Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells.
We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.


The myeloid zinc finger gene, MZF-1, regulates the CD34 promoter in vitro.
MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.


Online object oriented Monte Carlo computational tool for the needs of biomedical optics.
Conceptual engineering design and optimization of laser-based imaging techniques and optical diagnostic systems used in the field of biomedical optics requires a clear understanding of the light-tissue interaction and peculiarities of localization of the detected optical radiation within the medium. The description of photon migration within the turbid tissue-like media is based on the concept of radiative transfer that forms a basis of Monte Carlo (MC) modeling. An opportunity of direct simulation of influence of structural variations of biological tissues on the probing light makes MC a primary tool for biomedical optics and optical engineering. Due to the diversity of optical modalities utilizing different properties of light and mechanisms of light-tissue interactions a new MC code is typically required to be developed for the particular diagnostic application. In current paper introducing an object oriented concept of MC modeling and utilizing modern web applications we present the generalized online computational tool suitable for the major applications in biophotonics. The computation is supported by NVIDEA CUDA Graphics Processing Unit providing acceleration of modeling up to 340 times.


Genotype-specific Trp53 mutational analysis in ultraviolet B radiation-induced skin cancers in Xpc and Xpc Trp53 mutant mice.
We have examined the mutational spectrum in the Trp53 gene from UVB radiation-induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5-8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals.


Hormonal and metabolic regulation of human chondrosarcoma in vitro.
Prostaglandin A1 has a profound inhibitory effect on uridine incorporation into RNA of normal cartilage whereas N6-monobutyryladenosine 3',5'-cyclic monophosphate is either stimulatory or without an effect. Sera from intact and growth hormone-treated hypophysectomized rats stimulate RNA synthesis but serum from untreated hypophysectomized rats does not. The present study investigated the in vitro regulation of [3H]uridine incorporation into RNA of six human chondrosarcomas to determine if malignant human chondrocytes are under similar metabolic and hormonal regulation. Prostaglandin A1 (25 micrograms/ml) markedly inhibited uridine incorporation in all six tumors (56 to 80%). N6-Monobutyryladenosine 3',5'-cyclic monophosphate (1 mM) inhibited uridine incorporation in five tumors (20 to 50%). Uridine incorporation was stimulated by growth hormone-dependent serum factors in one tumor and by growth hormone-independent serum factors in two tumors. Two tumors were more responsive to serum from growth hormone-treated hypophysectomized rats than to serum from intact rats, and one tumor was unresponsive to serum stimulation. The data indicate that: (a) prostaglandin A1 is a very potent inhibitor of RNA synthesis in human chondrosarcomas; (b) N6-monobutyryladenosine 3',5'-cyclic monophosphate affects human chondrosarcomas differently than it does normal cartilage; and (c) responses of human chondrosarcomas to serum growth factors vary among individual tumors.


Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2+ entry in both cultured and in situ mouse astrocytes.
Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.


Does core stability exercise improve lumbopelvic stability (through endurance tests) more than general exercise in chronic low back pain? A quasi-randomized controlled trial.
The aim was to compare core stability and general exercises (GEs) in chronic low back pain (LBP) patients based on lumbopelvic stability (LPS) assessment through three endurance core stability tests. There is a controversy about preference of core stability exercise (CSE) over other types of exercise for chronic LBP. Studies which have compared these exercises used other outcomes than those related to LPS. As it is claimed that CSE enhances back stability, endurance tests for LPS were used. A 16-session CSE program and a GE program with the same duration were conducted for two groups of participants. Frequency of interventions for both groups was three times a week. Forty-three people (aged 18-60 years) with chronic non-specific LBP were alternately allocated to core stability (n = 22) or GE group (n = 21) when admitted. The primary outcomes were three endurance core stability tests including: (1) trunk flexor; (2) trunk extensor; and (3) side bridge tests. Secondary outcomes were disability and pain. Measurements were taken at baseline and the end of the intervention. After the intervention, test times increased and disability and pain decreased within groups. There was no significant difference between two groups in increasing test times (p = 0.23 to p = 0.36) or decreasing disability (p = 0.16) and pain (p = 0.73). CSE is not more effective than GE for improving endurance core stability tests and reducing disability and pain in chronic non-specific LBP patients.


Pili and lipopolysaccharide of Pseudomonas aeruginosa bind to the glycolipid asialo GM1.
This study investigated which adhesins of Pseudomonas aeruginosa interact with the glycolipid asialo GM1, using solid-phase binding and thin-layer chromatography assays. Radioiodinated pili and flagella contaminated with lipopolysaccharide (LPS) bound to the glycolipid. When LPS was reduced to acceptable levels in pilus and flagellum samples, only pili specifically bound to the glycolipid. Commercial, radiolabeled LPS as well as whole bacteria of strain ATCC 19660 also bound to asialo GM1. Binding was specific, competitive, and saturable. Organ cultures of whole mouse eyes and scanning electron microscopy techniques were used also, and strain ATCC 19660 was inhibited from corneal binding by exogenous pili or commercial LPS and inhibition was concentration dependent for both. Binding of radiolabeled strain ATCC 19660 bacteria to neutral lipids extracted from bovine corneal epithelial tissue showed that the bacteria bound to a glycolipid which migrated at a position similar to that of an asialo GM1 standard and that the glycolipid stained positively with an antibody specific for asialo GM1. The data provide evidence that pili (reduced LPS) and LPS of P. aeruginosa bind to asialo GM1 glycolipid and that the glycolipid is not restricted to the mouse cornea.


Cranberries for preventing urinary tract infections.
Cranberries (particularly in the form of cranberry juice) have been used widely for several decades for the prevention and treatment of urinary tract infections. The aim of this review is to assess the effectiveness of cranberries in preventing such infections. To assess the effectiveness of cranberry juice and other cranberry products in preventing urinary tract infections in susceptible populations. Electronic databases and the Internet were searched using English and non English language terms; companies involved with the promotion and distribution of cranberry preparations were contacted; reference lists of review articles and relevant trials were searched. All randomised or quasi randomised controlled trials of cranberry juice/products for the prevention of urinary tract infections in susceptible populations. Trials of men, women or children were included. Reviewers RJ and LM independently assessed and extracted information using specially designed data extraction forms. For each included trial, information was collected on methods of the trial, participants, interventions and outcomes. We were unable to perform statistical analysis due to the nature of the data available for review. Four trials met the inclusion criteria (three cross-over, one parallel group). Three compared the effectiveness of cranberry juice versus placebo juice or water and one compared the effectiveness of cranberry capsules versus placebo. Two further trials were excluded. The outcomes of interest were number of urinary tract infections in each group (symptomatic and asymptomatic), side effects and adherence to therapy. Data from three out of the four trials indicated that cranberries were effective for at least one of the outcomes of interest. The quality of the four included trials was poor, however, and thus the reliability of the results must be questionable. The small number of poor quality trials gives no reliable evidence of the effectiveness of cranberry juice and other cranberry products. The large number of dropouts/withdrawals from the trials indicates that cranberry juice may not be acceptable over long periods of time. Other cranberry products such as cranberry capsules may be more acceptable. On the basis of the available evidence, cranberry juice cannot be recommended for the prevention of urinary tract infections in susceptible populations. Further properly designed trials with relevant outcomes are needed.


Buccal mucosal graft urethroplasty in male cats with traumatic complete urethral rupture.
To evaluate the feasibility of buccal mucosal graft urethroplasty for repairing complete urethral rupture in cats. 15 male domestic shorthair cats with traumatic complete urethral rupture. In each cat, a section of buccal mucosa was harvested, sutured, and formed into a tubule by use of an 8F indwelling catheter as support. This tubular graft was connected to both ruptured ends of the urethra to renew the urinary passage. The catheter was left in place until the absence of leakage was confirmed by positive contrast retrograde urethrography. After spontaneous urination was confirmed, cats were discharged from the hospital. Six months later, urethrography was repeated and owners were asked to score their cats' urinary function and quality of life. 13 cats recovered well following surgery, with no complications in the oral cavity or surgical site and no signs of difficulty or discomfort when urinating. Urethrography 2 weeks and 6 months after surgery revealed no stricture or leakage in the abdominal cavity. The 2 remaining cats developed a urethral stricture and underwent second surgery with a successful outcome. At the 6-month follow-up, 14 cats had only mild urinary signs, and 1 cat had incontinency. Owners indicated they were delighted (n = 14) or pleased (1) with their cats' quality of life. Buccal mucosa was found to be a good source of graft tissue for performance of urethroplasty in male cats, yielding satisfactory outcomes with few postoperative complications. The described technique may be suitable for severe and complicated cases of urethral rupture in male cats.


The TSX Gives a Short Course in Health Economics: It's the Prices, Stupid!
The fall in Shoppers Drug Mart shares last April 8 gave a crystal-clear demonstration of the link between health expenditures and health incomes. Reacting (finally) to the excessive retail prices of generic drugs, the Ontario government effectively halved the rate of reimbursement of ingredient costs and banned the "professional allowances" (kickbacks) paid to pharmacies by generic manufacturers. Taxpayers and private payers will save hundreds of millions of dollars, and pharmacy revenues will fall by an equivalent amount. Patients will still get their drugs, with no loss of quantity, quality or even convenience; no one's health is threatened. But investor profits will fall. There are similar savings opportunities throughout the health system. Health costs are primarily a political, not an economic, problem.


Malpractice Litigation in Cornea and Refractive Surgery: A Review of the WestLaw Database.
To report and analyze the causes and outcomes of malpractice litigation in cornea and refractive surgery. The WestLaw database was reviewed for all malpractice litigation related to ophthalmology in the United States between 1930 and 2014. Search terms included ophthalmology or ophthalmologist and malpractice anywhere in the retrieved results. All cases involving cornea and refractive surgery were included in this analysis, and results were compared with ophthalmology as a whole. One hundred fifty-nine cornea and refractive surgery cases between the years 1964 and 2014 were included. Ninety-three cases (58.5%) were resolved through a jury trial, 21.5% of which were associated with plaintiff verdicts with a median adjusted jury award of $588,896 (mean $1,518,686). Nineteen cases (11.9%) resulted in settlements with a median adjusted indemnity of $782,533 (mean $761,963). Commonly litigated scenarios included laser-assisted in situ keratomileusis (74.2%) and corneal transplantation (8.2%). Overall, 88.7% of cases involved surgical or procedural claims, 8.8% involved noninterventional claims, and 2.5% involved medical claims only. Cornea and refractive surgery is a high-risk subspecialty of ophthalmology. Laser-assisted in situ keratomileusis and corneal transplant claims were the most commonly litigated entities in this series. Many cases focus on failure to achieve patients' desired expectations after elective refractive procedures or the negligent performance of refractive surgery on patients with contraindications to surgery, both highlighting the importance of detailed informed consent discussions, realistic goal setting with patients, and thorough examinations and preoperative evaluation.


Tracking implementation strategies in the randomized rollout of a Veterans Affairs national opioid risk management initiative.
In 2018, the Department of Veterans Affairs (VA) issued Notice 2018-08 requiring facilities to complete "case reviews" for Veterans identified in the Stratification Tool for Opioid Risk Mitigation (STORM) dashboard as high risk for adverse outcomes among patients prescribed opioids. Half of the facilities were randomly assigned to a Notice version including additional oversight. We evaluated implementation strategies used, whether strategies differed by randomization arm, and which strategies were associated with case review completion rates. Facility points of contact completed a survey assessing their facility's use of 68 implementation strategies based on the Expert Recommendations for Implementing Change taxonomy. We collected respondent demographic information, facility-level characteristics, and case review completion rates (percentage of high-risk patients who received a case review). We used Kruskal-Wallis tests and negative binomial regression to assess strategy use and factors associated with case reviews. Contacts at 89 of 140 facilities completed the survey (64%) and reported using a median of 23 (IQR 16-31) strategies. The median case review completion rate was 71% (IQR 48-95%). Neither the number or types of strategies nor completion rates differed by randomization arm. The most common strategies were using the STORM dashboard (97%), working with local opinion leaders (80%), and recruiting local partners (80%). Characteristics associated with case review completion rates included respondents being ≤ 35 years old (incidence rate ratio, IRR 1.35, 95% CI 1.09-1.67) and having < 5 years in their primary role (IRR 1.23; 95% CI 1.01-1.51), and facilities having more prior academic detailing around pain and opioid safety (IRR 1.40, 95% CI 1.12-1.75). Controlling for these characteristics, implementation strategies associated with higher completion rates included (1) monitoring and adjusting practices (adjusted IRR (AIRR) 1.40, 95% CI 1.11-1.77), (2) identifying adaptations while maintaining core components (AIRR 1.28, 95% CI 1.03-1.60), (3) conducting initial training (AIRR 1.16, 95% CI 1.02-1.50), and (4) regularly sharing lessons learned (AIRR 1.32, 95% CI 1.09-1.59). In this national evaluation of strategies used to implement case reviews of patients at high risk of opioid-related adverse events, point of contact age and tenure in the current role, prior pain-related academic detailing at the facility, and four specific implementation strategies were associated with case review completion rates, while randomization to additional centralized oversight was not. This project is registered at the ISRCTN Registry with number ISRCTN16012111. The trial was first registered on May 3, 2017.


Natural variation and the capacity to adapt to ocean acidification in the keystone sea urchin Strongylocentrotus purpuratus.
A rapidly growing body of literature documents the potential negative effects of CO2 -driven ocean acidification (OA) on marine organisms. However, nearly all this work has focused on the effects of future conditions on modern populations, neglecting the role of adaptation. Rapid evolution can alter demographic responses to environmental change, ultimately affecting the likelihood of population persistence, but the capacity for adaptation will differ among populations and species. Here, we measure the capacity of the ecologically important purple sea urchin Strongylocentrotus purpuratus to adapt to OA, using a breeding experiment to estimate additive genetic variance for larval size (an important component of fitness) under future high-pCO2 /low-pH conditions. Although larvae reared under future conditions were smaller than those reared under present-day conditions, we show that there is also abundant genetic variation for body size under elevated pCO2 , indicating that this trait can evolve. The observed heritability of size was 0.40 ± 0.32 (95% CI) under low pCO2 , and 0.50 ± 0.30 under high-pCO2 conditions. Accounting for the observed genetic variation in models of future larval size and demographic rates substantially alters projections of performance for this species in the future ocean. Importantly, our model shows that after incorporating the effects of adaptation, the OA-driven decrease in population growth rate is up to 50% smaller, than that predicted by the 'no-adaptation' scenario. Adults used in the experiment were collected from two sites on the coast of the Northeast Pacific that are characterized by different pH regimes, as measured by autonomous sensors. Comparing results between sites, we also found subtle differences in larval size under high-pCO2 rearing conditions, consistent with local adaptation to carbonate chemistry in the field. These results suggest that spatially varying selection may help to maintain genetic variation necessary for adaptation to future OA.


Dynamic PCr and pH imaging of human calf muscles during exercise and recovery using (31) P gradient-Echo MRI at 7 Tesla.
Simultaneous acquisition of spatially resolved (31) P-MRI data for evaluation of muscle specific energy metabolism, i.e., PCr and pH kinetics. A three-dimensional (3D) gradient-echo sequence for multiple frequency-selective excitations of the PCr and Pi signals in an interleaved sampling scheme was developed and tested at 7 Tesla (T). The pH values were derived from the chemical shift-induced phase difference between the resonances. The achieved spatial resolution was ∼2 mL with image acquisition time below 6 s. Ten healthy volunteers were studied performing plantar flexions during the delay between (31) P-MRI acquisitions, yielding a temporal resolution of 9-10 s. Signal from anatomically matched regions of interest had sufficient signal-to-noise ratio to allow single-acquisition PCr and pH quantification. The Pi signal was clearly detected in voxels of actively exercising muscles. The PCr depletions were in gastrocnemius 42 ± 14% (medialis), 48 ± 17% (lateralis) and in soleus 20 ± 11%. The end exercise pH values were 6.74 ± 0.18 and 6.65 ± 0.27 for gastrocnemius medialis and lateralis, respectively, and 6.96 ± 0.12 for soleus muscle. Simultaneous acquisition of PCr and Pi images with high temporal resolution, suitable for measuring PCr and pH kinetics in exercise-recovery experiments, was demonstrated at 7T. This study presents a fast alternative to MRS for quantifying energy metabolism of posterior muscle groups of the lower leg. Magn Reson Med 75:2324-2331, 2016. © 2015 Wiley Periodicals, Inc.


Increased adherence to prenatal group B streptococcal screening guidelines through a paired electronic reminder and education intervention.
The 2010 Centers for Disease Control and Prevention (CDC) update on perinatal group B streptococcal (GBS) prevention advises universal vaginal-rectal GBS screening of pregnant women in the 35th through 37th week of gestation. Because GBS colonization is transient, a test performed more than 5 weeks before delivery may not have sufficient negative predictive value to be clinically useful. Our objective was to increase rates of quality-improved, CDC-adherent GBS screening and decrease repeat screening. A reminder for maternal vaginal-rectal GBS testing was added to the physicians' electronic ordering screen, and family medicine physicians and residents were educated about screening guidelines through standardized, in-person presentations. Retrospective chart review was performed before and after these interventions. Univariate or bivariate analysis was performed for demographic factors, timing of first screen, rates of CDC-adherent screening (the newly defined quality-improved screen and the usual screen), and rates of repeat and unnecessary screens. Multivariate analysis was performed with quality-improved and usual screening as dependent variables. Bivariate analysis showed that post-intervention rates of quality-improved screening increased from 30 to 62 % (P < .001), usual screening increased from 69 to 84 % (P = .005), and repeat GBS screening decreased from 20 to 8 % (P = .007). Multivariate analysis showed increased post-intervention odds of quality-improved screening [odds ratio (OR) 3.59; 95 % CI 2.07-6.34] and usual screening (OR 2.67; 95 % CI 1.40-5.25). Low-cost, reproducible quality improvement interventions (electronic order reminder, educational sessions) have the potential to increase guideline adherence for GBS screening in pregnant women and decrease repeat screening.


Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion.
Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.


Should the IDC-9 Trauma Mortality Prediction Model become the new paradigm for benchmarking trauma outcomes?
Optimum quantification of injury severity remains an imprecise science with a need for improvement. The accuracy of the criterion standard Injury Severity Score (ISS) worsens as a patient's injury severity increases, especially among patients with penetrating trauma. The objective of this study was to comprehensively compare the mortality prediction ability of three anatomic injury severity indices: the ISS, the New ISS (NISS), and the DRG International Classification of Diseases-9th Rev.-Trauma Mortality Prediction Model (TMPM-ICD-9), a recently developed contemporary injury assessment model. Retrospective analysis of patients in the National Trauma Data Bank from 2007 to 2008. The TMPM-ICD-9 values were computed and compared with the ISS and NISS for each patient using in-hospital mortality after trauma as the outcome measure. Discrimination and calibration were compared using the area under the receiver operator characteristic curve. Subgroup analysis was performed to compare each score across varying ranges of injury severity and across different types of injury. A total of 533,898 patients were identified with a crude mortality rate of 4.7%. The ISS and NISS performed equally in the groups with minor (ISS, 1-8) and moderate (ISS, 9-15) injuries, regardless of the injury type. However, in the populations with severe (ISS, 16-24) and very severe (ISS, ≥ 25) injuries for all injury types, the NISS predicted mortality better than the ISS did. The TMPM-ICD-9 outperformed both the NISS and ISS almost consistently. The NISS and TMPM-ICD-9 are both superior predictors of mortality as compared with the ISS. The immediate adoption of NISS for evaluating trauma outcomes using trauma registry data is recommended. The TMPM-ICD-9 may be an even better measure of human injury, and its use in administrative or nonregistry data is suggested. Further research on its attributes is recommended because it has the potential to become the basis for benchmarking trauma outcomes. Prognostic study, level III.


Analysis Tool Web Services from the EMBL-EBI.
Since 2004 the European Bioinformatics Institute (EMBL-EBI) has provided access to a wide range of databases and analysis tools via Web Services interfaces. This comprises services to search across the databases available from the EMBL-EBI and to explore the network of cross-references present in the data (e.g. EB-eye), services to retrieve entry data in various data formats and to access the data in specific fields (e.g. dbfetch), and analysis tool services, for example, sequence similarity search (e.g. FASTA and NCBI BLAST), multiple sequence alignment (e.g. Clustal Omega and MUSCLE), pairwise sequence alignment and protein functional analysis (e.g. InterProScan and Phobius). The REST/SOAP Web Services (http://www.ebi.ac.uk/Tools/webservices/) interfaces to these databases and tools allow their integration into other tools, applications, web sites, pipeline processes and analytical workflows. To get users started using the Web Services, sample clients are provided covering a range of programming languages and popular Web Service tool kits, and a brief guide to Web Services technologies, including a set of tutorials, is available for those wishing to learn more and develop their own clients. Users of the Web Services are informed of improvements and updates via a range of methods.


Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella.
We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.


KLF family members regulate intrinsic axon regeneration ability.
Neurons in the central nervous system (CNS) lose their ability to regenerate early in development, but the underlying mechanisms are unknown. By screening genes developmentally regulated in retinal ganglion cells (RGCs), we identified Krüppel-like factor-4 (KLF4) as a transcriptional repressor of axon growth in RGCs and other CNS neurons. RGCs lacking KLF4 showed increased axon growth both in vitro and after optic nerve injury in vivo. Related KLF family members suppressed or enhanced axon growth to differing extents, and several growth-suppressive KLFs were up-regulated postnatally, whereas growth-enhancing KLFs were down-regulated. Thus, coordinated activities of different KLFs regulate the regenerative capacity of CNS neurons.


Semiconductor wires and ribbons for high-performance flexible electronics.
This article reviews the properties, fabrication and assembly of inorganic semiconductor materials that can be used as active building blocks to form high-performance transistors and circuits for flexible and bendable large-area electronics. Obtaining high performance on low temperature polymeric substrates represents a technical challenge for macroelectronics. Therefore, the fabrication of high quality inorganic materials in the form of wires, ribbons, membranes, sheets, and bars formed by bottom-up and top-down approaches, and the assembly strategies used to deposit these thin films onto plastic substrates will be emphasized. Substantial progress has been made in creating inorganic semiconducting materials that are stretchable and bendable, and the description of the mechanics of these form factors will be presented, including circuits in three-dimensional layouts. Finally, future directions and promising areas of research will be described.


Mechanisms of steric and cooperative actions of alcuronium on cardiac muscarinic acetylcholine receptors.
Kinetics of the interactions between the neuromuscular blocker alcuronium, the specific muscarinic antagonist N-[methyl-3H] methyl scopolamine ([3H]NMS), and muscarinic receptors were investigated in homogenates of rat heart atria. Two effects of alcuronium on the binding of [3H]NMS could be distinguished. (a) Alcuronium concentration-dependently slowed the association of [3H]NMS with receptors and the dissociation of [3H]NMS from receptors so that, at high alcuronium concentrations, equilibrium binding could not be reached, even after 20 hr, without special precautions. (b) Alcuronium increased the affinity of receptors for [3H]NMS, which was manifested by a decrease of the apparent Kd (> 3-fold) with no change in the Bmax for [3H]NMS binding. The effects of alcuronium on the rates of [3H]NMS association and dissociation can be explained only by a reaction mechanism in which [3H]NMS binds only to free receptors (not occupied by alcuronium), whereas alcuronium binds both to free receptors and to receptors occupied by [3H]NMS. Similarly, [3H]NMS cannot dissociate from receptors as long as alcuronium is attached to them. Experimental data agree with corresponding mathematical models. It is proposed that alcuronium blocks entry to the pocket containing the [3H]NMS binding site. In addition to this blocking effect, alcuronium has a positive allosteric effect on [3H]NMS binding, presumably by inducing a conformational change of the orthosteric muscarinic binding site. Earlier observations suggesting that, at high concentrations, alcuronium also competes for [3H]NMS binding sites can be explained by insufficient equilibration of the system.


Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans.
The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.


Alpha-2 adrenergic modulation of sleep: time-of-day-dependent pharmacodynamic profiles of dexmedetomidine and clonidine in the rat.
Alpha adrenergic agonists such as clonidine are widely used for their antihypertensor effects, but they also cause sedation. The mechanisms underlying soporific effects of such compounds are poorly understood, but appear to involve the alpha-2 adrenergic receptor sub-type. To further investigate the role of this receptor in sleep-wake regulation, rats received injections i.p. either during their peak of activity (circadian time CT-18: 6 hr after lights out) or near the mid-point of their sleep-dominated phase (CT-5: 5 hr after lights on) with either the highly selective alpha-2 agonist dexmedetomidine (dMED) 0.02 to 0.04 mg/kg or the less selective alpha-2 agonist, clonidine 0.04 to 0.08 mg/kg, or vehicle. Clonidine and dMED showed remarkable overall similarities in their soporific profiles. Except for the lower dose of clonidine, both CT-5 and CT-18 treatments increased the percent of time spent in non-REM (NREM) sleep. The increase in NREM was followed by a reduction of NREM sleep that was accompanied by locomotor activity and body temperature above control levels. After CT-5 treatments, this period of reduced NREM sleep was followed by a secondary increase in NREM 7 to 10 hr posttreatment. REM sleep was markedly reduced for 9 to 10 hr after all treatments at both times of day, with elevated REM levels 18 to 30 hr posttreatment. Pre-treatment with the selective alpha-2 antagonist atipamezole (0.5 mg/kg) reversed the effects of CT-18 dMED 0.04 mg/kg except REM sleep suppression, which was only partially reversed. The NREM-inducing potency of dMED 0.02 mg/kg was greater when administered at CT-18 than at CT-5. Taken together with other evidence, these findings suggest that the profound NREM-inducing effects of dMED may be mediated by postsynaptic alpha-2 adrenoceptors. Furthermore, the pharmacodynamic action of alpha-2 adrenergic agonists, like many other sedative hypnotics (e.g., benzodiazepines), produce a hysteresis in sleep-wake regulation characterized by "rebound" waking after drug-induced sleep.


Tocotrienol: the natural vitamin E to defend the nervous system?
Vitamin E is essential for normal neurological function. It is the major lipid-soluble, chain-breaking antioxidant in the body, protecting the integrity of membranes by inhibiting lipid peroxidation. Mostly on the basis of symptoms of primary vitamin E deficiency, it has been demonstrated that vitamin E has a central role in maintaining neurological structure and function. Orally supplemented vitamin E reaches the cerebrospinal fluid and brain. Vitamin E is a generic term for all tocopherols and their derivatives having the biological activity of RRR-alpha-tocopherol, the naturally occurring stereoisomer compounds with vitamin E activity. In nature, eight substances have been found to have vitamin E activity: alpha-, beta-, gamma- and delta-tocopherol; and alpha-, beta-, gamma- and delta-tocotrienol. Often, the term vitamin E is synonymously used with alpha-tocopherol. Tocotrienols, formerly known as zeta, , or eta-tocopherols, are similar to tocopherols except that they have an isoprenoid tail with three unsaturation points instead of a saturated phytyl tail. Although tocopherols are predominantly found in corn, soybean, and olive oils, tocotrienols are particularly rich in palm, rice bran, and barley oils. Tocotrienols possess powerful antioxidant, anticancer, and cholesterol-lowering properties. Recently, we have observed that alpha-tocotrienol is multi-fold more potent than alpha-tocopherol in protecting HT4 and primary neuronal cells against toxicity induced by glutamate as well as by a number of other toxins. At nanomolar concentration, tocotrienol, but not tocopherol, completely protected neurons by an antioxidant-independent mechanism. Our current work identifies two major targets of tocotrienol in the neuron: c-Src kinase and 12-lipoxygenase. Dietary supplementation studies have established that tocotrienol, fed orally, does reach the brain. The current findings point towards tocotrienol as a potent neuroprotective form of natural vitamin E.


Risk factors for arterial ischemic stroke in childhood.
Stroke affects up to 13 of 100,000 children, is more common in boys and African Americans, and is associated with considerable cognitive and psychiatric morbidity, as well as motor disability. Around half are hemorrhagic and half are ischemic. Underlying conditions include sickle cell disease, cardiac abnormalities, chromosomal abnormalities (eg, Down syndrome), and neurocutaneous conditions (eg, neurofibromatosis), but up to half the patients with ischemic stroke have no previously diagnosed condition. Although there is almost certainly an important genetic component to stroke risk, head trauma, infections, drugs and radiation appear to play an etiological role in some patients. The majority of the patients with infarction in an arterial distribution have associated cerebrovascular disease. Vascular pathologies include carotid or vertebrobasilar dissection, intracranial vasculopathy affecting the middle and anterior cerebral arteries, which is often transient, and moyamoya. Intermediate risk factors may include hypertension, hypoxia, and poor nutrition leading, for example, to iron deficiency and hyperhomocysteinemia. Some chronic conditions may directly influence the child's behavior and stroke recurrence risk, although large cohorts and randomized controlled trials will be needed before strategies for modification can be evidence-based.


Thalassaemia Trait with Gaucher Disease: A Diagnostic Dilemma.
Gaucher Disease is an autosomal recessive disease caused by the accumulation of glucocerebrosidase due to deficiency in lysosomal glucocerebrosidase. Thalassaemia trait is asymptomatic and is usually an incidental diagnosis. Both thalassaemia and Gaucher disease can have similar haematologic manifestations and hence, their coexistence causes diagnostic dilemma. Our patient presented at one-and-a-half years with weakness, pallor, failure to thrive and massive hepatosplenomegaly. Clinical examination and history pointed to a lipid storage disease. Peripheral smear revealed microcytic hypochromic cells and nucleated red cells with haemolytic blood picture. Thalassaemia trait was indicated on haemoglobin variant analysis using High Performance Liquid Chromatography. Liver biopsy, bone marrow aspirate and therapeutic splenectomy revealed Gaucher-like cells. Type 1 Gaucher disease can be clinically asymptomatic as well as present with massive liver and spleen enlargement and involvement of bone marrow. Anaemia, splenomegaly and thrombocytopenia are the usual presentations at diagnosis, similar to the haemoglobinopathies. Gaucher-like cells with normal beta-glucocerebrosidase (pseudo-Gaucher cells) are seen in thalassaemia, leukaemia, mycobacterial infections and myeloma. Gaucher disease coexisting with thalassaemia trait is uncommon. We report the occurrence of thalassaemia trait and Gaucher disease in a child, which resulted in confusion regarding the haematological diagnosis. This report highlights the necessity of independent establishment of the diagnosis in every patient so that appropriate management decisions are taken.


Wegner's granulomatosis developing for the first time in a patient eight years after starting maintenance hemodialysis.
Wegener's granulomatosis is a serious autoimmune disorder characterized by necrotizing small-vessel vasculitis. It is a multisystem disease that primarily affects the lung and kidneys. Previous studies indicated few relapses of vasculitis after hemodialysis due to uremic immunosuppression. Our case report describes an end-stage renal failure patient who had developed non-caseating lung granulomata with giant cell formation and fibrinoid necrosis of arterial media that is consistent with Wegner's granulomatosis for the first time and eight years after initiation of maintenance hemodialysis. We believe that such a phenomenon has rarely been reported.


Peer support to improve diabetes care: an implementation evaluation of the Australasian Peers for Progress Diabetes Program.
Several studies have now demonstrated the benefits of peer support in promoting diabetes control. The aim of this study is to evaluate the implementation of a cluster randomised controlled trial of a group-based, peer support program to improve diabetes self-management and thereby, diabetes control in people with Type 2 Diabetes in Victoria, Australia. The intervention program was designed to address four key peer support functions i.e. 1) assistance in daily management, 2) social and emotional support, 3) regular linkage to clinical care, and 4) ongoing and sustained support to assist with the lifelong needs of diabetes self-care management. The intervention participants attended monthly group meetings facilitated by a trained peer leader for 12 months. Data was collected on the intervention's reach, participation, implementation fidelity, groups' effectiveness and participants' perceived support and satisfaction with the intervention. The RE-AIM and PIPE frameworks were used to guide this evaluation. The trial reached a high proportion (79%) of its target population through mailed invitations. Out of a total of 441 eligible individuals, 273 (61.9%) were willing to participate. The intervention fidelity was high (92.7%). The proportion of successful participants who demonstrated a reduction in 5 years cardiovascular disease risk score was 65.1 and 44.8% in the intervention and control arm respectively. Ninety-four percent (94%) of the intervention participants stated that the program helped them manage their diabetes on a day to day basis. Overall, attending monthly group meetings provided 'a lot of support' to 57% and 'moderate' support to 34% of the participants. Peer support programs are feasible, acceptable and can be used to supplement treatment for patients motivated to improve behaviours related to diabetes. However, program planners need to focus on the participation component in designing future programs. The use of two evaluation frameworks allowed a comprehensive evaluation of the trial from the provider-, participant- and public health perspective. The learnings gained from this evaluation will guide and improve future implementation by improving program feasibility for adoption and acceptability among participants, and will ultimately increase the likelihood of program effectiveness for the participants. Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12609000469213 . Registered 16 June 2009.


A quantitative assay for a Clara cell-specific protein and its application in the study of development of pulmonary airways in the rat.
Rat lung lavage contains a 10 kDa protein that has been shown by immunocytochemistry to be specific for Clara cells. An inhibition enzyme-linked immunosorbent assay was established for this protein using rabbit antibody to the 10 kDa Clara cell protein. The assay has a sensitivity of about 3.0 ng/ml and a working range of about 5 to 50 ng/ml. Quantitation of the 10 kDa protein in amniotic fluid revealed an increase of about 4-fold at day 20 of gestation. The 10 kDa protein content of lung homogenate increased steadily from day 18 of gestation to 1 wk after birth, after which a decline was observed. Nearly 60-fold increase in the concentration of the 10 kDa Clara cell protein in lungs was noted from day 18 of gestation to birth and a further about 7-fold increase was noted from the day of birth to 1 wk of age. A progressive increase in the 10 kDa protein, with increasing age, was also noted on immunoblot analysis of lung homogenates. As judged from the immunoblots of lung homogenates, stained with rabbit antirat 10 kDa protein antiserum, the content of an antigenically similar 200 kDa Clara cell protein was negligible. The quantitative results for 10 kDa Clara cell protein parallel the results of immunocytochemistry and quantitation of the volume density of Clara cell granules indicating that quantitation for the 10 kDa protein could be used to monitor the development of Clara cells and that of the pulmonary airways.


Revision Stapedectomy with Necrosis of the Long Process of the Incus: Does the Degree of Necrosis Matter? A Retrospective Clinical Study.
To discuss the different modalities for managing necrosis of the long process of the incus in revision stapedectomy on the basis of the degree of necrosis and compare the results with those reported in the literature. Thirty-six patients underwent revision stapedectomy with the necrosis of the long process of the incus from 2009 to 2016. The patients were divided into three groups on the basis of the degree of necrosis. For group A (minimal necrosis), augmentation technique with bone cement was performed. For group B (partial necrosis), the cement plug technique was performed. For group C (sever necrosis), malleus relocation with malleovestibulopexy was performed using reshaped necrosed incus. Air and bone conduction thresholds at frequencies of 500-3000 Hz were reviewed pre- and postoperatively using conventional audiometry. The air-bone gap (ABG) and bone conduction thresholds were measured. Postoperative ABG was reduced to <10 dB in 28 cases (77.8%) and <20 dB in all cases (100%). There was no significant change in postoperative bone conduction thresholds. The mean patient follow-up duration was 23 (range, 18-36) months. The cement plug technique was used in 75% of cases. Managing necrosis of the long process of the incus in revision stapedectomy should be considered according to the degree of necrosis. The cement plug technique is considered to be a reasonable option in most cases. Malleus relocation with malleovestibulopexy is an effective alternative to prosthesis.


Understanding the interactions between iron supplementation, infectious disease and adverse birth outcomes is essential to guide public health recommendations.
Pregnant women are highly susceptible to anaemia and iron deficiency due to the increased demands of pregnancy as well as other factors. Iron supplementation is recommended in pregnancy, yet the benefits on newborn outcomes are variable between populations, most likely due to the heterogeneity in the prevalence of iron deficiency, detrimental birth outcomes and infectious diseases. Furthermore, there are concerns regarding iron supplementation in malaria-endemic areas due to reports of increased risk of malaria in those receiving iron. This is compounded by limited knowledge of how iron deficiency, anaemia, malaria, and other infections may interact to influence birth outcomes. In a recent cohort study in Papua New Guinea, where there is a high burden of infections and iron deficiency, we found that iron deficiency in pregnancy was associated with a reduced risk of adverse birth outcomes. However, this effect could not be wholly explained by interactions between iron deficiency and malaria. We proposed that iron deficiency may confer a degree of protection against other infectious pathogens, which in turn caused improvements in birthweight. We argue that further studies in multiple populations are crucial to elucidate interactions between iron status, iron supplementation and birthweight as well as to understand the context-specific benefits of iron supplementation in pregnancy and inform public policy. Focus should be given to haematological studies on anaemia, haemodilution and iron absorption, as well as investigating infectious diseases and other nutritional deficiencies. This is a particular priority in resource-constrained settings where the prevalence of iron deficiency, poor nutrition, infections and poor birth outcomes are high. While current recommendations of iron supplementation and malaria prophylaxis to reduce the burden of poor pregnancy outcomes should be supported, the strength of evidence underpinning these must be improved and new insights should be garnered in order to maximise improvements in maternal and child health.Please see related article: https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-018-1146-z .Please see related article: https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-019-1375-9 .


Agile Co-Creation for Robots and Aging (ACCRA) Project: new technological solutions for older people.
Worldwide population is getting older. The older persons want to stay independent and wish to increase their engagement in social activities to tackle loneliness, depression, and isolation. Starting from these assumptions, we developed the ACCRA project (Agile Co-Creation for Robots and Aging) with the aim to enable the development of advanced ICT Robotics-based solutions for extending active and healthy aging in daily life by defining, developing and demonstrating an agile co-creation development process. ACCRA robotics solutions will be designed and developed to be tested in three different domains: mobility, daily life, socialization support in four countries (i.e., France, Netherlands, Italy, and Japan). The proposed approach identifies four different phases: (1) needs analysis, (2) agile co-creation, (3) experimentation, and (4) sustainability analysis. Currently, the first two phases were almost completed. For the needs phase, we have used the following recruitment criteria: (1) for mobility: age ≥ 60 years, the and presence of mobility issues assessed by Older Mobility Scale (EMS) with a score > 13; (2) for daily life: age ≥ 60 years, and the presence of difficulties engaging in housework assessed by Autonomie Gérontologie Groupes Iso-Ressources (AGGIR) with a GIR score ≥ 4; (3) for socialization support: age ≥ 60 years, and the absence or mild level of cognitive impairment assessed by Mini Mental State Examination (MMSE) with a score ≥ 24. The needs analysis and first co-creation sessions focus attention on the experience of older in the four countries. Preliminary results showed how, in all the pilot sites, many expectations were raised from older, formal and informal caregivers about the application of the technology into their life. Minor concerns existed about privacy, real efficacy and modularity in a real-world environment. Overall, a good attitude was recorded towards the use of technologies to support life and promote independent living. Moreover, the older engaged in our studies showed a great interest to be actively involved in the developing phase of something built based on their needs. The availability of new solutions to increase independence and quality of life in a sustainable manner appears to be mandatory in the actual society considering the actual socio-economic situation over the industrial countries.


How to recruit and retain health workers in underserved areas: the Senegalese experience.
Significant regional disparities in human resources for health deployment in Senegal weaken the country's health system and compromise population health. In recent years, the Ministry of Health adopted measures to improve the posting, recruitment and retention of health workers in rural and remote areas. One was the introduction of a special contracting system to recruit health workers. Health workers in Senegal are concentrated in specific urban centres, particularly Dakar. Whereas the Dakar region has 0.2 physicians per 1000 population, the Fatick, Kaolack, Kolda and Matam regions have fewer than 0.04. The density of midwives and, to a lesser extent, of nurses also varies considerably among different regions in Senegal. Between 2006 and 2008, the introduction of the special contracting system contributed to the successful recruitment of health workers in remote and rural regions and the reopening of health outposts. The introduction of a special contracting system for health workers was a successful approach to reopening health posts in regions with low health workforce density in Senegal. However, the long-term sustainability of such an approach, particularly in fiscal terms, must be considered, as a single policy intervention may not be enough to address the diverse and complex challenges in human resources for health facing different regions of Senegal.


The Impact of Childhood Maltreatment on Intravenous Ketamine Outcomes for Adult Patients with Treatment-Resistant Depression.
Childhood maltreatment is associated with a poor treatment response to conventional antidepressants and increased risk for treatment-resistant depression (TRD). The N-methyl-D-aspartate receptor (NDMAR) antagonist ketamine has been shown to rapidly improve symptoms of depression in patients with TRD. It is unknown if childhood maltreatment could influence ketamine's treatment response. We examined the relationship between childhood maltreatment using the Childhood Trauma Questionnaire (CTQ) and treatment response using the Quick Inventory of Depressive Symptoms-Self Report (QIDS-SR) in TRD patients receiving intravenous ketamine at a community outpatient clinic. We evaluated treatment response after a single infusion (n = 115) and a course of repeated infusions (n = 63). Repeated measures general linear models and Bayes factor (BF) showed significant decreases in QIDS-SR after the first and second infusions, which plateaued after the third infusion. Clinically significant childhood sexual abuse, physical abuse, and cumulative clinically significant maltreatment on multiple domains (maltreatment load) were associated with better treatment response to a single and repeated infusions. After repeated infusions, higher load was also associated with a higher remission rate. In contrast to conventional antidepressants, ketamine could be more effective in TRD patients with more childhood trauma burden, perhaps due to ketamine's proposed ability to block trauma-associated behavioral sensitization.


Evolutionarily Successful Asian 1 Dengue Virus 2 Lineages Contain One Substitution in Envelope That Increases Sensitivity to Polyclonal Antibody Neutralization.
The 4 dengue virus serotypes (DENV-1-4) cause the most prevalent mosquito-borne viral disease of humans worldwide. DENV-2 Asian 1 (A1) genotype viruses replaced the Asian-American (AA) genotype in Vietnam and Cambodia, after which A1 viruses containing Q or M at envelope (E) residue 160 became more prevalent than those with residue 160K in both countries (2008-2011). We investigated whether these substitutions conferred a fitness advantage by measuring neutralizing antibody titer against reporter virus particles (RVPs) representing AA, A1-160K, A1-160Q, and A1-160M, using patient sera from Vietnam and a well-characterized Nicaraguan cohort. Surprisingly, we found that A1-160Q and A1-160M RVPs were better neutralized by heterologous antisera than A1-160K. Despite this, Vietnamese patients infected with A1-160Q or A1-160M viruses had higher viremia levels than those infected with A1-160K. We thus found that independent lineages in Vietnam and Cambodia acquired a substitution in E that significantly increased polyclonal neutralization but nonetheless were successful in disseminating and infecting human hosts.


Imprinting on Clinical Rotations: Multisite Survey of High- and Low-Value Medical Student Behaviors and Relationship with Healthcare Intensity.
Physician behaviors are important to high-value care, and the learning environment medical students encounter on clinical clerkships may imprint their developing practice patterns. To explore potential imprinting on clinical rotations by (a) describing high- and low-value behaviors among medical students and (b) examining relationships with regional healthcare intensity (HCI). Multisite cross-sectional survey PARTICIPANTS: Third- and fourth-year students at nine US medical schools MAIN MEASURES: Survey items measured high-value (n = 10) and low-value (n = 9) student behaviors. Regional HCI was measured using Dartmouth Atlas End-of-Life Chronic Illness Care data (ratio of physician visits per decedent compared with the US average, hospital care intensity index, ratio of medical specialty to primary care physician visits per decedent). Associations between regional HCI and student behaviors were examined using unadjusted and adjusted (controlling for age, sex, and year in school) logistic regression analyses, using median item ratings to summarize reported engagement in high- and low-value behaviors. Of 2623 students invited, 1304 (50%) responded. Many reported trying to determine healthcare costs (1085/1234, 88%), but only 45% (571/1257) reported including cost details in case presentations. Students acknowledged suggesting tests solely to anticipate what their supervisor would want (1143/1220, 94%), show off their ability to generate a broad differential diagnosis (1072/1218, 88%), satisfy curiosity (958/1217, 79%), protect the team from liability (938/1215, 77%), and build clinical experience (533/1217, 44%). Students in higher intensity regions reported significantly more low-value behaviors: each one-unit increase in the ratio of physician visits per decedent increased the odds of reporting low-value behaviors by 20% (OR 1.20, 95% CI 1.04-1.38; P = 0.01). Third- and fourth-year medical students report engaging in both high- and low-value behaviors, which are related to regional HCI. This underscores the importance of the clinical learning environment and suggests imprinting is already underway during medical school.


Human sodium channel gating defects caused by missense mutations in S6 segments associated with myotonia: S804F and V1293I.
1. Missense mutations in the alpha-subunit of the human skeletal muscle sodium channel (hSkM1) have been detected in some heritable forms of myotonia. By recording Na+ currents from cells transfected with cDNA encoding either wild-type or mutant hSkM1, we characterized the functional consequences of two myotonia-associated mutations that lie at the cytoplasmic end of the sixth transmembrane segment in domain II (S804F) or domain III (V1293I). 2. Both mutations caused modest, but unequivocal, alterations in the voltage-dependent gating behaviour of hSkM1. For S804F, the abnormalities were limited to fast inactivation: the persistent Na+ current at the end of a 50 ms depolarization was increased 3-fold, the rate of inactivation from the open state was slowed 2-fold, and the voltage dependence of fast inactivation (h) was shifted by +3 mV. V1293I also disrupted fast inactivation, as evidenced by a 3-fold faster rate of recovery at hyperpolarized potentials (-70 mV). Activation was altered as well for V1293I: the voltage dependence was shifted by -6 mV (hyperpolarized). 3. Slow inactivation was not altered by S804F or V1293I. 4. We conclude that S804F and V1293I are not benign polymorphisms. Either mutation causes detectable alterations in channel gating and, in model simulations, the magnitude of the defects is sufficient to produce runs of myotonic discharges.


Review article: anti-adhesion therapies for inflammatory bowel disease.
A high proportion of patients with inflammatory bowel disease (IBD) do not achieve clinical remission with the current therapies including mesalazine (mesalamine), immunossupresants (IMS) and antibodies against tumour necrosis factor (anti-TNF). Moreover, IMS and anti-TNF involve a nonnegligible risk for infections and/or malignancies. The anti-adhesion molecules are one of the most interesting new treatments because of their gut-selectivity. To review the physiopathology of the adhesion molecules and the current drugs targeting this mechanism. We performed a literature review in PubMed and in clinicaltrials.gov using the terms 'anti-adhesion molecules', 'inflammatory bowel disease', 'natalizumab', 'vedolizumab', 'AMG181', 'Etrolizumab', 'PF-00547659', 'AJM300', 'Alicaforsen' and 'CCX282-B' up to November 2013. A total of eight drugs were found including those targeting the α4β1, α4β7 or αEβ7 integrins as well as the ICAM-1 and MAdCAM-1 addressins and the chemokine receptor 9. The rationale for these drugs is the blockade of gut-homing T lymphocytes and the ones targeting the α4β7/MAdCAM-1 interaction presented the most promising results in luminal disease. Vedolizumab, an α4β7 antibody, has completed phase 3 trials with very positive results especially for ulcerative colitis. However, many questions remain unanswered such as the effect of these therapies in perianal disease and extraintestinal manifestations. The blockade of the α4β7/MAdCAM-1 interaction and especially vedolizumab is an effective and safe gut-specific treatment for IBD. Further studies are needed to clarify the efficacy and safety of the other anti-adhesion drugs and to define the specific indications of these therapies in the different scenarios of IBD.


Mean Recency Period for Estimation of HIV-1 Incidence with the BED-Capture EIA and Bio-Rad Avidity in Persons Diagnosed in the United States with Subtype B.
HIV incidence estimates are used to monitor HIV-1 infection in the United States. Use of laboratory biomarkers that distinguish recent from longstanding infection to quantify HIV incidence rely on having accurate knowledge of the average time that individuals spend in a transient state of recent infection between seroconversion and reaching a specified biomarker cutoff value. This paper describes five estimation procedures from two general statistical approaches, a survival time approach and an approach that fits binomial models of the probability of being classified as recently infected, as a function of time since seroconversion. We compare these procedures for estimating the mean duration of recent infection (MDRI) for two biomarkers used by the U.S. National HIV Surveillance System for determination of HIV incidence, the Aware BED EIA HIV-1 incidence test (BED) and the avidity-based, modified Bio-Rad HIV-1/HIV-2 plus O ELISA (BRAI) assay. Collectively, 953 specimens from 220 HIV-1 subtype B seroconverters, taken from 5 cohorts, were tested with a biomarker assay. Estimates of MDRI using the non-parametric survival approach were 198.4 days (SD 13.0) for BED and 239.6 days (SD 13.9) for BRAI using cutoff values of 0.8 normalized optical density and 30%, respectively. The probability of remaining in the recent state as a function of time since seroconversion, based upon this revised statistical approach, can be applied in the calculation of annual incidence in the United States.


Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.
Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.


Developmental course of hypertension and regional cerebral blood flow in stroke-prone spontaneously hypertensive rats.
Regional cerebral blood flow (rCBF) was repeatedly measured by the hydrogen clearance method in the frontal cortex of stroke-prone spontaneously hypertensive rats (SHRSP) at the age of 50 days and thereafter. When SHRSP rats developed severe hypertension (over 200 mg Hg at the age of 60 days) rCBF began to decrease abruptly in the frontal cortex--one of the three predilection sites of stroke in these rats. In contrast, such a reduction in rCBF was not noted in either stroke-resistant spontaneously hypertensive rats (SHRSR) which developed moderate hypertension (under 200 mg Hg), or in Wistar-Kyoto rats (WK) with normal blood pressure (under 15 mm Hg). A similar marked reduction of rCBF with severe hypertension (over 200 mm Hg) was also detected in apoplectic gene-free renal infarction hypertensive rats (RHR) experimentally produced from age-matched WK animals. Blood samples were obtained through an implanted femoral artery canula without disturbing the nonanesthetized SHRSP, SHRSR and WK rats. Arterial blood gas analysis (PaCO2, PaO2 and pH) showed no significant differences at the age of 5 months in any of these rats. Chemical cerbrovascular reactivity, that is, an increase in rCBF in response to CO2 inhalation, showed no significant difference among SHRSP rats from the age of 50 days to 5 months. However, it markedly decreased in SHRSP rats at the age of 9 months and thereafter (the average age of male SHRSP rats which develop stroke is 9 months). The present study showed stroke did not occur in antihypertensive agent-treated SHRSP rats. In these SHRSP rats rCBF did not decrease as long as blood pressure was well-controlled.


Bet-hedging as a mechanism for the evolution of polyandry, revisited.
Females that mate with multiple males (polyandry) may reduce the risk that their eggs are fertilized by a single unsuitable male. About 25 years ago it was hypothesized that bet-hedging could function as a mechanism favoring the evolution of polyandry, but this idea is controversial because theory indicates that bet-hedging via polyandry can compensate the costs of mating only in small populations. Nevertheless, populations are often spatially structured, and even in the absence of spatial structure, mate-choice opportunity can be limited to a few potential partners. We examined the effectiveness of bet-hedging in such situations with simulations carried out under two scenarios: (1) intrinsic male quality, with offspring survival determined by male phenotype (male's ability to generate viable offspring), and (2) genetic incompatibility (offspring fitness determined nonadditively by parental genotypes). We find higher fixation probabilities for a polyandrous strategy compared to a monandrous strategy if complete reproductive failure due to male effects or parental incompatibility is pervasive in the population. Our results also indicate that bet-hedging polyandry can delay the extinction of small demes. Our results underscore the potential for bet-hedging to provide benefits to polyandrous females and have valuable implications for conservation biology.


Platelets and vascular integrity: how platelets prevent bleeding in inflammation.
Platelets play a central role in primary hemostasis by forming aggregates that plug holes in injured vessels. Half a century ago, detailed studies of the microvasculature by electron microscopy revealed that under inflammatory conditions that do not induce major disruption to vascular structure, individual platelets are mobilized to the vessel wall, where they interact with leukocytes and appear to seal gaps that arise between endothelial cells. Recent developments in genetic engineering and intravital microscopy have allowed further molecular and temporal characterization of these events. Surprisingly, it turns out that platelets support the recruitment of leukocytes to sites of inflammation. In parallel, however, they exercise their hemostatic function by securing the integrity of inflamed blood vessels to prevent bleeding from sites of leukocyte infiltration. It thus appears that platelets not only serve in concert as building blocks of the hemostatic plug but also act individually as gatekeepers of the vascular wall to help preserve vascular integrity while coordinating host defense. Variants of this recently appreciated hemostatic function of platelets that we refer to as "inflammation-associated hemostasis" are engaged in different contexts in which the endothelium is challenged or dysfunctional. Although the distinguishing characteristics of these variants and the underlying mechanisms of inflammation-associated hemostasis remain to be fully elucidated, they can differ notably from those supporting thrombosis, thus presenting therapeutic opportunities.


Can the NICE "end-of-life premium" be given a coherent ethical justification?
In 2009 the UK National Institute for Health and Clinical Excellence (NICE) announced that its health technology appraisal committees would henceforth give special additional weight to health gains from life-extending end-of-life treatments. This was a response to mounting concern from NICE's stakeholders that effective new drugs for end-stage cancer often fail NICE's standard test of cost effectiveness. This change of policy may be justifiable on procedural grounds as the result of a democratic political process responding to stakeholder concerns. However, according to the "accountability for reasonableness" framework proposed by the philosopher Norman Daniels and endorsed by NICE, there also needs to be transparency about the substantive ethical grounds for public health care resource allocation decisions. In that spirit, I analyze eleven potentially relevant justifications for the NICE "end-of-life premium," drawn from the economics and philosophy literature: (1) rule of rescue, (2) fair chances, (3) ex post willingness to pay, (4) caring externality, (5) financial protection, (6) symbolic value, (7) diminishing marginal value of future life years, (8) concentration of benefits, (9) dread, (10) time to set your affairs in order, and (11) severity of illness. I conclude that none of them yields a coherent ethical justification for the NICE end-of-life premium.


Lysosome Fusion Maintains Phagosome Integrity during Fungal Infection.
Phagosomes must maintain membrane integrity to exert their microbicidal function. Some microorganisms, however, survive and grow within phagosomes. In such instances, phagosomes must expand to avoid rupture and microbial escape. We studied whether phagosomes regulate their size to preserve integrity during infection with the fungal pathogen Candida albicans. Phagosomes release calcium as C. albicans hyphae elongate, inducing lysosome recruitment and insertion, thereby increasing the phagosomal surface area. As hyphae grow, the expanding phagosome consumes the majority of free lysosomes. Simultaneously, lysosome biosynthesis is stimulated by activation of TFEB, a transcriptional regulator of lysosomal biogenesis. Preventing lysosomal insertion causes phagosomal rupture, NLRP3 inflammasome activation, IL-1β secretion and host-cell death. Whole-genome transcriptomic analysis demonstrate that stress responses elicited in C. albicans upon engulfment are reversed if phagosome expansion is prevented. Our findings reveal a mechanism whereby phagosomes maintain integrity while expanding, ensuring that growing pathogens remain entrapped within this microbicidal compartment.


VA/Q inhomogeneity and AaDO2 in man during exercise: effect of SF6 breathing.
Pulmonary gas exchange was studied in five normal subjects both at rest and during moderate steady-state exercise on a bicycle ergometer while breathing a) room air and b) a mixture of 20.9% O2-balance sulfur hexafluoride (SF6). The alveolar-arterial oxygen pressure differences (AaDO2) widened significantly from rest to exercise. Breathing the O2-SF6 mixture reduced the AaDo2 significantly from 10.9 to 4.2 Torr at rest and from 15.5 to 10.1 Torr during exercise (P less than or equal to 0.01). There were no concurrent changes in metabolism, cardiac output, or heart rate during the SF6 breathing. Possible changes in the anatomic shunt fraction, alveolar-end-capillary equilibration, or the distribution of blood flow cannot account for these observations. We conclude that the AaDO2 increase during exercise reflects an inhomogeneity of ventilation-perfusion ratios (VA/Q) most probably arising within regions of the lung (intraregional inhomogeneity) rather than between regions (interregional inhomogeneity).


Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.
CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.


Upregulation of MMP-2 by HMGA1 promotes transformation in undifferentiated, large-cell lung cancer.
Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1-MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors.


Palladium-catalyzed Suzuki-Miyaura coupling of amides by carbon-nitrogen cleavage: general strategy for amide N-C bond activation.
The first palladium-catalyzed Suzuki-Miyaura cross-coupling of amides with boronic acids for the synthesis of ketones by sterically-controlled N-C bond activation is reported. The transformation is characterized by operational simplicity using bench-stable, commercial reagents and catalysts, and a broad substrate scope, including substrates with electron-donating and withdrawing groups on both coupling partners, steric-hindrance, heterocycles, halides, esters and ketones. The scope and limitations are presented in the synthesis of >60 functionalized ketones. Mechanistic studies provide insight into the catalytic cycle of the cross-coupling, including the first experimental evidence for Pd insertion into the amide N-C bond. The synthetic utility is showcased by a gram-scale cross-coupling and cross-coupling at room temperature. Most importantly, this process provides a blueprint for the development of a plethora of metal catalyzed reactions of typically inert amide bonds via acyl-metal intermediates. A unified strategy for amide bond activation to enable metal insertion into N-C amide bond is outlined ().


Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts.
Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.


Cigarette smoking and appendectomy: effect on clinical course of diverticulosis.
To investigate the effect of appendectomy and cigarette smoking on the clinical course of diverticulosis. A retrospective case-control study of 207 consecutive patients (45.8% male mean age 64.0 years), 150 with asymptomatic diverticulosis, and 57 with acute diverticulitis. Diagnosis of diverticulosis was defined on the basis of clinical and colonoscopic criteria, diverticulitis was defined by means of clinical, colonoscopic and computerised tomography criteria. Logistic regression function was used to define the relationship between the dependent variable (diverticulitis) and several covariates: sex, age, body mass index, smoking habit, and history of appendectomy. According to the final model, the risk of diverticulitis was 4.94-fold higher (95% confidence interval: 1.98-12.37) in patients with a history of appendectomy with emergency resection, compared to patients not submitted to appendectomy or with a history of elective resection (P < 0.001); and 2.79-fold higher (95% confidence interval: 1.30-5.96) in smokers than in non-smokers (P = 0.008). The effects of the two determinants were found to be independent, thus the cumulative risk of diverticulitis was 13.78-fold higher for smokers with a history of emergency surgical treatment. Smoking and emergency appendectomy are important predictive factors for the clinical course of diverticulosis.


Anti-cytokine therapy for rheumatoid arthritis.
Tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) are important in mediating inflammation in rheumatoid arthritis (RA). Randomized phase II and III clinical trials of anti-TNF reagents (infliximab and etanercept) have demonstrated an acceptable safety profile and marked clinical efficacy in cases of RA that have not responded adequately to conventional therapy. Combination therapy with methotrexate (MTX) appears to be particularly effective in patients whose disease activity persists despite prior disease-modifying antirheumatic drugs (DMARDs) and ongoing MTX monotherapy. DMARD-recalcitrant disease may become the main indication for the use of anti-TNF drugs in patients with RA. Trials of IL-1 receptor antagonist show a relatively modest anti-inflammatory effect and a possible retardation of joint damage. Whether anti-TNF therapy protects joints from structural damage is under investigation. One anti-TNF reagent has already been approved in the United States for the treatment of RA, and other cytokine antagonists or agonists are under development.


Photoinduced protein modifications by methylene blue and naproxen.
HPLC and emission spectroscopy were used to investigate UVA photosensitization of methylene blue (MB) or naproxen (NAP) towards bovine serum albumin (BSA). In addition, time resolved singlet oxygen measurements were carried out. The most stable drug : protein adducts stoichiometry of MB-BSA (1 : 1) and NAP-BSA (9 : 1) were verified by means of binding constant determination. UVA photosensitization of MB or NAP on BSA was studied by monitoring tryptophan (Trp) residue integrity. The sensitized photodegradation of the BSA resulted in different degrees of Trp damage. Thus, protein damage was determined by quantitative measurements of the different Trp (photo)-products. Indeed, many of these Trp derivatives are diagnostic for the photosensitization mechanism and some of them, for the first time in this work, were obtained by UVA photosensitization in proteins. The analysis of quantum yields of the photoproduct distribution allowed to weigh up the type I/II contribution to the UVA photosensitization mechanism. As expected, additional experiments in deuterated solvent resulted in an increase of the photodegradation quantum yields for those species where a singlet oxygen mechanism was involved. The UVA mediated generation of these Trp derivatives is consistent with the occurrence of singlet oxygen formation (almost dominant in MB), and photoionization (significant in NAP) within the protein matrix. Additional experiments at lower NAP concentration, as well as with human serum albumin (which differs for Trp content and, partially, localization), support further the molecular mechanism of photosensitization proposed. The results obtained in the case of this more complex system are in agreement with the free Trp model, even if, in almost all cases, the Trp photoproduct formation quantum yields are lower, due to the higher number of sensitization targets in the proteins.


An inhibitor of nuclear scaffold protease blocks chemical transformation of fibroblasts.
A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)


Dynamics of Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus spores in urban catch basins after simultaneous application against mosquito larvae.
Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (± 0.08) and 4.13 (± 0.09) log ml(-1) in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 10(5) g(-1) were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area.


Regulation and binding properties of S fimbriae cloned from E. coli strains causing urinary tract infection and meningitis.
S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin (sfa) gene cluster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and PC, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promoters via SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, PA, located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes.