Cost effectiveness and resource allocation of Plasmodium falciparum malaria control in Myanmar: a modelling analysis of bed nets and community health workers.
Funding for malaria control and elimination in Myanmar has increased markedly in recent years. While there are various malaria control tools currently available, two interventions receive the majority of malaria control funding in Myanmar: (1) insecticide-treated bed nets and (2) early diagnosis and treatment through malaria community health workers. This study aims to provide practical recommendations on how to maximize impact from investment in these interventions. A simple decision tree is used to model intervention costs and effects in terms of years of life lost. The evaluation is from the perspective of the service provider and costs and effects are calculated in line with standard methodology. Sensitivity and scenario analysis are undertaken to identify key drivers of cost effectiveness. Standard cost effectiveness analysis is then extended via a spatially explicit resource allocation model. Community health workers have the potential for high impact on malaria, particularly where there are few alternatives to access malaria treatment, but are relatively costly. Insecticide-treated bed nets are comparatively inexpensive and modestly effective in Myanmar, representing a low risk but modest return intervention. Unlike some healthcare interventions, bed nets and community health workers are not mutually exclusive nor are they necessarily at their most efficient when universally applied. Modelled resource allocation scenarios highlight that in this case there is no "one size fits all" cost effectiveness result. Health gains will be maximized by effective targeting of both interventions.


Epstein-Barr Virus-Negative Diffuse Large B-Cell Post-transplant Lymphoma in an Epstein-Barr Virus-Positive Recipient.
Post-transplant lymphoproliferative disease (PTLD) can arise as a complication after solid organ transplantation. Epstein-Barr virus (EBV) infection is a known risk factor and is known to drive disease manifestation. PTLD can occur in EBV-negative recipients and with EBV-negative donor organs, however, EBV-negative PTLD pathogenesis is unknown. Here, we present PTLD presenting as intussusception in a patient with a historic simultaneous pancreas-kidney transplant (SPK). This case study presents the first documented case of EBV-negative post-transplant lymphoproliferative disease in an EBV-seropositive SPK recipient from an EBV-positive donor. Here we describe the diagnosis and management of this patient, discuss the differences between EBV positive and negative driven post-transplant lymphoproliferative disease, and highlight areas of research opportunity in the latter.


Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase.
The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU), which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, are unstable in solution and decay nonstereospecifically to allantoin. In addition, nonenzymatic racemization of allantoin has been shown to occur at physiological pH. Since the further breakdown of allantoin is catalyzed by allantoinase, an enzyme that is specific for (S)-allantoin, an allantoin racemase is necessary for complete and efficient catabolism of uric acid. In this work, we characterize the structure and activity of allantoin racemase from Klebsiella pneumoniae (KpHpxA). In addition to an unliganded structure solved using selenomethionyl single-wavelength anomalous dispersion, structures of C79S/C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are presented. These structures reveal several important features of the active site including an oxyanion hole and a polar binding pocket that interacts with the ureido tail of allantoin and serves to control the orientation of the hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-enantiomers of allantoin is demonstrated, and analysis of the steady-state kinetics of KpHpxA yielded a k(cat)/K(m) of 6.0 × 10(5) M(-1) s(-1). Mutation of either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this enzyme. The data presented provide new insights into the activity and substrate specificity of this enzyme and enable us to propose a mechanism for catalysis that is consistent with the two-base mechanism observed in other members of the aspartate/glutamate family.


Steroid hormone receptors and their regulation by phosphorylation.
The steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors encompasses not only the receptors for steroids, thyroid hormone, retinoids and vitamin D, but also a large number of proteins whose functions and/or ligands are unknown and which are thus termed orphan receptors. Recent studies have highlighted the importance of phosphorylation in receptor function. Although most of the phosphorylation sites are serine and threonine residues, a few of the family members are also phosphorylated on tyrosine. Those steroid receptor family members that are bound to heat-shock proteins in the absence of ligand typically are basally phosphorylated and exhibit increases in phosphorylation upon ligand binding. Most of these sites contain Ser-Pro motifs, and there is evidence that cyclin-dependent kinases and MAP kinases (mitogen-activated protein kinases) phosphorylate subsets of these sites. In contrast, phosphorylation sites identified thus far in members of the family that bind to DNA in the absence of hormone typically do not contain Ser-Pro motifs and are frequently casein kinase II or protein kinase A sites. Phosphorylation has been implicated in DNA binding, transcriptional activation and stability of the receptors. The finding that some of the steroid receptor family members can be activated in the absence of ligand by growth factors or neurotransmitters that modulate kinase and/or phosphatase pathways underscores the role of phosphorylation in receptor function. Hence this family of transcription factors integrates signals from ligands as well as from signal transduction pathways, resulting in alterations in mRNA and protein expression that are unique to the complex signals received.


Genetic susceptibility to dental implant failure: a critical review.
The observation that clinical factors alone do not explain why some patients develop implant loss; the understanding of the osseointegrated implant failure as a complex, multifactorial process; and the observed aggregation of repetitive failure in certain individuals raise interesting questions related to host susceptibility to dental implant failure. Genetic analysis applied to dental implants began in the late 1990s, and since then, increased interest in genetic susceptibility to the phenotype has been demonstrated by several studies. These studies, however, have been based on and limited to candidate gene association analysis and were intended to find associations between specific alleles and/or genotypes of genetic markers and susceptibility to implant failure. The aim of this review is to provide a brief description of the current methodology for genetic analysis of complex traits, followed by a comprehensive review of the literature related to genetic susceptibility to dental implant failure and a discussion of different aspects of the applied methodology. Moreover, a novel approach of genome wide, case-control analysis is discussed as an alternative method to access genetic influence to dental implant failure mechanisms. Advances toward the elucidation of the genetic basis of dental implant loss may contribute to the understanding of why some patients do not respond to currently available treatments while others do and provide potential targets for effective screening, prevention, and treatment. For example, clinicians might be able to estimate, before the elective surgical procedure, the risk of a given patient to develop a negative individual host response.


Acceptance or decline of requests to review manuscripts: A gender-based approach from a public health journal.
Peer review in the scientific publication is widely used as a method to identify valuable knowledge. Editors have the task of selecting appropriate reviewers. We assessed the reasons given by potential reviewers for declining a request to review, and the factors associated with acceptance, taking into account the difference in the sex of the reviewer. This is a descriptive study of the review requests from a public health journal (Gaceta Sanitaria) with an enforced gender policy. The dependent variables were requests, response to requests, reasons potential reviewers gave for declining requests and time to review. We carried out a descriptive analysis of these indicators and applied logistic regression to analyze factors (professional and research/review experience) associated with having done at least one review in 2014-2015. Results were stratified by sex. Journal editors sent 1,775 requests to 773 potential reviewers; 52.3% of whom reviewed at least one manuscript. Of the 396 declined requests (22.3%), the most common reasons were lack of time and of experience (88.1%). No differences were observed by sex. In the multivariate analysis, having reviewed for the journal in previous years showed the strongest association with acceptance. Specific analyses of data on requests reviewers may be useful for improving the acceptance rates to review. This study did not show gender differences in several indicators of the reviewing process.


Sources of bias in artificial intelligence that perpetuate healthcare disparities-A global review.
While artificial intelligence (AI) offers possibilities of advanced clinical prediction and decision-making in healthcare, models trained on relatively homogeneous datasets, and populations poorly-representative of underlying diversity, limits generalisability and risks biased AI-based decisions. Here, we describe the landscape of AI in clinical medicine to delineate population and data-source disparities. We performed a scoping review of clinical papers published in PubMed in 2019 using AI techniques. We assessed differences in dataset country source, clinical specialty, and author nationality, sex, and expertise. A manually tagged subsample of PubMed articles was used to train a model, leveraging transfer-learning techniques (building upon an existing BioBERT model) to predict eligibility for inclusion (original, human, clinical AI literature). Of all eligible articles, database country source and clinical specialty were manually labelled. A BioBERT-based model predicted first/last author expertise. Author nationality was determined using corresponding affiliated institution information using Entrez Direct. And first/last author sex was evaluated using the Gendarize.io API. Our search yielded 30,576 articles, of which 7,314 (23.9%) were eligible for further analysis. Most databases came from the US (40.8%) and China (13.7%). Radiology was the most represented clinical specialty (40.4%), followed by pathology (9.1%). Authors were primarily from either China (24.0%) or the US (18.4%). First and last authors were predominately data experts (i.e., statisticians) (59.6% and 53.9% respectively) rather than clinicians. And the majority of first/last authors were male (74.1%). U.S. and Chinese datasets and authors were disproportionately overrepresented in clinical AI, and almost all of the top 10 databases and author nationalities were from high income countries (HICs). AI techniques were most commonly employed for image-rich specialties, and authors were predominantly male, with non-clinical backgrounds. Development of technological infrastructure in data-poor regions, and diligence in external validation and model re-calibration prior to clinical implementation in the short-term, are crucial in ensuring clinical AI is meaningful for broader populations, and to avoid perpetuating global health inequity.


Responses of water accumulation and solute metabolism in tomato fruit to water scarcity and implications for main fruit quality variables.
Fruit is important for human health, and applying deficit irrigation in fruit production is a strategy to regulate fruit quality and support environmental sustainability. Responses of different fruit quality variables to deficit irrigation have been widely documented, and much progress has been made in understanding the mechanisms of these responses. We review the effects of water shortage on fruit water accumulation considering water transport from the parent plant into the fruit determined by hydraulic properties of the pathway (including xylem water transport and transmembrane water transport regulated by aquaporins) and the driving force for water movement. We discuss water relations and solute metabolism that affect the main fruit quality variables (e.g. size, flavour, nutrition, and firmness) at the cellular level under water shortage. We also summarize the most recent advances in the understanding of responses of the main fruit quality variables to water shortage, considering the effects of variety, the severity of water deficit imposed, and the developmental stage of the fruit. We finally identify knowledge gaps and suggest avenues for future research. This review provides new insights into the stress physiology of fleshy fruit, which will be beneficial for the sustainable production of high-quality fruit under deficit irrigation.


Experiences of the first 16 hospitals using copper-silver ionization for Legionella control: implications for the evaluation of other disinfection modalities.
Hospital-acquired legionnaires' disease can be prevented by disinfection of hospital water systems. This study assessed the long-term efficacy of copper-silver ionization as a disinfection method in controlling Legionella in hospital water systems and reducing the incidence of hospital-acquired legionnaires' disease. A standardized, evidence-based approach to assist hospitals with decision making concerning the possible purchase of a disinfection system is presented. The first 16 hospitals to install copper-silver ionization systems for Legionella disinfection were surveyed. Surveys conducted in 1995 and 2000 documented the experiences of the hospitals with maintenance of the system, contamination of water with Legionella, and occurrence of hospital-acquired legionnaires' disease. All were acute care hospitals with a mean of 435 beds. All 16 hospitals reported cases of hospital-acquired legionnaires' disease prior to installing the copper-silver ionization system. Seventy-five percent had previously attempted other disinfection methods including superheat and flush, ultraviolet light, and hyperchlorination. By 2000, the ionization systems had been operational from 5 to 11 years. Prior to installation, 47% of the hospitals reported that more than 30% of distal water sites yielded Legionella. In 1995, after installation, 50% of the hospitals reported 0% positivity, and 43% still reported 0% in 2000. Moreover, no cases of hospital-acquired legionnaires' disease have occurred in any hospital since 1995. This study represents the final step in a proposed 4-step evaluation process of disinfection systems that includes (1) demonstrated efficacy of Legionella eradication in vitro using laboratory assays, (2) anecdotal experiences in preventing legionnaires' disease in individual hospitals, (3) controlled studies in individual hospitals, and (4) validation in confirmatory reports from multiple hospitals during a prolonged time (5 to 11 years in this study). Copper-silver ionization is now the only disinfection modality to have fulfilled all four evaluation criteria.


Multigenerational: Households and the School Readiness of Children Born to Unmarried Mothers.
Following the ongoing increase in nonmarital fertility, policy makers have looked for ways to limit the disadvantages faced by children of unmarried mothers. Recent initiatives included marriage promotion and welfare-to-work programs. Yet policy might also consider the promotion of three generational households. We know little about whether multigenerational households benefit children of unwed mothers, although they are mandated for unmarried teen mothers applying for welfare benefits. Multigenerational households are also becoming increasingly common. Thus, using data from the Panel Study of Income Dynamics (N = 217), this study examines whether grandparent-headed coresidential households benefit preschool-aged children's school readiness, employing propensity score techniques to account for selection into these households. Findings reveal living with a grandparent is not associated with child outcomes for families that select into such arrangements but is positively associated with reading scores and behavior problems for families with a low propensity to coreside. The implications of these findings for policy are discussed.


Single-dose gadolinium with magnetization transfer versus triple-dose gadolinium in the MR detection of multiple sclerosis lesions.
To compare the efficacy of single-dose gadolinium with magnetization transfer contrast (MTC) with that of triple-dose gadolinium in detecting enhancing multiple sclerosis lesions. Twenty-one patients with multiple sclerosis were examined with MR imaging first with 0.1 mmol/kg gadolinium (single dose) and then, after 24 to 72 hours, with 0.3 mmol/kg gadolinium (triple dose). T2-weighted fast spin-echo and T1-weighted spin-echo MR images with and without MTC were obtained before contrast administration followed by either T1-weighted spin-echo images with MTC (single dose) or conventional T1-weighted spin-echo images (triple dose), starting 5, 17, and 29 minutes after contrast administration. All images were evaluated in a blinded fashion and scored in random order by two readers. Outcome parameters included number of enhancing lesions, number of active MR examinations (those containing at least one enhancing lesion), contrast ratio (signal intensity of enhancing lesion divided by signal intensity of normal-appearing white matter), and size of enhancing lesions. Eighty-one percent more enhancing lesions and 49% more active MR examinations were detected when a triple dose of gadolinium was used as compared with a single dose. The level of agreement between readers as to the number of enhancing lesions was significantly higher for triple-dose than for single-dose gadolinium. With triple-dose gadolinium, contrast ratios and areas of enhancement increased by 10% and 33%, respectively. Delayed imaging increased the size of the lesion by 11% on single-dose MTC images and by 18% on triple-dose images. Triple-dose gadolinium is more effective (higher sensitivity and interobserver agreement) than single-dose gadolinium in combination with MTC in detecting enhancing multiple sclerosis lesions.


Testing Immune-Related Adverse Events in Cancer Immunotherapy.
Immune-targeted therapeutics are being used in cancer. Immune "checkpoint inhibition" provides promise for prolonged disease-free patient survival. Use of immune checkpoint inhibitors in cancer has coincided with the onset of immune-related adverse events (irAEs). irAEs are caused by a break in host self-tolerance, which can be deadly. Acute management of irAEs is complicated by difficulty making a prompt clinical diagnosis. The goal is to maximize anticancer benefit while minimizing irAE risk. We currently lack diagnostic tools to assess pretreatment irAE risk and facilitate diagnosis. Current immunologic understanding of irAEs is discussed with an emphasis on how patients with congenital syndromes of T-cell activation may inform this understanding. The prospects of improving diagnostics for and treatment of irAEs are discussed.


An investigation of Rubens flame tube resonances.
The Rubens flame tube is a century-old teaching demonstration that allows observers to visualize acoustic standing wave behavior [H. Rubens and O. Krigar-Menzel, (1905). Ann. Phys. 17, 149-164]. Flammable gas inside the tube flows through holes drilled along the top, and flames are then lit above. The tube is closed at one end and driven with a loudspeaker at the other end. When the tube is driven at one of its resonance frequencies, flames form a visual standing wave pattern as they vary in height according to the pressure amplitude in the tube. Although the basic performance of the tube has been explained [G. Ficken and C. Stephenson, (1979). Phys. Teach. 17, 306-310], this paper discusses a previously unreported characteristic of the tube: a shift of the tube's resonance frequencies away from those predicted by simple introductory physics. Results from an equivalent circuit model of the tube and agreement between experiments and the model suggest that the shift is caused by the presence of the holes. For teachers and educators seeking to better understand and explain the tube to students, this article serves as a resource regarding the basic phenomena affecting the behavior of the tube.


Cancer-induced immunosuppression: IL-18-elicited immunoablative NK cells.
During cancer development, a number of regulatory cell subsets and immunosuppressive cytokines subvert adaptive immune responses. Although it has been shown that tumor-derived interleukin (IL)-18 participates in the PD-1-dependent tumor progression in NK cell-controlled cancers, the mechanistic cues underlying this immunosuppression remain unknown. Here, we show that IL-18 converts a subset of Kit(-) (CD11b(-)) into Kit(+) natural killer (NK) cells, which accumulate in all lymphoid organs of tumor bearers and mediate immunoablative functions. Kit(+) NK cells overexpressed B7-H1/PD-L1, a ligand for PD-1. The adoptive transfer of Kit(+) NK cells promoted tumor growth in two pulmonary metastases tumor models and significantly reduced the dendritic and NK cell pools residing in lymphoid organs in a B7-H1-dependent manner. Neutralization of IL-18 by RNA interference in tumors or systemically by IL-18-binding protein dramatically reduced the accumulation of Kit(+)CD11b(-) NK cells in tumor bearers. Together, our findings show that IL-18 produced by tumor cells elicits Kit(+)CD11b(-) NK cells endowed with B7-H1-dependent immunoablative functions in mice.


An awareness-raising E-learning approach for children living in a high diabetic population.
Recent epidemiological studies have shown an increased incidence of diabetes worldwide. In United Arab Emirates (UAE), where this study on Emirati children was conducted, over 20% of the population has diabetes. With such a high incidence, there is a need to raise the awareness of the disease in order to reduce the growing number of cases and manage the disease more effectively. This research aims to identify the needs of affected children and proposes a design for an E-learning prototype that can pedagogically raise their awareness and knowledge of the disease. The use of a prototype was chosen in order to validate and refine the usability of the system, and to quickly evaluate user-interface designs without the need for an expensive working model to help refine and develop the system design. The system requirements were identified through a set of interviews with kindergarten teachers, curriculum design experts in UAE, and diabetes nutrition specialists and clinicians. The rationale behind the interview was to identify the optimal age group, describe the appropriate level of the instructional materials and activities, and propose a suitable learning approach that could facilitate and improve diabetes awareness among this age group. The prototype was evaluated by children, teachers, parents (or guardians) and nutrition specialists. We followed a three-stage software development based on a user-informed approach model for stepwise refinement that ranged from prototype to final design. The evaluation results indicate that the proposed computer-supported learning approach can generate positive learning and behavior in children while reducing the time needed to complete awareness tasks when compared to traditional methods; thus making learning more engaging and allowing children to learn at their own pace.


Reliever-Triggered Inhaled Glucocorticoid in Black and Latinx Adults with Asthma.
Black and Latinx patients bear a disproportionate burden of asthma. Efforts to reduce the disproportionate morbidity have been mostly unsuccessful, and guideline recommendations have not been based on studies in these populations. In this pragmatic, open-label trial, we randomly assigned Black and Latinx adults with moderate-to-severe asthma to use a patient-activated, reliever-triggered inhaled glucocorticoid strategy (beclomethasone dipropionate, 80 μg) plus usual care (intervention) or to continue usual care. Participants had one instructional visit followed by 15 monthly questionnaires. The primary end point was the annualized rate of severe asthma exacerbations. Secondary end points included monthly asthma control as measured with the Asthma Control Test (ACT; range, 5 [poor] to 25 [complete control]), quality of life as measured with the Asthma Symptom Utility Index (ASUI; range, 0 to 1, with lower scores indicating greater impairment), and participant-reported missed days of work, school, or usual activities. Safety was also assessed. Of 1201 adults (603 Black and 598 Latinx), 600 were assigned to the intervention group and 601 to the usual-care group. The annualized rate of severe asthma exacerbations was 0.69 (95% confidence interval [CI], 0.61 to 0.78) in the intervention group and 0.82 (95% CI, 0.73 to 0.92) in the usual-care group (hazard ratio, 0.85; 95% CI, 0.72 to 0.999; P = 0.048). ACT scores increased by 3.4 points (95% CI, 3.1 to 3.6) in the intervention group and by 2.5 points (95% CI, 2.3 to 2.8) in the usual-care group (difference, 0.9; 95% CI, 0.5 to 1.2); ASUI scores increased by 0.12 points (95% CI, 0.11 to 0.13) and 0.08 points (95% CI, 0.07 to 0.09), respectively (difference, 0.04; 95% CI, 0.02 to 0.05). The annualized rate of missed days was 13.4 in the intervention group and 16.8 in the usual-care group (rate ratio, 0.80; 95% CI, 0.67 to 0.95). Serious adverse events occurred in 12.2% of the participants, with an even distribution between the groups. Among Black and Latinx adults with moderate-to-severe asthma, provision of an inhaled glucocorticoid and one-time instruction on its use, added to usual care, led to a lower rate of severe asthma exacerbations. (Funded by the Patient-Centered Outcomes Research Institute and others; PREPARE ClinicalTrials.gov number, NCT02995733.).


Survey on intraoperative temperature management in Europe.
Inadvertent perioperative hypothermia causes serious morbidity in surgical patients. However, recent reports suggest that patients might still be hypothermic after elective surgery. We thus surveyed intraoperative temperature monitoring and management practices in Europe. Postal survey of 801 representative hospitals from 17 European countries on the same day. The questions addressed the number of surgical procedures and type of anaesthesia performed, mode and site of temperature monitoring and method of patient warming. Mean and standard error of the mean or count and percentage were calculated. The t-test or contingency table analysis with the Fisher's exact test were used. Eight thousand and eighty-three surgical procedures were assessed from 316 responding hospitals (39.4%). Overall, patient temperature monitored in 19.4% and 38.5% of the patients were actively warmed. Under general anaesthesia, body temperature was monitored in 25% and during regional anaesthesia in 6%, P = 0.0005. Nasopharyngeal temperature was most often taken under general anaesthesia, while tympanic temperature was preferred during regional anaesthesia. Under general anaesthesia, 43% of patients were actively warmed as compared to 28% with regional anaesthesia, P = 0.0005. Forced-air warming was the method of choice for both general and regional anaesthesia. Intraoperative temperature monitoring is still uncommon and hence active patient warming is not a standard of care in Europe. Awareness of perioperative hypothermia is critical to its prevention, and thus temperature monitoring is a pre-requisite. The objective is to maintain normothermia in patients throughout surgery. A European practice guideline for perioperative patient temperature management is warranted.


Real time displacement measurement in sinusoidal phase modulating interferometry.
A new signal processing system for real time displacement measurement in sinusoidal phase modulating interferometry is described. Although sinusoidal phase modulating interferometry is effective in measuring with high accuracy the displacement of an object, conventional signal processing takes a long time. In this method, detection of the object's displacement is easily achieved by sampling the interference signal at those times that satisfy certain conditions and by processing the sampled signals with electric circuits in real time. The delay time of this signal processing system is <45 micros.


Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo.
Anthrax toxin, a major virulence factor of Bacillus anthracis, gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8- and CMG2-null mice and TEM8/CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.


Old Cryptococcus neoformans cells contribute to virulence in chronic cryptococcosis.
Does cell age matter in virulence? The emergence of persister cells during chronic infections is critical for persistence of infection, but little is known how this occurs. Here, we demonstrate for the first time that the replicative age of the fungal pathogen Cryptococcus neoformans contributes to persistence during chronic meningoencephalitis. Generationally older C. neoformans cells are more resistant to hydrogen peroxide stress, macrophage intracellular killing, and antifungal agents. Older cells accumulate in both experimental rat infection and in human cryptococcosis. Mathematical modeling supports the concept that the presence of older C. neoformans cells emerges from in vivo selection pressures. We propose that advanced replicative aging is a new unanticipated virulence trait that emerges during chronic fungal infection and facilitates persistence. Therapeutic interventions that target old cells could help in the clearance of chronic infections. Our findings that the generational age of Cryptococcus neoformans cells matters in pathogenesis introduces a novel concept to eukaryotic pathogenesis research. We propose that emerging properties of aging C. neoformans cells and possibly also other fungal pathogens contribute to persistence and virulence. Whereas the replicative life span of strains may not matter for virulence per se, age-related resilience and thus the generational age of individual C. neoformans cells within a pathogen population could greatly affect persistence of the pathogen population and therefore impact outcome.


Molecular evolution of immune genes in the malaria mosquito Anopheles gambiae.
As pathogens that circumvent the host immune response are favoured by selection, so are host alleles that reduce parasite load. Such evolutionary processes leave their signature on the genes involved. Deciphering modes of selection operating on immune genes might reveal the nature of host-pathogen interactions and factors that govern susceptibility in host populations. Such understanding would have important public health implications. We analyzed polymorphisms in four mosquito immune genes (SP14D1, GNBP, defensin, and gambicin) to decipher selection effects, presumably mediated by pathogens. Using samples of Anopheles arabiensis, An. quadriannulatus and four An. gambiae populations, as well as published sequences from other Culicidae, we contrasted patterns of polymorphisms between different functional units of the same gene within and between populations. Our results revealed selection signatures operating on different time scales. At the most recent time scale, within-population diversity revealed purifying selection. Between populations and between species variation revealed reduced differentiation (GNBP and gambicin) at coding vs. noncoding- regions, consistent with balancing selection. McDonald-Kreitman tests between An. quadriannulatus and both sibling species revealed higher fixation rate of synonymous than nonsynonymous substitutions (GNBP) in accordance with frequency dependent balancing selection. At the longest time scale (>100 my), PAML analysis using distant Culicid taxa revealed positive selection at one codon in gambicin. Patterns of genetic variation were independent of exposure to human pathogens. Purifying selection is the most common form of selection operating on immune genes as it was detected on a contemporary time scale on all genes. Selection for "hypervariability" was not detected, but negative balancing selection, detected at a recent evolutionary time scale between sibling species may be rather common. Detection of positive selection at the deepest evolutionary time scale suggests that it occurs infrequently, possibly in association with speciation events. Our results provided no evidence to support the hypothesis that selection was mediated by pathogens that are transmitted to humans.


Carisoprodol, meprobamate, and driving impairment.
This paper considers the pharmacology of the centrally acting muscle relaxant carisoprodol, and its metabolite meprobamate, which is also administered as an anxiolytic in its own right. Literature implicating these drugs in impaired driving is also reviewed. A series of 104 incidents in which these drugs were detected in the blood of drivers involved in accidents or arrested for impaired driving was considered, with respect to the analytical toxicology results, patterns of drug use in these subjects, the driving behaviors exhibited, and the symptoms observed in the drivers. Symptomatology and driving impairment were consistent with other CNS depressants, most notably alcohol. Reported driving behaviors included erratic lane travel, weaving, driving slowly, swerving, stopping in traffic, and hitting parked cars and other stationary objects. Drivers on contact by the police displayed poor balance and coordination, horizontal gaze nystagmus, bloodshot eyes, unsteadiness, slurred speech, slow responses, tendency to doze off or fall asleep, difficulty standing, walking or exiting their vehicles, and disorientation. Many of these cases had alcohol or other centrally acting drugs present also, making difficult the attribution of the documented impairment specifically to carisoprodol and meprobamate. In 21 cases, however, no other drugs were detected, and similar symptoms were present. Impairment appeared to be possible at any concentration of these two drugs; however, the most severe driving impairment and most overt symptoms of intoxication were noted when the combined concentration exceeded 10 mg/L, a level still within the normal therapeutic range.


A novel type of regulatory element is required for promoter-specific activity of the PDGF-B intronic enhancer region.
We have previously described a non-classical, promoter-specific enhancer for the human Platelet-Derived Growth Factor B (PDGF-B) gene. In JEG-3 choriocarcinoma cells the activity of the enhancer depends upon co-operation with a sequence (the Enhancer-Dependent cis Co-activator "EDC" element) within the promoter. The PDGF-B enhancer fails to activate heterologous promoters, indicating that promoter-specificity depends on an element within the enhancer that can recognise a target sequence within the promoter. Here we identify a sequence within the enhancer of the PDGF-B gene which directs activation of the PDGF-B promoter by distal cis-acting elements. This specifies the wild-type PDGF-B promoter as the target for the enhancer and has been designated the EDC specificity element (EDCse). The cell-type specific nature of this interaction is extended by the observation that the EDCse is also dispensable for enhancer activity in breast-cancer cells (ZR-75). Concomitant to this observation, JEG-3 and ZR-75 cells differ in the binding of nuclear factors to the EDCse. We discuss the relevance of the EDC/EDCse system in regulation of gene expression.


Comparison of Four Methods for Percutaneous Achilles Tendon Lengthening: A Cadaveric Study.
Percutaneous Achilles tendon lengthening can result in Achilles tendon rupture. This complication has been controversially linked to torsion effects in the Achilles tendon. Routine percutaneous triple-hemisection techniques (group A), rotary triple-hemisection (group B), distal double-hemisection (group C), and proximal double-hemisection (group D) were compared in cadaveric specimens to provide insights into the mechanism of uneven incision lengthening and inadvertent Achilles tendon rupture. The degree of Achilles tendon torsion on various planes was measured in 20 lower limb pairs from fresh cadavers. The increase in postoperative maximum ankle joint dorsiflexion degree and the length of the lengthened Achilles tendon were greater in group B (p < .05) and group C (p < .05) compared with the routine percutaneous triple-hemisection technique (group A). The width of the tensile gap of the distal incision was significantly greater in group B (p < .05) and group C (p < .05) compared with that in group A. Rotary triple-hemisection was shown to eliminate the effect of Achilles tendon torsion on percutaneous Achilles tendon lengthening. Because proximal double-hemisection is performed away from the distal Achilles tendon where the fibers rotate sharply, the technique results in more even extension of the incisions and achieves a greater increase in the maximum degree of ankle joint dorsiflexion. Uneven incision lengthening was observed with the routine percutaneous triple-hemisection and distal double-hemisection techniques. Achilles tendon torsion affected the surgical outcomes. Rotary triple-hemisection and proximal double-hemisection techniques resulted in more even extension of the incisions and achieved a greater increase in the degree of maximum ankle joint dorsiflexion.


Evolutionary insights from suffix array-based genome sequence analysis.
Gene and protein sequence analyses, central components of studies in modern biology are easily amenable to string matching and pattern recognition algorithms. The growing need of analysing whole genome sequences more efficiently and thoroughly, has led to the emergence of new computational methods. Suffix trees and suffix arrays are data structures, well known in many other areas and are highly suited for sequence analysis too. Here we report an improvement to the design of construction of suffix arrays. Enhancement in versatility and scalability, enabled by this approach, is demonstrated through the use of real-life examples. The scalability of the algorithm to whole genomes renders it suitable to address many biologically interesting problems. One example is the evolutionary insight gained by analysing unigrams, bi-grams and higher n-grams, indicating that the genetic code has a direct influence on the overall composition of the genome. Further, different proteomes have been analysed for the coverage of the possible peptide space, which indicate that as much as a quarter of the total space at the tetra-peptide level is left un-sampled in prokaryotic organisms, although almost all tri-peptides can be seen in one protein or another in a proteome. Besides, distinct patterns begin to emerge for the counts of particular tetra and higher peptides, indicative of a 'meaning' for tetra and higher n-grams. The toolkit has also been used to demonstrate the usefulness of identifying repeats in whole proteomes efficiently. As an example, 16 members of one COG,coded by the genome of Mycobacterium tuberculosis H37Rv have been found to contain a repeating sequence of 300 amino acids.


What makes an ideal hospital-based medical leader? Three views of healthcare professionals and managers: A case study.
Medical leadership is an increasingly important aspect of hospital management. By engaging physicians in leadership roles, hospitals aim to improve their clinical and financial performances. Research has revealed numerous factors that are regarded as necessary for 'medical leaders' to master, however we lack insights into their relative importance. This study investigates the views of healthcare professionals and managers on what they consider the most important factors for medical leadership. Physicians (n = 11), nurses (n = 10), laboratory technicians (n = 4) and managers (n = 14) were interviewed using Q methodology. Participants ranked 34 statements on factors elicited from the scientific literature, including personal features, context-specific features, activities and roles. By-person factor analysis revealed three distinct views of medical leadership. The first view represents a strategic leader who prioritizes the interests of the hospital by participating in hospital strategy and decision making. The second view describes a social leader with strong collaboration and communication skills. The third view reflects an accepted leader among peers that is guided by a clear job description. Despite these differences, all respondents agreed upon the importance of personal skills in collaboration and communication, and having integrity and a clear vision. We find no differences in views related to particular healthcare professionals, managers, or departments as all views were defined by a mixture of departments and participants. The findings contribute to increased calls from both practice and literature to increase conceptual clarity by eliciting the relative importance of medical leadership-related factors. Hospitals that wish to increase the engagement of physicians in improving clinical and financial performances through medical leadership should focus on selecting and developing leaders who are strong strategists, socially skilled and accepted by clinical peers.


Epidemiology of acute myocardial infarction with the emphasis on patients who did not reach the coronary care unit and non-AMI admissions.
To describe the characteristics and outcome of patients with acute myocardial infarction (AMI) in a community, with particular emphasis on those who never reached a Coronary Care Unit (CCU) and those in whom the primary diagnosis was something other than a heart attack. Patients hospitalised in the city of Göteborg, Sweden, and discharged (dead or alive) with a diagnosis of AMI. Among 1423 patient admissions the mean overall age was 75 years (81 years and 79 years in the two subsets). Among all patients, 33% had a history of heart failure and 20% had a history of cerebrovascular disease. The figures were even higher in the two subsets which were evaluated. In overall terms, an invasive strategy (coronary angiography) was used in 32% (in 5% and 9% in the two subsets respectively). The overall one-year and three-year mortality rate was 30% and 44% respectively. The three-year mortality rate among patients not admitted to a CCU was 65% and, among patients with no suspicion of a heart attack on admission, it was 68%. Even in the 21st century, patients with AMI who reach hospital alive run a high risk of death and nearly half are dead within the first three years. In overall terms, patients are characterised by high age and high co-morbidity. Among patients who do not reach a CCU and among patients with no suspicion of AMI on admission, approximately two thirds are dead within the subsequent three years.


C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases.
The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.


Cell adhesion property affected by cyclooxygenase and lipoxygenase: Opto-electric approach.
Expression of cyclooxygenases (COX) and lipoxygenases (LOX) has been linked to many pathophysiological phenotypes, including cell adhesion. However, many current approaches to measure cellular changes are performed only in a fixed-time point. Since cells dynamically move in conjunction with the cell matrix, there is a pressing need for dynamic or time-dependent methods for the investigation of cell properties. In the presented study, we used stable human colorectal cancer cell lines ectopically expressing COX-1, COX-2, and 15LOX-1, to investigate whether expression of COX-1, COX-2, or 15LOX-1 would affect cell adhesion using our opto-electric methodology. In a fixed-time point experiment, only COX-1- and COX-2-expressing cells enhanced phosphorylation of focal adhesion kinase, but all the transfected cells showed invasion activity. However, in a real-time experiment using opto-electric approaches, transmitted cellular morphology was much different with tight adhesion being shown in COX-2 expressing cells, as imaged by differential interference contrast microscopy (DICM) and interference reflection contrast microscopy (IRCM). Furthermore, micro-impedance measurements showed a continued increase in both resistance and reactance of COX- and LOX-transfected cells, consistent with the imaging data. Our data indicate that both COX- and LOX-expressing cells have strong cell-to-cell and cell-to-substrate adhesions, and that cell imaging analysis with cell impedance data generates fully reliable results on cell adhesion measurement.


VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex.
The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.


Improving hindlimb locomotor function by Non-invasive AAV-mediated manipulations of propriospinal neurons in mice with complete spinal cord injury.
After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI.


A study on the feasibility of suppressing ovarian activity following the end of postpartum amenorrhoea by increasing the frequency of suckling.
The aim of the study was to test the effect of increasing the frequency of breastfeeding upon ovarian function following the end of postpartum amenorrhoea. Women exclusively breastfeeding (n = 14) who experienced their first postpartum menstruation between the third and fifth month after delivery were randomly allocated to an experimental (n = 7) and a control (n = 7) group and studied during the next two cycles. Women in the experimental group agreed to increase the number of breastfeeding episodes per day throughout the second cycle. Blood samples were drawn twice a week from the first to the third postpartum menstruation for prolactin, oestradiol and progesterone determinations. The number of breastfeeding episodes per day and night and the length of each episode were recorded daily. An average frequency of 11 nursing episodes per 24 h was reported during the first study cycle in both groups. The experimental group increased by 50% the breastfeeding frequency after the second menstruation while the control group kept its spontaneous feeding pattern. The increase of suckling frequency occurred mainly during day hours. The total breastfeeding time per 24 h was not increased. The length of the menstrual cycle was not altered by the increased suckling frequency. The endocrine pattern differed neither between the first and second cycle of the experimental group nor between the experimental and control group, with the exception of PRL levels which were higher in the experimental group throughout both cycles. Twenty cycles (71%) showed progesterone values compatible with ovulation (greater than 9.5 nmol/l). The highest progesterone values and the highest oestradiol values observed in the ovulatory cycles of lactating women were within the range found in non-nursing fertile women. Plasma levels of PRL were significantly elevated in all cycles but one, in comparison with PRL levels in non-nursing women. It is concluded that if cyclic ovarian function is reestablished early in fully nursing women, a mere increase in the number of breastfeeding episodes, without augmenting total suckling time and the frequency of nocturnal episodes, does not provide a sufficient stimulus to resuppress the pituitary-ovarian axis.


Re-using Mini-Sentinel data following rapid assessments of potential safety signals via modular analytic programs.
The U.S. Food and Drug Administration (FDA)'s Mini-Sentinel pilot has created a distributed data system with over 125 million lives and nearly 350 million person-years of observation time. The pilot allows the FDA to use modular analytic programs to assess suspected safety signals quickly. The FDA convened a committee to assess the implications of such rapid assessments on subsequent analyses of the same product-outcome pair using the same data. The committee offers several non-binding recommendations based on the strength of the knowledge of the suspected association before running the analysis: signal generation (an analysis with no prior), signal refinement (an analysis with a weak or moderate prior), and signal evaluation (an analysis with a strong prior). The committee believes that modular programs (MPs) are most useful for signal refinement. If MPs are used for analyses with no or weak/moderate priors, the committee members generally agree that the data may be re-used if certain conditions are met. When there is a strong prior, the committee recommends that a protocol-based assessment be used; Mini-Sentinel data may be analyzed by MPs and re-used only under very uncommon circumstances. The committee agrees that any subsequent assessment of the same product-outcome pair that follows an MP analysis should not be interpreted as independent confirmation of the association, such as would be established via replication of the same product-outcome association in two different populations. Instead, the follow-up assessment should be interpreted as an analysis that has reduced insofar as possible systematic errors that may have been present or residual in the original MP analysis. The committee also discussed how this general framework may apply to two completed rapid assessments of dabigatran and bleeding risk and of olmesartan and celiac disease risk.


An assessment of the distribution and spread of the tick Hyalomma marginatum in the western Palearctic under different climate scenarios.
We applied a process-driven model to evaluate the impact of climate scenarios for the years 2020, 2050, and 2080 on the life cycle of Hyalomma marginatum ticks in the western Palearctic. The net growth rate of the tick populations increased in every scenario tested compared to the current climate baseline. These results support the expectations of increased tick survival and increased population turnover in future climate scenarios. We included a basic evaluation of host movement based on rules connected to altitude, slope, size of the near patches, and inter-patch distances in the real landscape over the target area. Data on landscape were obtained from medium-resolution MODIS satellite imagery, which allowed us to test the potential spread of the populations. Such a model of host dispersal linked to the process-driven life cycle model demonstrated that eastern (Turkey, Russia, and Balkans) populations of H. marginatum currently are well separated and have little mixing with western (Italy, Spain, and northern Africa) populations. The northern limit is marked by the cold areas in the Balkans, Alps, and Pyrenees. Under the warmer conditions predicted by the climate scenarios, the exchange of ticks throughout new areas, previously free of the vector, is expected to increase, mainly in the Balkans and southern Russia, over the limit of the mountain ranges. Therefore, the northern limit of the tick range would increase. Additional studies are necessary to understand the implications of host changes in range and abundance for H. marginatum and Crimean-Congo hemorrhagic fever virus.


A Rare Case of Radiculopathy: Monostotic Fibrous Dysplasia of the Sacrum.
Fibrous dysplasia is a bone disease characterized by an osteoblastic dysfunction resulting in a fibrous replacement of the normal medullary bone. We describe the case of a 33-year-old who presented with low back pain irradiating to her right leg. Both the computed tomography scan and magnetic resonance imaging showed an osteolytic, multicystic lesion of the right hemi-sacrum with invasion of the right S1 foramen. She underwent foraminotomy and curettage of the lesion. Histological diagnosis was fibrous dysplasia, without features of malignant transformation. Three years after surgery the patient is asymptomatic and imaging is stable. This is the fifth known case of monostotic fibrous dysplasia involving the sacrum, a rare entity that must be considered in the differential diagnosis when approaching patients with sacral lesions.


Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation.
In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.


Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST) and Microsatellite Instability in Patients with Colorectal Cancer and Its Clinical Features.
Recently, a different type of microsatellite instability (MSI) instability designated 'elevated microsatellite alterations at selected tetranucleotide repeats' (EMAST) has been reported in several neoplasms, but its clinical implications remain unclear. We aimed to determine the relationships among EMAST, MSI and clinicopathologic characteristics, including oncologic outcomes, in colorectal cancer (CRC). We evaluated 100 sporadic CRC cases subjected to surgery using five markers (MYCL1, D9S242, D20S85, D8S321, and D20S82) for EMAST and the Bethesda panel for MSI status. Immunohistochemical detection of hMSH3, c-erbB2, EGFR and thymidylate synthase was performed. Clinical characteristics and prognostic relevance were assessed. We identified 22 EMAST-positive tumors (22.0%) and 32 MSI-high (MSI-H) tumors (32.0%). EMAST was more frequent in colon cancer than rectal cancer (p=0.033), and associated with MSI-H phenotype (p<0.001), low expression of hMSH3 (p=0.004), and overexpression of thymidylate synthase (p=0.006). Among the 38 MSI-L tumors, only one (4.5%) showed EMAST. Long-term oncologic results in terms of overall and disease-free survival were similar between EMAST and non-EMAST tumors. EMAST is more closely related to MSI-H than MSI-L or MSS status. The clinical and molecular characteristics of EMAST were distinct in terms of tumor location, thymidylate synthase expression, MSI status and hMSH3 expression. Our preliminary findings support the utility of EMAST as a new potential classifier in CRC.


Effect of a hypocaloric diet, increased protein intake and resistance training on lean mass gains and fat mass loss in overweight police officers.
We compare the effects of a moderate hypocaloric, high-protein diet and resistance training, using two different protein supplements, versus hypocaloric diet alone on body compositional changes in overweight police officers. A randomized, prospective 12-week study was performed comparing the changes in body composition produced by three different treatment modalities in three study groups. One group (n = 10) was placed on a nonlipogenic, hypocaloric diet alone (80% of predicted needs). A second group (n = 14) was placed on the hypocaloric diet plus resistance exercise plus a high-protein intake (1.5 g/kg/day) using a casein protein hydrolysate. In the third group (n = 14) treatment was identical to the second, except for the use of a whey protein hydrolysate. We found that weight loss was approximately 2.5 kg in all three groups. Mean percent body fat with diet alone decreased from a baseline of 27 +/- 1.8 to 25 +/- 1.3% at 12 weeks. With diet, exercise and casein the decrease was from 26 +/- 1.7 to 18 +/- 1.1% and with diet, exercise and whey protein the decrease was from 27 +/- 1.6 to 23 +/- 1.3%. The mean fat loss was 2. 5 +/- 0.6, 7.0 +/- 2.1 and 4.2 +/- 0.9 kg in the three groups, respectively. Lean mass gains in the three groups did not change for diet alone, versus gains of 4 +/- 1.4 and 2 +/- 0.7 kg in the casein and whey groups, respectively. Mean increase in strength for chest, shoulder and legs was 59 +/- 9% for casein and 29 +/- 9% for whey, a significant group difference. This significant difference in body composition and strength is likely due to improved nitrogen retention and overall anticatabolic effects caused by the peptide components of the casein hydrolysate.


Structural basis on the dityrosyl-diiron radical cluster and the functional differences of human ribonucleotide reductase small subunits hp53R2 and hRRM2.
Ribonucleotide reductase (RNR) is an enzyme for the de novo conversion of ribonucleotides to deoxyribonucleotides. The two human RNR small subunits hRRM2 and hp53R2 share 83% sequence homology but show distinct expression patterns and function. Structural analyses of the oxidized form of hRRM2 and hp53R2 indicate that both proteins contain a conserved Gln127-hp53R2/Gln165-hRRM2 close to the dinuclear iron center and the essential tyrosine residue Tyr124-hp53R2/Tyr162-hRRM2 forms hydrogen bonds with the tyrosine and iron ligands, implying a critical role for the glutamine residue in assembling the dityrosyl-diiron radical cofactor. The present work also showed that Tyr221 in hRRM2, which is replaced by Phe183 in hp53R2, forms a hydrogen bond with Tyr162 to extend the hydrogen bond network from Gln165-hRRM2. Mutagenesis and spectroscopic experiments suggested that the tyrosine-to-phenylalanine switch at Phe183-hp53R2/Tyr221-hRRM2 could lead to differences in radical generation or enzymatic activity for hp53R2 and hRRM2. This study correlates the distinct catalytic mechanisms of the small subunits hp53R2 and hRRM2 with a hydrogen-bonding network and provides novel directions for designing and developing subunit-specific therapeutic agents for human RNR enzymes.


Functional and structural characterization of the 2/2 hemoglobin from Synechococcus sp. PCC 7002.
Cyanobacterium Synechococcus sp. PCC 7002 contains a single gene (glbN) coding for GlbN, a protein of the 2/2 hemoglobin lineage. The precise function of GlbN is not known, but comparison to similar 2/2 hemoglobins suggests that reversible dioxygen binding is not its main activity. In this report, the results of in vitro and in vivo experiments probing the role of GlbN are presented. Transcription profiling indicated that glbN is not strongly regulated under any of a large number of growth conditions and that the gene is probably constitutively expressed. High levels of nitrate, used as the sole source of nitrogen, and exposure to nitric oxide were tolerated better by the wild-type strain than a glbN null mutant, whereas overproduction of GlbN in the null mutant background restored the wild-type growth. The cellular contents of reactive oxygen/nitrogen species were elevated in the null mutant under all conditions and were highest under NO challenge or in the presence of high nitrate concentrations. GlbN overproduction attenuated these contents significantly under the latter conditions. The analysis of cell extracts revealed that the heme of GlbN was covalently bound to overproduced GlbN apoprotein in cells grown under microoxic conditions. A peroxidase assay showed that purified GlbN does not possess significant hydrogen peroxidase activity. It was concluded that GlbN protects cells from reactive nitrogen species that could be encountered naturally during growth on nitrate or under denitrifying conditions. The solution structure of covalently modified GlbN was determined and used to rationalize some of its chemical properties.


Efficacy of a prevascularized expanded polytetrafluoroethylene solid support system as a transplantation site for pancreatic islets.
An intraperitoneally located and prevascularized expanded polytetrafluoroethylene solid support is potentially a suitable transplantation site for encapsulated pancreatic islets, because it allows for both the implantation of a large volume islet graft in the immediate vicinity of blood vessels, and its complete removal. The present study investigates the efficacy of such solid supports for the implantation of nonencapsulated islet isografts in streptozotocin diabetic rat recipients. These solid supports were always coated with acidic fibroblast growth factor, because we found that this growth factor enhances the neovascularization. The success rates of 5-microl (group A) and 10-microl (group B) islet isografts in solid supports were compared with the success rates of 5-microl (group C) and 10-microl (group D) islet isografts implanted in the unmodified peritoneal cavity. Four of seven rats in group A and all seven rats in group B became normoglycemic for at least 6 months. Only two of eight rats in group C and four of eleven rats in group D showed normoglycemia. The normoglycemia lasted for at least 6 months in zero of two animals in group C and in three of four animals in group D. Because of the low success rates in groups C and D, intravenous and oral glucose testing were restricted to the successful recipients in groups A and B. Glucose tolerance was found to be proportional to the grafted islet volume but, expectedly, in both groups the glucose tolerance and the insulin responses were somewhat lower than in controls. Thus, the prevascularized expanded polytetrafluoroethylene solid support, rather than the unmodified peritoneal cavity, is an efficacious transplantation site, potentially suitable for encapsulated islets.


Specific regulation of RGS2 messenger RNA by angiotensin II in cultured vascular smooth muscle cells.
The effects of angiotensin II (Ang II) are mediated primarily by Ang II type 1 receptors, which in turn are coupled to heterotrimeric G proteins. After receptor activation, the G(alpha) and G(betagamma) subunits dissociate, contributing to the signaling cascades involving protein kinase C (PKC) activation. Regulators of G protein signaling (RGS proteins) comprise a class of proteins that have been shown to negatively regulate the G(alpha) subunit. We examined which RGS sequences were expressed in vascular smooth muscle cells and which of these were regulated by Ang II. Reverse transcription-polymerase chain reaction showed that of 16 RGS sequences screened, six RGS transcripts (RGS2, 3, 10, 11, and 12 and GAIP) were present. Northern blot analysis demonstrated that RGS3, 10, and 12 and GAIP were not regulated by Ang II at the mRNA level. In contrast, RGS2 mRNA was rapidly and dose dependently increased (395 +/- 24% peak, 45 min) by Ang II but returned to baseline level by 6 to 8 h. Phorbol-12-myristate-13-acetate, a PKC activator, robustly increased RGS2. This signal was attenuated by the PKC inhibitor GF 109203X (50 +/- 4%) and by phorbol-12, 13-dibutyrate-mediated down-regulation of PKC (48 +/- 13%). Tyrosine kinase inhibition and calcium deprivation did not affect the up-regulation of RGS2 mRNA after Ang II stimulation. Actinomycin D treatment inhibited both Ang II- and phorbol-12-myristate-13-acetate-stimulated RGS2 up-regulation, suggesting activation of transcription by these agonists. The stability of RGS2 mRNA did not appear to be affected by Ang II. Thus, RGS2 is a likely candidate for negative regulation of the G proteins coupled to the Ang II type 1 receptor in vascular smooth muscle cells. Regulation of this protein may be of critical importance in modulating the role of Ang II in vascular disease.


Notch signaling regulates immunosuppressive tumor-associated macrophage function in pancreatic cancer.
Pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. The poor survival of patients with PDA has been attributed to a high rate of early metastasis and low efficacy of current therapies, which partly result from its complex immunosuppressive tumor microenvironment. Previous studies from our group and others have shown that tumor-associated macrophages (TAMs) are instrumental in maintaining immunosuppression in PDA. Here, we explored the role of Notch signaling, a key regulator of immune response, within the PDA microenvironment. We identified Notch pathway components in multiple immune cell types within human and mouse pancreatic cancer. TAMs, the most abundant immune cell population in the tumor microenvironment, expressed high levels of Notch receptors, with cognate ligands such as JAG1 expressed on tumor epithelial cells, endothelial cells, and fibroblasts. TAMs with activated Notch signaling expressed higher levels of immunosuppressive mediators, suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. Genetic inhibition of Notch in myeloid cells led to reduced tumor size and decreased macrophage infiltration in an orthotopic PDA model. Combination of pharmacological Notch inhibition with PD-1 blockade resulted in increased cytotoxic T-cell infiltration, tumor cell apoptosis, and smaller tumor size. Our work implicates macrophage Notch signaling in the establishment of immunosuppression and indicates that targeting the Notch pathway may improve the efficacy of immune-based therapies in PDA patients.


Professionally Responsible Disclosure of Genomic Sequencing Results in Pediatric Practice.
Genomic sequencing is being rapidly introduced into pediatric clinical practice. The results of sequencing are distinctive for their complexity and subsequent challenges of interpretation for generalist and specialist pediatricians, parents, and patients. Pediatricians therefore need to prepare for the professionally responsible disclosure of sequencing results to parents and patients and guidance of parents and patients in the interpretation and use of these results, including managing uncertain data. This article provides an ethical framework to guide and evaluate the professionally responsible disclosure of the results of genomic sequencing in pediatric practice. The ethical framework comprises 3 core concepts of pediatric ethics: the best interests of the child standard, parental surrogate decision-making, and pediatric assent. When recommending sequencing, pediatricians should explain the nature of the proposed test, its scope and complexity, the categories of results, and the concept of a secondary or incidental finding. Pediatricians should obtain the informed permission of parents and the assent of mature adolescents about the scope of sequencing to be performed and the return of results.


Biophysiologically plausible implementations of the maximum operation.
Visual processing in the cortex can be characterized by a predominantly hierarchical architecture, in which specialized brain regions along the processing pathways extract visual features of increasing complexity, accompanied by greater invariance in stimulus properties such as size and position. Various studies have postulated that a nonlinear pooling function such as the maximum (MAX) operation could be fundamental in achieving such selectivity and invariance. In this article, we are concerned with neurally plausible mechanisms that may be involved in realizing the MAX operation. Different canonical models are proposed, each based on neural mechanisms that have been previously discussed in the context of cortical processing. Through simulations and mathematical analysis, we compare the performance and robustness of these mechanisms. We derive experimentally verifiable predictions for each model and discuss the relevant physiological considerations.


A Mixed Methods Study of Change Processes Enabling Effective Transition to Team-Based Care.
Team-based care is considered central to achieving value in primary care, yet results of large-scale primary care transformation initiatives have been mixed. We explore how underlying change processes influence the effectiveness of transition to team-based care. We studied 12 academically affiliated primary care practices participating in a learning collaborative, using longitudinal staff survey data to measure progress toward team-based care and qualitative interviews with practice staff to understand practice transformation. Transformation efforts focused on team formation and capacity building for quality improvement. Using thematic analysis, we explored types of change processes undertaken and the relationship between change processes and effective team-based care. We identified three prototypical approaches to change: pursuing functional and cultural change processes, functional only, and cultural only. Practice sites prioritizing both change processes formed the most effective teams: simultaneous functional and cultural change spurred a mutually reinforcing virtuous cycle. We describe implications for research, practice, and policy.


Role of reactive oxygen species (ROS) in CDDO-Me-mediated growth inhibition and apoptosis in colorectal cancer cells.
CDDO-Me, an oleanane synthetic triterpenoid has shown strong antitumorigeic activity towards diverse cancer cell types including colorectal cancer cells. In the present study, we investigated the role of free radicals in the growth inhibitory and apoptosis-inducing activity of CDDO-Me in colorectal cancer cells lines. Results demonstrated that CDDO-Me potently inhibited the growth of colorectal cancer cells and pretreatment of cancer cells with small-molecule antioxidant N-acetylcysteine (NAC) completely blocked the growth inhibitory activity of CDDO-Me. CDDO-Me caused the generation of reactive oxygen species, which was inhibited by NAC and mitochondrial chain 1 complex inhibitors DPI and rotenone. CDDO-Me induced apoptosis as demonstrated by the cleavage of PARP-1, activation of procaspases -3, -8, and -9 and mitochondrial depolarization and NAC blocked the activation of these apoptosis related processes. Furthermore, induction of apoptosis by CDDO-Me was associated with the inhibition of antiapoptotic/ prosurvival Akt, mTOR and NF-kappaB signaling proteins and the inhibition of these signaling molecules was blocked by NAG. Together these studies provided evidence that CDDO-Me is a potent anticancer agent, which imparts growth inhibition and apoptosis in colorectal cancer cells through the generation of free radicals.


A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient cavalier king charles spaniels is amenable to exon 51 skipping.
Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.


Polycyclic aromatic hydrocarbons (PAHs): a possible cause of lung cancer mortality among nickel/copper smelter and refinery workers.
A retrospective industrial hygiene investigation was undertaken to explain the cause of a statistically significant excess lung cancer mortality observed in a subset of a large cohort of nickel workers involved in mining, smelting, and refining of nickel and copper in Ontario. The focus of this paper is to demonstrate how an industrial hygiene follow-up assessment of an epidemiologic finding can help to identify a likely cause. Polycyclic aromatic hydrocarbons (PAHs) alone or in association with particulate and gaseous contaminants (e.g., SO2) were likely the causative agents of the excess lung cancer observed among the lead welders, cranemen, and arc furnace workers of the copper refinery.


Genetic bases of estrogen-induced pituitary tumorigenesis: identification of genetic loci determining estrogen-induced pituitary growth in reciprocal crosses between the ACI and Copenhagen rat strains.
Estrogens stimulate proliferation and enhance survival of the prolactin (PRL)-producing lactotroph of the anterior pituitary gland and induce development of PRL-producing pituitary tumors in certain inbred rat strains but not others. The goal of this study was to elucidate the genetic bases of estrogen-induced pituitary tumorigenesis in reciprocal intercrosses between the genetically related ACI and Copenhagen (COP) rat strains. Following 12 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), pituitary mass, an accurate surrogate marker of absolute lactotroph number, was increased 10.6-fold in ACI rats and 4.5-fold in COP rats. Composite interval mapping analyses of the phenotypically defined F(2) progeny from the reciprocal crosses identified six quantitative trait loci (QTL) that determine the pituitary growth response to DES. These loci reside on chromosome 6 [Estrogen-induced pituitary tumor (Ept)1], chromosome 3 (Ept2 and Ept6), chromosome 10 (Ept9), and chromosome 1 (Ept10 and Ept13). Together, these six Ept loci and one additional suggestive locus on chromosome 4 account for an estimated 40% of the phenotypic variance exhibited by the combined F(2) population, while 34% of the phenotypic variance was estimated to result from environmental factors. These data indicate that DES-induced pituitary mass behaves as a quantitative trait and provide information that will facilitate identification of genes that determine the tumorigenic response of the pituitary gland to estrogens.


Mechanism and Mitigation of the Decomposition of an Oxorhenium Complex-Based Heterogeneous Catalyst for Perchlorate Reduction in Water.
A biomimetic heterogeneous catalyst combining palladium nanoparticles and an organic ligand-coordinated oxorhenium complex on activated carbon, Re(hoz)2-Pd/C, was previously developed and shown to reduce aqueous perchlorate (ClO4-) with H2 at a rate ∼100 times faster than the first generation ReOx-Pd/C catalyst prepared from perrhenate (ReO4-). However, the immobilized Re(hoz)2 complex was shown to partially decompose and leach into water as ReO4-, leading to an irreversible loss of catalytic activity. In this work, the stability of the immobilized Re(hoz)2 complex is shown to depend on kinetic competition between three processes: (1) ReV(hoz)2 oxidation by ClO4- and its reduction intermediates ClOx-, (2) ReVII(hoz)2 reduction by Pd-activated hydrogen, and (3) hydrolytic ReVII(hoz)2 decomposition. When ReV(hoz)2 oxidation is faster than ReVII(hoz)2 reduction, the ReVII(hoz)2 concentration builds up and leads to hydrolytic decomposition to ReO4- and free hoz ligand. Rapid ReV(hoz)2 oxidation is mainly promoted by highly reactive ClOx- formed from the reduction of ClO4-. To mitigate Re(hoz)2 decomposition and preserve catalytic activity, ruthenium (Ru) and rhodium (Rh) were evaluated as alternative H2 activators to Pd. Rh showed superior activity for reducing the ClO3- intermediate to Cl-, thereby preventing ClOx- buildup and lowering Re complex decomposition in the Re(hoz)2-Rh/C catalyst. In contrast, Ru showed the lowest ClO3- reduction activity and resulted in the most Re(hoz)2 decomposition among the Re(hoz)2-M/C catalysts. This work highlights the importance of using mechanistic insights from kinetic and spectroscopic tests to rationally design water treatment catalysts for enhanced performance and stability.


Reducing care-resistant behaviors during oral hygiene in persons with dementia.
Nursing home residents with dementia are often dependent on others for mouth care, yet will react with care-resistant behavior when receiving assistance. The oral health of these elders deteriorates in the absence of daily oral hygiene, predisposing them to harmful systemic problems such as pneumonia, hyperglycemia, cardiac disease, and cerebral vascular accidents. The purpose of this study is to determine whether care-resistant behaviors can be reduced, and oral health improved, through the application of an intervention based on the neurobiological principles of threat perception and fear response. The intervention, called Managing Oral Hygiene Using Threat Reduction, combines best mouth care practices with a constellation of behavioral techniques that reduce threat perception and thereby prevent or de-escalate care-resistant behaviors. Using a randomized repeated measures design, 80 elders with dementia from 5 different nursing homes will be randomized at the individual level to the experimental group, which will receive the intervention, or to the control group, which will receive standard mouth care from research team members who receive training in the proper methods for providing mouth care but no training in resistance recognition or prevention/mediation. Oral health assessments and care-resistant behavior measurements will be obtained during a 7-day observation period and a 21-day intervention period. Individual growth models using multilevel analysis will be used to estimate the efficacy of the intervention for reducing care-resistant behaviors in persons with dementia, and to estimate the overall efficacy of the intervention using oral health outcomes. Activity-based costing methods will be used to determine the cost of the proposed intervention. At the conclusion of this study, the research team anticipates having a proven intervention that prevents and reduces care-resistant within the context of mouth care. Long-term objectives include testing the effect of the intervention on systemic illnesses among persons with dementia; examining the transferability of this intervention to other activities of daily living; and disseminating threat reduction interventions to nursing home staff, which may radically change the manner in which care is provided to persons with dementia. ClinicalTrials.gov: NCT01363258.


Health-related behaviour in Europe - A comparison of selected indicators for Germany and the European Union.
Demographic change, new health threats, but also inequalities in health and health care provision in and between European Union (EU) member states pose major albeit similar challenges to European health systems. Regular information on health and health-related behaviour is essential if member states' health systems are to respond and develop appropriately to these challenges. The 'European Health Interview Survey' (EHIS) is a vital source of data for indicators of health status and health-related behaviour in the EU. This article presents a comparative review of health-related behaviour at the European level. Health-related behaviour is of particular relevance because an unhealthy diet, physical inactivity, obesity, smoking, and harmful use of alcohol are among the most important determinants associated with non-communicable chronic diseases. Eurostat has used data from EHIS Wave 2 to publish details about the current prevalence of obesity, daily fruit and vegetable intake, health-enhancing aerobic physical activity, smoking and heavy episodic drinking for the EU's member states. In the following, the figures for Germany are compared to the European average. A wide range of prevalences exists between the various EU member states, in some cases stretching to more than 50 percentage points. In Germany, the prevalence of obesity and smoking remains relatively close to the EU average. Moreover, the results on physical activity are especially welcome. In particular, the proportion of women and men who undertake adequate levels of physical activity decreases more slowly with increasing age compared to the EU average. Nevertheless, the low fruit and vegetable intake, especially among younger generations, and the high proportion of women and men who drink six or more alcoholic beverages on one occasion (heavy episodic drinking) at least once a month pose problems for Germany. In summary, the results provided by EHIS offer a basis for sharing experiences between EU member states regarding effective measures in health promotion and disease prevention.


A KNOX family TALE.
Knotted1 defined the first homeobox gene family to be isolated in plants and was identified from dominant maize mutants that produced 'knots' of mis-specified tissue on the leaf. The Knotted1-like homeobox (KNOX) gene family expanded last year to include members lacking the defining homeobox with exciting implications for KNOX gene regulation and function. Recent evidence for direct KNOX regulation by myb-related ARP proteins and epigenetic silencing by polycomb repressive complexes have also shed light on the mechanisms defining KNOX gene expression.


The need for a European hepatitis C programme monitoring resistance to direct-acting antiviral agents in real life to eliminate hepatitis C.
The World Health Organization (WHO) has declared that hepatitis C virus (HCV) should be eliminated as a public health threat. A key recommendation to reach this elimination goal, is to reduce new infections by 90% and liver-related mortality by 65%. Highly effective direct-acting antiviral agents (DAA) play a major role in this elimination. Unfortunately, DAA treatment fails approximately 2.5-5% of patients, often in the presence of resistance-associated substitutions (RAS). This could eventually lead to a total number of 1.8-3.6 million first-line DAA failures. RAS may jeopardise the elimination goals for several reasons; most importantly, virus transmission and infection progression will continue. More data are required to handle RAS adequately and identify mutational patterns causing resistance. Currently, sample sizes are small, data are scattered and methods heterogenic. Collaboration is therefore key and a European collaboration, such as HEPCARE, should provide a solution.


Sea urchin reproductive performance in a changing ocean: poor males improve while good males worsen in response to ocean acidification.
Ocean acidification (OA) is predicted to be a major driver of ocean biodiversity change. At projected rates of change, sensitive marine taxa may not have time to adapt. Their persistence may depend on pre-existing inter-individual variability. We investigated individual male reproductive performance under present-day and OA conditions using two representative broadcast spawners, the sea urchins Lytechinus pictus and Heliocidaris erythrogramma. Under the non-competitive individual ejaculate scenario, we examined sperm functional parameters (e.g. swimming speed, motility) and their relationship with fertilization success under current and near-future OA conditions. Significant inter-individual differences in almost every parameter measured were identified. Importantly, we observed strong inverse relationships between individual fertilization success rate under current conditions and change in fertilization success under OA. Individuals with a high fertilization success under current conditions had reduced fertilization under OA, while individuals with a low fertilization success under current conditions improved. Change in fertilization success ranged from -67% to +114% across individuals. Our results demonstrate that while average population fertilization rates remain similar under OA and present-day conditions, the contribution by different males to the population significantly shifts, with implications for how selection will operate in a future ocean.


Posttraumatic stress reactions in volunteer firefighters.
Volunteer firefighters in NSW were surveyed for experiences of posttraumatic stress. Firefighters were asked to describe their experiences of stress and indices were obtained of psychological disturbance. Findings indicated that most firefighters felt that their safety had been threatened. One-quarter of firefighters indicated that they experienced significant levels of posttraumatic stress, although many respondents attributed their stress to personal events. Posttraumatic stress was associated with multiple and recent critical incidents. Findings are discussed in terms of etiological factors of posttraumatic stress and the need for appropriate intervention.


Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes.
Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein 1 light chain 3 (LC3). Although studies have indicated that L. monocytogenes induces autophagy, the significance of this process in the infectious cycle and the mechanisms involved remain poorly understood. Here, we examined the role of the autophagy adaptor optineurin (OPTN), the phosphorylation of which by the TANK binding kinase 1 (TBK1) enhances its affinity for LC3 and promotes autophagosomal degradation, during L. monocytogenes infection. In LC3- and OPTN-depleted host cells, intracellular replicating L. monocytogenes increased, an effect not seen with a mutant lacking the pore-forming toxin listeriolysin O (LLO). LLO induced the production of OPTN. In host cells expressing an inactive TBK1, bacterial replication was also inhibited. Our studies have uncovered an OPTN-dependent pathway in which L. monocytogenes uses LLO to restrict bacterial growth. Hence, manipulation of autophagy by L. monocytogenes, either through induction or evasion, represents a key event in its intracellular life style and could lead to either cytosolic growth or persistence in intracellular vacuolar structures.


Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity.
Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a "wild-type" scFv (scFv#E4-4) composed of V(H) and V(L) domains from a mouse anti-E(2) antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by "CDR-shuffling". Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by "shuffling" into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(L29)Val] that had 5-fold higher affinity (K(a) = 2.6 x 10(8) M(-1)) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V(H) and V(L) of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K(a) = 6.3 x 10(8) M(-1); Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E(2) radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E(2)-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.


Do we care about high-cost patients? Estimating the savings on health spending by integrated care.
A recent integrated health care initiative in Belgium supports 12 regional pilot projects scattered across the country and representing 21% of the population. As in shared savings programs, part of the estimated savings in health spending are paid out to the projects to reinvest in new actions. Short-term savings are expected in particular from cost reductions among high-cost patients. We estimate the effect of the projects on spending using a difference-in-difference model. The sensitivity of the results to the right-skewness of spending is commonly addressed by removing or top-coding high-cost cases. However, this leads to an underestimation of realized savings at the top end of the distribution, therefore, lowering incentives for cost reduction. We show that this trade-off can be weakened by an alternative approach in which cost categories that fall out of the scope of the projects' interventions are excluded from the dependent variable. We find that this approach leads to improvements in precision and model fit that are of the same magnitude as excluding high-cost cases altogether. At the same time, it sharpens the incentives for cost reduction because the model better reflects the costs that projects can affect.


Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.
The most widely cultivated species of cotton, Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome species G. herbaceum and G. arboreum, the D-genome species G. raimondii and G. thurberi, and the AD tetraploid G. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S-28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six new G. hirsutum 18S-28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybridization signal at these loci allowed us to designate them as major, intermediate, or minor 18S-28S loci. Using genomic painting with labeled A-genome DNA, five 18S-28S loci were localized to the G. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S-28S rDNA loci in G. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species had three loci and both D-genome species had four. G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S-28S rDNA loci. All four of the diploid genomes we examined contained a single 5S locus. In g. herbaceum (A1) and G. thurberi (D1), the 5S locus is syntenic to a major 18S-28S locus, but in G. arboreum (A2) and G. raimondii (D5), the proposed D-genome progenitor of G. hirsutum, the 5S loci are syntenic to minor and intermediate 18S-28S loci, respectively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S-28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repeats can differ widely.