particularly in resource-poor settings. To evaluate a brief educational intervention and periodic follow-up contacts (BIC) for suicide attempters in five culturally different sites (Campinas, Brazil; Chennai, India; Colombo, Sri Lanka; Karaj, Islamic Republic of Iran; and Yuncheng, People's Republic of China) as part of the WHO Multisite Intervention Study on Suicidal Behaviors (SUPRE-MISS). Among the 1,867 suicide attempters enrolled in the emergency departments of the participating sites, 922 (49.4%) were randomly assigned to a brief intervention and contact (BIC) group and 945 (50.6%) to a treatment as usual (TAU) group. Repeated suicide attempts over the 18 months following the index attempt - the secondary outcome measure presented in this paper - were identified by follow-up calls or visits. Subsequent completed suicide - the primary outcome measure - has been reported in a previous paper. Overall, the proportion of subjects with repeated suicide attempts was similar in the BIC and TAU groups (7.6% vs. 7.5%, chi(2) = 0.013; p = .909), but there were differences in rates across the five sites. This study from five low- and middle-income countries does not confirm the effectiveness of brief educational intervention and follow-up contacts for suicide attempters in reducing subsequent repetition of suicide attempts up to 18 months after discharge from emergency departments.


LfrR is a repressor that regulates expression of the efflux pump LfrA in Mycobacterium smegmatis.
The lfrA gene of Mycobacterium smegmatis encodes an efflux pump which mediates resistance to different fluoroquinolones, cationic dyes, and anthracyclines. The deletion of the lfrR gene, coding for a putative repressor and localized upstream of lfrA, increased the lfrA expression. In this study, reverse transcription-PCR experiments showed that the two genes are organized as an operon, and lacZ reporter fusions were used to identify the lfrRA promoter region. The lfrRA promoter assignment was verified by mapping the transcription start site by primer extension. Furthermore, we found that some substrates of the multidrug transporter LfrA, e.g., acriflavine, ethidium bromide, and rhodamine 123, enhance lfrA expression at a detectable level of transcription. LfrR protein was purified from Escherichia coli as a fusion protein with a hexahistidine tag and found to bind specifically to a fragment 143 bp upstream of lfrR by gel shift analysis. Furthermore, acriflavine was able to cause the dissociation of the LfrR from the promoter, thus suggesting that this molecule interacts directly with LfrR, inducing lfrA expression. These results suggest that the LfrR repressor is able to bind to different compounds, which allows induction of LfrA multidrug efflux pump expression in response to these ones. Together, all data suggest that the LfrA pump is tightly regulated and that the repression and induction can be switched about a critical substrate concentration which is toxic for the cell.


Does extracorporeal shock wave lithotripsy-related pain get affected by menstrual cycle and menopause?
The objective of the study was to investigate the effect of the menstrual cycle and menopause on extracorporeal shock wave lithotripsy (ESWL)-related pain outcome. Since March 2017, we evaluated a total of 145 women who underwent the first session of ESWL for renal or ureteral stones. Patients were divided into two groups, as menstruating and menopaused women. For menstruating women, the number of days between the last day of mens and ESWL was noted and women were separated as in the follicular phase (1-14 days) or in the luteal phase (15-30 days) of menstruation. To control these two female groups, 149 men of similar age were included in the study. After the procedure, the experienced pain was recorded on a ten-point visual analog scale (VAS) by the patient and they also rated the severity of pain as no, mild, tolerable, and intolerable on the pain questionnaire. The mean age of the patients was 43 ± 15 years for the female group and 42 ± 13 years for the male group. While stone burden was not different between the female and male groups (p = 0.459), VAS score was not statistically different between genders (p = 0.293). However, men reported a higher rate of mild pain, while women reported a higher rate of tolerable pain (p = 0.008) in the pain questionnaire. Mean VAS score was significantly lower for the menopaused women group than menstruating women, young and old men (p = 0.001). In a subgroup analysis, menopaused women group reported lower VAS score and better pain questionnaire result than menstruating women (p < 0.001). There was no statistically significant difference between the follicular and luteal phase of the menstrual cycle in terms of mean VAS score and pain questionnaire results (p = 0.891 and 0.441, respectively). When compared with the young men group, the only significant difference was pain questionnaire results between women in the luteal phase (p = 0.014). Multiple regression analysis showed that only menstruation (β = 0.639, p < 0.001) was an independent factor for VAS score. Menstrual cycle phase had no effect on pain perception during the ESWL session and menopaused women felt less pain than menstruating women during this procedure. The control male group showed that the reduction of ESWL-related pain in menopause was not related to aging.


Focal transnasal approach to the upper, middle, and lower clivus.
Carefully tailoring the transclival approach to the involved parts of the upper, middle, or lower clivus requires a precise understanding of the focal relationships of the clivus. To develop an optimal classification of the upper, middle, and lower clivus and to define the extra and intracranial relationships of each clival level. Ten cadaveric heads and 10 dry skulls were dissected using the surgical microscope and endoscope. The clivus is divided into upper, middle, and lower thirds by 2 endocranial landmarks: the dural pori of the abducens nerves and the dural meati of the glossopharyngeal nerves. Useful surgical landmarks exposed in the transnasal approach that aid in locating the junction of the clival divisions are the lower limit of the paraclival segment of the internal carotid artery, which is located 4.9 mm above the posterior opening of the vidian canal, and the pharyngeal tubercle. The upper, middle, and lower clival approaches provide access to the anterior midline parts of the previously described upper, middle, and lower neurovascular complexes in the posterior fossa. The nasal and nasopharyngeal relationships important in expanding the transnasal approach to the borders of the clivus are reviewed. The transclival approach can be carefully tailored to expose focal lesions in the anterior part of the posterior fossa.