Repeated vulvovaginal fungal infections cause persistent pain in a mouse model of vulvodynia.
Provoked vestibulodynia, the most common form of vulvodynia (unexplained pain of the vulva), is a prevalent, idiopathic pain disorder associated with a history of recurrent candidiasis (yeast infections). It is characterized by vulvar allodynia (painful hypersensitivity to touch) and hyperinnervation. We tested whether repeated, localized exposure of the vulva to a common fungal pathogen can lead to the development of chronic pain. A subset of female mice subjected to recurrent Candida albicans infection developed mechanical allodynia localized to the vulva. The mice with allodynia also exhibited hyperinnervation with peptidergic nociceptor and sympathetic fibers (as indicated by increased protein gene product 9.5, calcitonin gene-related peptide, and vesicular monoamine transporter 2 immunoreactivity in the vaginal epithelium). Long-lasting behavioral allodynia in a subset of mice was also observed after a single, extended Candida infection, as well as after repeated vulvar (but not hind paw) inflammation induced with zymosan, a mixture of fungal antigens. The hypersensitivity and hyperinnervation were both present at least 3 weeks after the resolution of infection and inflammation. Our data show that infection can cause persistent pain long after its resolution and that recurrent yeast infection replicates important features of human provoked vulvodynia in the mouse.


Quantum dots to monitor RNAi delivery and improve gene silencing.
A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and 'off target' effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell-cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.


Organic field-effect transistor-based flexible sensors.
Flexible electronic devices have attracted a great deal of attention in recent years due to their flexibility, reduced complexity and lightweight. Such devices can conformably attach themselves to any bendable surface and can possess diverse transduction mechanisms. Consequently, with continued emphasis on innovation and development, major technological breakthroughs have been achieved in this area. This review focuses on the advancements of using organic field-effect transistors (OFETs) in flexible electronic applications in the past 10 years. In addition, to the above mentioned features, OFETs have multiple advantages such as low-cost, readout integration, large-area coverage, and power efficiency, which yield synergy. To begin with, we have introduced organic semiconductors (OSCs), followed by their applications in various device configurations and their mechanisms. Later, the use of OFETs in flexible sensor applications is detailed with multiple examples. Special attention is paid to discussing the effects induced on physical parameters of OFETs with respect to variations in external stimuli. The final section provides an outlook on the mechanical aspects of OSCs, activation and revival processes of sensory layers, small area analysis, and pattern recognition techniques for electronic devices.


The activity view of inner speech.
We distinguish two general approaches to inner speech (IS)-the "format" and the "activity" views-and defend the activity view. The format view grounds the utility of IS on features of the representational format of language, and is related to the thesis that the proper function of IS is to make conscious thinking possible. IS appears typically as a product constituted by representations of phonological features. The view also has implications for the idea that passivity phenomena in cognition may be misattributed IS. The activity view sees IS as a speaking activity that does not have a proper function in cognition. It simply inherits the array of functions of outer speech. We argue that it is methodologically advisable to start from this variety of uses, which suggests commonalities between internal and external activities. The format view has several problems; it has to deny "unsymbolized thinking"; it cannot easily explain how IS makes thoughts available to consciousness, and it cannot explain those uses of IS where its format features apparently play no role. The activity view not only lacks these problems but also has explanatory advantages: construing IS as an activity allows it to be integrally constituted by its content; the view is able to construe unsymbolized thinking as part of a continuum of phenomena that exploit the same mechanisms, and it offers a simple explanation for the variety of uses of IS.


'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis.
Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.


Plasma observations at Io with the Galileo spacecraft.
Plasma measurements made during the flyby of Io on 7 December 1995 with the Galileo spacecraft plasma analyzers reveal that the spacecraft unexpectedly passed directly through the ionosphere of Io. The ionosphere is identified by a dense plasma that is at rest with respect to Io. This plasma is cool relative to those encountered outside the ionosphere. The composition of the ionospheric plasmas includes O++, O+ and S++, S+, and SO2+ ions. The plasma conditions at Io appear to account for the decrease in the magnetic field, without the need to assume that Io has a magnetized interior.


Crohn's disease and ulcerative colitis are associated with the DNA repair gene MLH1.
The purpose of this study was to determine whether there is an association between Crohn's disease and ulcerative colitis with MLH1. Identification of genes involved in the etiology of inflammatory bowel disease may lead to the development of markers that objectively can define disease and permit therapy. The treatment of Crohn's disease of the colon and ulcerative colitis also is complicated by difficulties in differentiating the two conditions. The DNA and clinical data were obtained on 126 unrelated individuals (45 Crohn's disease, 36 ulcerative colitis, and 45 control subjects without intestinal disease). Polymerase chain reaction products were analyzed by single-strand conformation polymorphisms (MLH1 exons 9, 11, 14, 15, and 16) and polyacrylamide gel electrophoresis (markers D3S1611 and D3S1768). All comparisons were analyzed by chi square test. The association between single haplotypes and disease was expressed as relative odds. MLH1 exons 9, 11, 14, and 16 were monomorphic. Two, four, and six alleles were detected in MLH1 exon 15, D3S1611, and D3S1768, respectively. Significant associations were observed for MLH1 exon 15/D3S1611 haplotypes AB (OR = 5.5; p = 0.007) and BA (p = 0.002) with Crohn's disease and for haplotypes AB (OR = 4.0; p = 0.042), BA (p = 0.035), and BC (OR = 6.1; p = 0.016) with ulcerative colitis. Family history of inflammatory bowel disease was associated with D3S1768/D3S1611 (p = 0.05) and MLH1 exon 15/D3S1611 haplotypes (p = 0.03). D3S1611/D3S1768 haplotype CD (OR = 11.3; p = 0.03) was associated with disease, whereas MLH1 exon 15/D3S1611 haplotype AA (OR = 0.25; p = 0.02) was protective. Comparisons of MLH1 exon 15/D3S1611 haplotypes of Crohn's colitis and patients with ulcerative colitis were significant (p = 0.037). This study identifies a novel genetic and clinical association between MLH1 and inflammatory bowel disease.


Cancer incidence among pesticide applicators exposed to metolachlor in the Agricultural Health Study.
Metolachlor is one of the most widely used herbicides in the United States. We evaluated the incidence of cancer among pesticide applicators exposed to metolachlor in the Agricultural Health Study, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina. A total of 50,193 pesticide applicators were included. Detailed information on pesticide exposure and lifestyle factors was obtained from self-administered enrollment questionnaires completed between 1993 and 1997; average length of follow-up was 7.33 years. Two metolachlor exposure metrics were used : (i) lifetime days personally mixed or applied metolachlor and (ii) intensity-weighted lifetime days (lifetime days x an intensity level). Poisson regression analysis was used to estimate relative risks (RR) and 95% confidence intervals (95%CI) for cancer subtypes by tertiles of metolachlor exposure. No clear risk for any cancer subtype was found for exposure to metolachlor. A significantly decreased RR was found for prostate cancer in the highest category of lifetime days exposure (RR = 0.59; 95%CI, 0.39-0.89) and in the second highest category of intensity-weighted lifetime days exposure (RR = 0.66; 95%CI, 0.45-0.97); however, the test for trend was not significant for either exposure metric. A nonsignificantly increased risk was found for lung cancer with lifetime days exposure in the highest category (RR = 2.37; 95%CI, 0.97-5.82, p-trend = 0.03) but not with intensity-weighted lifetime days. Given the widespread use of metolachlor and the frequent detection of metolachlor in both surface and ground water, future analyses of the AHS will allow further examination of long-term health effects, including lung cancer and the less common cancers.


The Role of Mediobasal Hypothalamic PACAP in the Control of Body Weight and Metabolism.
Body energy homeostasis results from balancing energy intake and energy expenditure. Central nervous system administration of pituitary adenylate cyclase activating polypeptide (PACAP) dramatically alters metabolic function, but the physiologic mechanism of this neuropeptide remains poorly defined. PACAP is expressed in the mediobasal hypothalamus (MBH), a brain area essential for energy balance. Ventromedial hypothalamic nucleus (VMN) neurons contain, by far, the largest and most dense population of PACAP in the medial hypothalamus. This region is involved in coordinating the sympathetic nervous system in response to metabolic cues in order to re-establish energy homeostasis. Additionally, the metabolic cue of leptin signaling in the VMN regulates PACAP expression. We hypothesized that PACAP may play a role in the various effector systems of energy homeostasis, and tested its role by using VMN-directed, but MBH encompassing, adeno-associated virus (AAVCre) injections to ablate Adcyap1 (gene coding for PACAP) in mice (Adcyap1MBHKO mice). Adcyap1MBHKO mice rapidly gained body weight and adiposity, becoming hyperinsulinemic and hyperglycemic. Adcyap1MBHKO mice exhibited decreased oxygen consumption (VO2), without changes in activity. These effects appear to be due at least in part to brown adipose tissue (BAT) dysfunction, and we show that PACAP-expressing cells in the MBH can stimulate BAT thermogenesis. While we observed disruption of glucose clearance during hyperinsulinemic/euglycemic clamp studies in obese Adcyap1MBHKO mice, these parameters were normal prior to the onset of obesity. Thus, MBH PACAP plays important roles in the regulation of metabolic rate and energy balance through multiple effector systems on multiple time scales, which highlight the diverse set of functions for PACAP in overall energy homeostasis.


Anesthesia of the sighthound.
The sighthounds are an ancient group of dog breeds that have been selectively bred for high-speed pursuit of prey by sight. Probably as a consequence of this selection process, these dogs have a number of idiosyncrasies that can potentially adversely affect their anesthetic management. These include (1) nervous demeanor which can lead to stress-induced clinical complications, such as hyperthermia; (2) lean body conformation with high surface-area-to-volume ratio, which predisposes these dogs to hypothermia during anesthesia; (3) hematological differences such as a higher packed cell volume and lower serum protein compared with other dog breeds which may complicate interpretation of preanesthetic blood work; (4) Impaired biotransformation of drugs by the liver resulting in prolonged recovery from certain intravenous anesthetics, especially thiopental; and increased risks of drug interactions. Safe anesthetic management of sighthounds should include sedative premedication and appropriate use of analgesic drugs to minimize perioperative stress. Thiopental, or any other thiobarbiturate, should not be used in these dogs. Propofol, ketamine/diazepam combination, and methohexital are recommended alternative intravenous anesthetics. Avoid coadministration of agents that inhibit drug biotransformation, such as chloramphenicol. Inhalation anesthesia using isoflurane is the preferred anesthetic maintenance technique. Core body temperature should be monitored closely and techniques to minimize hypothermia should be employed both during anesthesia and into the recovery period.


Conservative Treatment Seems the Best Choice in Adenocarcinoma In Situ of the Cervix Uteri.
To study diagnostic and therapeutic strategies, outcomes, and follow-up in a large series of women with adenocarcinoma in situ (AIS) of the uterine cervix and investigate if human papillomavirus (HPV) typing among women with negative cytology reports would have helped with early AIS detection. Records of 132 AIS cases diagnosed between 1989 and 2012 were retrieved. Clinical and pathological data were reviewed and analyzed. Mean age at diagnosis was 37 years. Seventy-two percent (n = 95) of all patients were asymptomatic; diagnosis was established using cytology and biopsy. Primary treatment for 124 patents was cold knife cone or loop electrosurgical excision procedure (LEEP). Positive margins were found in 18% of those women treated with CKC versus 40% in those treated with LEEP. The mean follow-up time was 62 months (range, 2-217 months; median, 46 months). Three recurrences were found after conservative treatment in 86 patients. High-risk HPV (hrHPV) positivity was detected in 115 (96%) of 120 patients, with HPV-18 being the most commonly occurring subtype (51%). There is a small risk of relapse after conservative therapy with cold knife cone or LEEP when resection margins are negative in women with AIS. Patients should be given the options of hysterectomy or conservative therapy with strict follow-up.


Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways.
The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.


Women drivers' behaviour, socio-demographic characteristics and accidents.
The purpose of this study was to examine factors which affect driving behaviour and accident rates in women in Australia. Two groups of women (aged 18-23 and 45-50 years) participating in the Australian Longitudinal Study on Women's Health, completed a mailed questionnaire on driver behaviour and road accidents. Self reported accident rates in the last 3 years were 1.87 per 100,000 km for the young drivers (n = 1199) and 0.59 per 100,000 km for the mid-age drivers (n = 1564); most accidents involved damage only, not injury. Mean scores for lapses obtained using the Driver Behaviour Questionnaire, were similar in the two age groups and similar to those found in other studies. In contrast, scores for errors and violations for the young women were higher than for the mid-age group and previous reports using the same instruments. Riskier driving behaviour among young women was associated with stress and habitual alcohol consumption. In the mid-age group, poorer driver behaviour scores were related to higher levels of education, feeling rushed, higher habitual alcohol consumption and lower life satisfaction scores. Accident rates in both groups were significantly related to lapses. Women born in non-English speaking countries had significantly higher risk of accidents compared to Australian-born women: relative risk = 3.40, 95% confidence interval (1.93, 5.98) for the young drivers; relative risk = 1.77, 95% confidence interval (1.11, 2.83) for mid-age drivers. These findings support the need for road safety campaigns targeted at young women to reduce dangerous driving practices, such as speeding, 'tail gating' and overtaking on the inside. There is also a need for further research to understand how lifestyle characteristics are associated with higher risk of accidents and to explore factors which might account for the higher risk for women drivers who were born overseas.


Physical Activity and Sports-Real Health Benefits: A Review with Insight into the Public Health of Sweden.
Positive effects from sports are achieved primarily through physical activity, but secondary effects bring health benefits such as psychosocial and personal development and less alcohol consumption. Negative effects, such as the risk of failure, injuries, eating disorders, and burnout, are also apparent. Because physical activity is increasingly conducted in an organized manner, sport's role in society has become increasingly important over the years, not only for the individual but also for public health. In this paper, we intend to describe sport's physiological and psychosocial health benefits, stemming both from physical activity and from sport participation per se. This narrative review summarizes research and presents health-related data from Swedish authorities. It is discussed that our daily lives are becoming less physically active, while organized exercise and training increases. Average energy intake is increasing, creating an energy surplus, and thus, we are seeing an increasing number of people who are overweight, which is a strong contributor to health problems. Physical activity and exercise have significant positive effects in preventing or alleviating mental illness, including depressive symptoms and anxiety- or stress-related disease. In conclusion, sports can be evolving, if personal capacities, social situation, and biological and psychological maturation are taken into account. Evidence suggests a dose-response relationship such that being active, even to a modest level, is superior to being inactive or sedentary. Recommendations for healthy sports are summarized.


Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.
Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington's disease. In view of these data and the involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF in the Golgi apparatus with respect to Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affected the KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75(NTR)). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.


Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198.
Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S.enterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S.enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. enterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. enterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.


Simulation training for dental foundation in oral and maxillofacial surgery - a new benchmark.
Simulation training involves reproducing the management of real patients in a risk-free environment. This study aims to assess the use of simulation training in the management of acutely ill patients for those in second year oral and maxillofacial surgery dental foundation training (DF2s). DF2s attended four full day courses on the recognition and treatment of acutely ill patients. These incorporated an acute life-threatening events: recognition and treatment (ALERT(™)) course, simulations of medical emergencies and case-based discussions on management of surgical inpatients. Pre- and post-course questionnaires were completed by all candidates. A maximum of 11 DF2s attended the course. The questionnaires comprised 1-10 rating scales and Likert scores. All trainees strongly agreed that they would recommend this course to colleagues and all agreed or strongly agreed that it met their learning requirements. All DF2s perceived an improvement in personal limitations, recognition of critical illness, communication, assessing acutely ill patients and initiating treatment. All participants felt their basic resuscitation skills had improved and that they had learned new skills to improve delivery of safety-critical messages. These techniques could be implemented nationwide to address the more complex educational needs for DF2s in secondary care. A new benchmark for simulation training for DF2 has been established.


Heavy-metal concentrations in small mammals from a diffusely polluted floodplain: importance of species- and location-specific characteristics.
The soil of several floodplain areas along large European rivers shows increased levels of heavy metals as a relict from past sedimentation of contaminants. These levels may pose risks of accumulation in food webs and toxicologic effects on flora and fauna. However, for floodplains, data on heavy-metal concentrations in vertebrates are scarce. Moreover, these environments are characterised by periodical flooding cycles influencing ecologic processes and patterns. To investigate whether the suggested differences in accumulation risks for insectivores and carnivores, omnivores, and herbivores are reflected in the actual heavy-metal concentrations in the species, we measured the current levels of Zn, Cu, Pb, and Cd in 199 specimens of 7 small mammal species (voles, mice, and shrews) and in their habitats in a diffusely polluted floodplain. The highest metal concentrations were found in the insectivorous and carnivorous shrew, Sorex araneus. Significant differences between the other shrew species, Crocidura russula, and the vole and mouse species was only found for Cd. The Cu concentration in Clethrionomys glareolus, however, was significantly higher than in several other vole and mouse species. To explain the metal concentrations found in the specimens, we related them to environmental variables at the trapping locations and to certain characteristics of the mammals. Variables taken into account were soil total and CaCl(2)-extractable metal concentrations at the trapping locations; whether locations were flooded or nonflooded; the trapping season; and the life stage; sex; and fresh weight of the specimens. Correlations between body and soil concentrations and location or specimen characteristics were weak. Therefore; we assumed that exposure of small mammals to heavy-metal contamination in floodplains is significantly influenced by exposure time, which is age related, as well as by dispersal and changes in foraging and feeding patterns under influence of periodic flooding.


Adenoviral vectors persist in vivo and maintain activated CD8+ T cells: implications for their use as vaccines.
CD8(+) T cell-numbers rapidly expand and then contract after exposure to their cognate antigen. Here we show that the sustained frequencies of transgene product-specific CD8(+) T cells elicited by replication-defective adenovirus vectors are linked to persistence of low levels of transcriptionally active adenovirus vector genomes at the site of inoculation, in liver, and lymphatic tissues. Continuously produced small amounts of antigen maintain fully active effector CD8(+) T cells, while also allowing for their differentiation into central memory cells. The long-term persistence of adenoviral vectors may be highly advantageous for their use as vaccines against pathogens for which T-cell-mediated protection requires both fully activated T cells for immediate control of virus-infected cells and central memory CD8(+) T cells that, because of their higher proliferative capacity, may be suited best to eliminate cells infected by pathogens that escaped the initial wave of effector T cells.


Genetically engineered structure-based vaccine for bluetongue disease.
At present the only vaccines used against bluetongue (BT) disease are live-attenuated virus vaccines. Since viruses with RNA genomes such as BT virus (BTV) have a high frequency of mutations, live virus vaccines could have breakthroughs (vaccine failures) and mutate into virulent strains. In addition, multiple BTV serotypes exist in nature which could potentially cause additional problems with live virus vaccines. The BTV genome is made up of 10 segments and therefore potentially could exchange these segments (or genes) randomly between different viruses including vaccine strains, generating novel viruses with mixed genes. Hence it is necessary to develop BTV vaccines that pose no such threat. Ideally BTV vaccines should be completely devoid of harmful genes. Recent protein expression technology has provided novel approaches for the development of intrinsically safe vaccines. The technology involves the synthesis of immunogenic proteins and particles that elicit highly protective immune responses. We have generated such vaccines, termed virus-like particle (VLP) vaccines. These vaccines (which do not carry either BTV or foreign genes) give the immune system information about viral structures so that it can generate a complete defence against the real virus infection very efficiently. A series of vaccine trials were undertaken outdoors under natural UV light using 50-200 BTV-susceptible sheep per trial. Vaccination trials of sheep showed that the VLPs were highly immunogenic, and protected sheep when animals were challenged with virulent virus even 15 months after the first immunisation. Moreover, a cocktail of five VLPs afforded protection against not only each of the homologous BTV serotypes but also against certain heterologous serotypes that are genetically related to some of these vaccine strains. VLPs representing a number of serotypes are currently available and can be produced fairly quickly if there is such a need. Based on our sequence data it can be predicted that a mixture of seven or eight types of VLPs (that are already available) will provide protection against at least 10 or more serotypes depending on their phylogenetic relationships. BTV VLPs offer particular advantages as potential vaccines over other systems. Large quantities of VLPs can be produced and easily purified using a one-step protocol based on the physical properties of the particle. More importantly, these particles are devoid of any nucleic acid and thus pose no threat by re-assortment or the re-emergence of virulence that attenuated vaccines can cause.


Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase.
Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.


How does performance of the Friedreich Ataxia Functional Composite compare to rating scales?
Progression of Friedreich ataxia (FRDA) is often measured using neurological rating scales such as the Friedreich Ataxia Rating Scale (FARS). Performance scales comprising functional measures have been used in other conditions due to their increased sensitivity and reproducibility and may replace examination-based measures. The aims of this study were to examine the relationship between the Friedreich Ataxia Functional Composite (FAFC) measures and characteristics of FRDA to determine if the FAFC is more sensitive to clinical change over time compared to its components. One hundred and twenty-two individuals completed the timed 25-foot walk (T25FW), 9-Hole Peg Test (9HPT) and the low-contrast letter acuity (LCLA) test at baseline, 63 at year 1, 34 at year 2 and 25 at year 3. Composite scores, Z2 (T25FW and 9HPT) and Z3 (T25FW, 9HPT and LCLA) were created. Correlation analyses were conducted. Change in FAFC components were examined over 1, 2, and 3 years. The FARS, Z2, Z3 and 9HPT showed significant change over all time points compared to baseline. The T25FW only demonstrated significant change over 3 years. The LCLA demonstrated no significant change over any of the time points. The FAFC shows significant change over time and indicates disease progression, however, this may result from individual components driving the differences. The LCLA showed no change over time, rendering Z3 redundant. The FAFC is of limited value in cohorts with non-ambulant individuals as it leads to skewing of the dataset and is better suited to less affected populations.


Effectiveness of surface protection for resin-modified glass-ionomer materials.
The purpose of this study was to evaluate the effectiveness of various surface treatments for resin-modified glass-ionomer restorative materials by determining dye uptake spectrophotometrically. Two hundred twenty-four specimens, 4.1 mm in diameter and 2.0 mm thick, were made of 3 materials: Vitremer, Fuji II LC, and Photac-Fil Aplicap. Specimens were divided into 15 groups. The positive and negative control specimens remained unprotected, while the experimental specimens were protected with Heliobond light-activated bonding resin, Colorama nail varnish, or surface coatings indicated by the manufacturers of the glass-ionomer materials: Finishing Gloss for Vitremer, Fuji Varnish for Fuji II LC, and Ketac Glaze for Photac-Fil. The disks were immersed in 0.05% methylene blue for 24 hours except for the negative control group, which was immersed in deionized water. After 24 hours, the disks were removed, washed, and individually placed in 1 mL of 65% nitric acid for 24 hours. The solutions were centrifuged and the spectrophotometric absorbance was determined at 606 nm. The dye uptake was expressed in micrograms of dye per milliliter, and the results were analyzed with the Kruskal-Wallis test. There were no differences in dye uptake among the 3 resin-modified glass-ionomer restorative materials; however, all of them required surface protection. The best surface protection for the 3 evaluated materials was obtained with Heliobond light-activated bonding resin.


Standard guidelines of care for vitiligo surgery.
Vitiligo surgery is an effective method of treatment for selected, resistant vitiligo patches in patients with vitiligo. PHYSICIAN'S QUALIFICATIONS: The physician performing vitiligo surgery should have completed postgraduate training in dermatology which included training in vitiligo surgery. If the center for postgraduation does not provide education and training in cutaneous surgery, the training may be obtained at the surgical table (hands-on) under the supervision of an appropriately trained and experienced dermatosurgeon at a center that routinely performs the procedure. Training may also be obtained in dedicated workshops. In addition to the surgical techniques, training should include local anesthesia and emergency resuscitation and care. Vitiligo surgery can be performed safely in an outpatient day care dermatosurgical facility. The day care theater should be equipped with facilities for monitoring and handling emergencies. A plan for handling emergencies should be in place, with which all nursing staff should be familiar. Vitiligo grafting for extensive areas may need general anesthesia and full operation theater facility in a hospital setting and the presence of an anesthetist is recommended in such cases. INDICATIONS FOR VITILIGO SURGERY: Surgery is indicated for stable vitiligo that does not respond to medical treatment. While there is no consensus on definitive parameters for stability, the Task Force suggests the absence of progression of disease for the past one year as a definition of stability. Test grafting may be performed in doubtful cases to detect stability. A detailed consent form elaborating the procedure and possible complications should be signed by the patient. The patient should be informed of the nature of the disease and that the determination of stability is only a vague guide. The consent form should specifically state the limitations of the procedure, about the possible future progression of disease and whether more procedures will be needed for proper results. The patient should be provided with adequate opportunity to seek information through brochures and one-to-one discussions. The need for concomitant medical therapy should be emphasized and the patient should understand that proper results take time (a few months to a year). Preoperative laboratory studies include hemogram including platelet counts, bleeding and clotting time (or prothrombin and activated partial thromboplastin time), and blood chemistry profile. Screening for antibodies for hepatitis B surface antigen and HIV is recommended depending on individual requirements. Lignocaine (2%) with or without adrenaline is generally used for anesthesia; infiltration and nerve block anesthesia are adequate in most cases. General anesthesia may be needed in patients with extensive lesions. Proper postoperative immobilization and care are very important to obtain satisfactory results.


Dot-blot hybridization and RT-PCR detection of extra small virus (XSV) associated with white tail disease of prawn Macrobrachium rosenbergii.
The availability of specific and reliable detection methods is essential for monitoring the health status of farmed species, particularly for viral diseases. Extra small virus (XSV), a virus-like particle, is associated with Macrobrachium rosenbergii Noda virus (MrNV) in white tail disease (WTD) of M. rosenbergii. We developed 2 genome-based detection methods for the identification of XSV, namely dot-blot hybridization and a single-step RT-PCR. Detection limits were established and are ca. 2.5 pg and 5 fg of viral RNA for dot-blot hybridization and RT-PCR, respectively. Application of the methods to field samples indicated that some animals positively diagnosed with MrNV did not contain XSV, at least within the detection limit of the methodology. This raises the question of the actual role of XSV and its interactions with MrNV in WTD of M. rosenbergii.


Hypoxanthine--guanine phosphoribosyltransferase activity in blood and skeletal muscles of normal and dystrophic mice.
1. Hypoxanthine--guanine phosphoribosyltransferase (HGPRT) activity was measured in erythrocyte haemolysates and quadriceps muscle extracts of normal and dystrophic 129 ReJ and C57 BL/6J mice with [8(-14)C]hypoxanthine as substrate and 5-phosphorylribose 1-pyrophosphate as a ribose 5-phosphate donor. [8(-14)C]Inosine monophosphate formed was separated by high-voltage electrophoresis and radioactivity was measured by liquid-scintillation counting. 2. In erythrocyte haemolysates, HGPRT activity was similar in normal and dystrophic C57 BL/6J mice but was significantly higher in dystrophic than in normal 129 ReJ mice. Elevated enzyme activity was observed only in mice that were clinically severely affected. 3. In muscle homogenates, HGPRT activity was significantly higher in dystrophic than in normal animals of both 129 ReJ and C57 BL/6J mice. Enzyme activity was not related to the severity of the disease. 4. It is suggested that changes in erythrocytes are secondary to the dystrophic process and that elevated HGPRT activity in skeletal muscle may be related to abnormal energy metabolism, possibly via the pentose monophosphate shunt.


Spaceflight and ageing: reflecting on Caenorhabditis elegans in space.
The prospect of space travel continues to capture the imagination. Several competing companies are now promising flights for the general population. Previously, it was recognized that many of the physiological changes that occur with spaceflight are similar to those seen with normal ageing. This led to the notion that spaceflight can be used as a model of accelerated ageing and raised concerns about the safety of individuals engaging in space travel. Paradoxically, however, space travel has been recently shown to be beneficial to some aspects of muscle health in the tiny worm Caenorhabditis elegans. C. elegans is a commonly used laboratory animal for studying ageing. C. elegans displays age-related decline of some biological processes observed in ageing humans, and about 35% of C. elegans' genes have human homologs. Space flown worms were found to have decreased expression of a number of genes that increase lifespan when expressed at lower levels. These changes were accompanied by decreased accumulation of toxic protein aggregates in ageing worms' muscles. Thus, in addition to spaceflight producing physiological changes that are similar to accelerated ageing, it also appears to produce some changes similar to delayed ageing. Here, we put forward the hypothesis that in addition to the previously well-appreciated mechanotransduction changes, neural and endocrine signals are altered in response to spaceflight and that these may have both negative (e.g. less muscle protein) and some positive consequences (e.g. healthier muscles), at least for invertebrates, with respect to health in space. Given that changes in circulating hormones are well documented with age and in astronauts, our view is that further research into the relationship between metabolic control, ageing, and adaptation to the environment should be productive in advancing our understanding of the physiology of both spaceflight and ageing.


A review of the research papers published by the international College of Applied Kinesiology from 1981 to 1987.
Applied Kinesiology (AK) is a diagnostic and therapeutic approach used by a large number of chiropractors. AK seminars are conducted worldwide; during these seminars mention is frequently made of the presence of supportive research. A review was undertaken of the type and scientific quality of 50 papers which had been published between 1981 and 1987 by the International College of Applied Kinesiology, 20 of which were classified as research papers. These were subjected to further scrutiny relating to criteria considered crucial in research methodology, namely, a clear identification of sample size, inclusion criteria, blind and naive subjects and statistical analysis. Although some papers satisfied several of these criteria, none satisfied all seven of them. As none of the papers included adequate statistical analyses, no valid conclusions could be drawn concerning their report of findings.


Measles eradication: recommendations from a meeting cosponsored by the World Health Organization, the Pan American Health Organization, and CDC.
Recent successes in interrupting indigenous transmission of measles virus in the Americas and in the United Kingdom prompted the World Health Organization (WHO), Pan American Health Organization (PAHO), and CDC to convene a meeting in July, 1996 to consider the feasibility of global measles eradication. Presentations at the meeting included an overview of global measles control and elimination efforts; detailed reviews of successful measles elimination efforts in Latin America, the English-speaking Caribbean, Canada, and the United States; surveillance for clinical disease; laboratory tools for antibody detection and virus identification; and other factors that might influence the feasibility of disease eradication. With this background information, meeting organizers asked participants to address five questions: 1) Is global measles eradication feasible? 2) Is measles eradication feasible with current vaccines? 3) What are the appropriate vaccination strategies for measles eradication? 4) How should surveillance for measles be carried out? 5) What role should outbreak control play in the strategy to eliminate measles? Participants agreed that measles eradication is technically feasible with available vaccines and recommended adoption of the goal of global eradication with a target date during 2005-2010, with the proviso that measles eradication efforts should not interfere with poliomyelitis eradication but should build on the successes of the global Poliomyelitis Eradication Initiative. Although existing vaccines are adequate for eradication, vaccination strategies that rely on administration of a single dose of vaccine are not. In the Americas, sustained interruption of indigenous measles virus transmission has been achieved through a three-tiered vaccination strategy that includes a) "catch-up" vaccination of all persons aged 1-14 years, regardless of disease history or vaccination status; b) "keep-up" vaccination of > or = 90% of children in each successive birth cohort at age 12 months and c) "follow-up" campaigns designed to vaccinate all persons within a specific age range whenever the number of susceptible persons in the preschool-aged population approximates the size of a typical birth cohort (in practice, every 3-5 years). In other regions, different strategies may be optimal. Surveillance, a critical component of any strategy to eliminate or eradicate measles, has two functions: to assess the effectiveness of the measles elimination strategy and to detect circulation of measles virus in a population. Systematic surveillance based on clinical diagnosis should be implemented early in any measles elimination program. In countries attempting to eliminate indigenous measles, all isolated cases of measles and at least one case in each chain of transmission should be confirmed by laboratory tests. Specimens for virus isolation (e.g., urine, nasopharyngeal swabs, or blood) should be collected in conjunction with field investigations. Vaccination campaigns generally have not proved to be effective responses to measles outbreaks. Outbreaks should be treated as opportunities to reinforce surveillance and to identify measures to prevent future outbreaks. The major obstacles to measles eradication are not technical but perceptual, political, and financial. Measles is often mistakenly perceived as a mild illness. This misperception, which is particularly prevalent in industrialized countries, can inhibit the development of public and political support for the allocation of resources required for an effective elimination effort. The disease burden imposed by measles should be documented, particularly in industrialized countries, so that this information can be used to educate parents, medical practitioners, public health workers, and political leaders about the benefits of measles eradication.


Nonparenchymal cells from regenerating rat liver generate interleukin-1alpha and -1beta: a mechanism of negative regulation of hepatocyte proliferation.
Following experimental partial hepatectomy of 70% in the rat, there is a semisynchronized surge of hepatocyte proliferation that ceases after 48 to 72 hours. Little is known about the determinants governing the termination of the proliferative phase, although transforming growth factor (TGF) beta has been implicated as an important inhibitor of hepatocyte replication in this model. We previously reported an additional non-TGF-beta inhibitor in medium conditioned by nonparenchymal cells isolated from regenerating liver (CM-NPC-Reg) between 24 and 48 hours after partial hepatectomy, but it was not found in medium conditioned by nonparenchymal cells from unoperated control liver. CM-NPC-Reg suppressed replicative DNA synthesis of primary rat hepatocytes in response to hepatocyte growth factor (HGF), epidermal growth factor (EGF), or TGF-alpha as assessed by 3H-thymidine incorporation. We now present evidence that interleukin (IL)-1 is the major inhibitor of hepatocyte DNA synthesis present in CM-NPC-Reg. IL-1 receptor antagonist abrogated the inhibition, as did antibodies to rat IL-1alpha and -beta; a combination of both antibodies was required, implicating both IL-1alpha and IL-1beta as active constituents in CM-NPC-Reg. To investigate in vivo changes in IL-1 expression, we assessed expression of IL-1alpha messenger RNA (mRNA) in whole rat liver following partial hepatectomy; mRNA was down-regulated at 10 hours in the pre-replicative phase of liver regeneration and up-regulated at 24 hours and 48 hours when proliferation is waning. Rat hepatocytes isolated from liver 24 hours after partial hepatectomy showed increased sensitivity to the inhibitory action of IL-1. Exogenous IL-1beta, administered parenterally to a group of rats at 0 and 12 hours after partial hepatectomy significantly reduced the incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), into hepatocytes at 18 hours. These data indicate that nonparenchymal cells isolated from regenerating rat liver elaborate IL-1, and support the hypothesis that IL-1 plays a role suppressing hepatocyte proliferation and terminating the surge of DNA synthesis induced after partial hepatectomy.


Adaptive and perceptual learning technologies in medical education and training.
Recent advances in the learning sciences offer remarkable potential to improve medical education and maximize the benefits of emerging medical technologies. This article describes 2 major innovation areas in the learning sciences that apply to simulation and other aspects of medical learning: Perceptual learning (PL) and adaptive learning technologies. PL technology offers, for the first time, systematic, computer-based methods for teaching pattern recognition, structural intuition, transfer, and fluency. Synergistic with PL are new adaptive learning technologies that optimize learning for each individual, embed objective assessment, and implement mastery criteria. The author describes the Adaptive Response-Time-based Sequencing (ARTS) system, which uses each learner's accuracy and speed in interactive learning to guide spacing, sequencing, and mastery. In recent efforts, these new technologies have been applied in medical learning contexts, including adaptive learning modules for initial medical diagnosis and perceptual/adaptive learning modules (PALMs) in dermatology, histology, and radiology. Results of all these efforts indicate the remarkable potential of perceptual and adaptive learning technologies, individually and in combination, to improve learning in a variety of medical domains.


Targeted projection NMR spectroscopy for unambiguous metabolic profiling of complex mixtures.
Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of (1)H and (13)C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D (1)H-(13)C TOCSY-HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane.


Some power allocation algorithms for cognitive uplink satellite systems.
Cognitive satellite communication (SatCom) is rapidly emerging as a promising technology to overcome the scarcity of the exclusive licensed band model in order to fulfill the increasing demand for high data rate services. The paper addresses power allocation methods for multi-operator multi-beam uplink satellite communication systems co-existing with a Ka-band terrestrial network, using cognitive radio paradigm. Such a scenario is especially challenging because of (i) the coexisting multiple SatCom operators over the cognitive band need to coordinate the use of their resources under limited inter-operator information exchange, and (ii) nonlinear onboard high power amplifier (HPA) which leads to nonlinear interference between users and beams. In order to tackle the first challenge, we propose distributed power allocation algorithms including the standard Alternate Direction Multiplier Method (ADMM); Regarding the HPA nonlinear impairment, we propose nonlinear-aware power allocation based on Signomial Programming. The proposed solutions outperform state-of-the-art in both cases.


Foot care in epidermolysis bullosa: evidence-based guideline.
This guideline was designed to provide service providers and users with an evidence-based set of current best practice guidelines for people and their families and carers, living with epidermolysis bullosa (EB). A systematic literature review relating to the podiatric care of patients with EB was undertaken. Search terms were used, for which the most recent articles relating to podiatric treatment were identified from as early as 1979 to the present day, across seven electronic search engines: MEDLINE, Wiley Online Library, Google Scholar, Athens, ResearchGate, Net and PubFacts.com. The Scottish Intercollegiate Guidelines Network (SIGN) methodology was used. The first guideline draft was analysed and discussed by clinical experts, methodologists and patients and their representatives at four panel meetings. The resulting document went through an external review process by a panel of experts, other healthcare professionals, patient representatives and lay reviewers. The final document will be piloted in three different centres in the U.K. and Australia. Following an EB community international survey the outcomes indicated six main areas that the community indicated as a priority to foot management. These include blistering and wound management, exploring the most suitable footwear and hosiery for EB, management of dystrophic nails, hyperkeratosis (callus), maintaining mobility and fusion of toes (pseudosyndactyly). The evidence here is limited but several interventions currently practised by podiatrists show positive outcomes.


Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.
Several groups have suggested that transplantation of marrow stromal cells (MSCs) promotes functional recovery in animal models of brain trauma. Recent studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit, as transplanted MSCs have only limited ability to replace injured central nervous system (CNS) tissue. To gain insight into the mechanisms responsible for such effects, we systematically investigated the therapeutic potential of MSCs for treatment of brain injury. Using in vitro studies, we detected the synthesis of various growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3). Enzyme-linked immunosorbent assay (ELISA) demonstrated that MSCs cultured in Dulbecco's modified Eagle medium (DMEM) produced substantial amounts of NGF for at least 7 weeks, whereas the levels of BDNF, GDNF and NT-3 remained unchanged. In studies in mice, after intraventricular injection of MSCs, NGF levels were increased significantly in cerebrospinal fluid by ELISA, confirming our cell culture results. Further studies showed that treatment of traumatic brain injury with MSCs could attenuate the loss of cholinergic neuronal immunostaining in the medial septum of mice. These studies demonstrate for the first time that by increasing the brain concentration of NGF, intraventricularly transplanted MSCs might play an important role in the treatment of traumatic brain injury.


Generation of genome-scale gene-associated SNPs in catfish for the construction of a high-density SNP array.
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing. Transcriptome sequencing was conducted for channel catfish and blue catfish using Illumina next generation sequencing technology. Approximately 220 million reads (15.6 Gb) for channel catfish and 280 million reads (19.6 Gb) for blue catfish were obtained by sequencing gene transcripts derived from various tissues of multiple individuals from a diverse genetic background. A total of over 35 billion base pairs of expressed short read sequences were generated. Over two million putative SNPs were identified from channel catfish and almost 2.5 million putative SNPs were identified from blue catfish. Of these putative SNPs, a set of filtered SNPs were identified including 342,104 intra-specific SNPs for channel catfish, 366,269 intra-specific SNPs for blue catfish, and 420,727 inter-specific SNPs between channel catfish and blue catfish. These filtered SNPs are distributed within 16,562 unique genes in channel catfish and 17,423 unique genes in blue catfish. For aquaculture species, transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. When multiple individuals with different genetic backgrounds are used, RNA-Seq is very effective for the identification of SNPs. The SNPs identified in this report will provide a much needed resource for genetic studies in catfish and will contribute to the development of a high-density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for genome association studies and whole genome-based selection in catfish.


Assessing Extravascular Lung Water in Critically Ill Patients Using Lung Ultrasound: A Systematic Review on Methodological Aspects in Diagnostic Accuracy Studies.
Lung ultrasound (LUS) is a non-invasive bedside method used to quantify extravascular lung water (EVLW). To evaluate the methodology and diagnostic accuracy of LUS in studies assessing EVLW in intensive care unit patients, PubMed and Embase were searched for studies comparing LUS with imaging modalities. In 14 relevant studies a wide variety of equipment used and training of examiners were noted. Four scoring systems were reported: (i) a binary score (the presence of three or more B-lines); (ii) a categorical score; (iii) a numerical score; (iv) a quantitative LUS score using software. The diagnostic accuracy of LUS varied: sensitivity ranged from 50%-98%, specificity from 76%-100% and r² from 0.20-0.91. Methodology and diagnostic accuracy varies substantially in published reports. Further research is needed to correlate methodological factors with diagnostic accuracy. Hospitals should standardize LUS methodology. Consensus is needed to harmonize LUS methodology for lung water assessment.


Murine neuronatin deficiency is associated with a hypervariable food intake and bimodal obesity.
Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.


Application of advanced quantification techniques in nanoparticle-based vaccine development with the Sf9 cell baculovirus expression system.
Nanoparticles generated by recombinant technologies are receiving increased interest in several applications, particularly the use of virus like particles (VLPs) for the generation of safer vaccines. The characterization and quantification of these nanoparticles with complex structures is very relevant for a better comprehension of the production systems and should circumvent the limitations of the most conventional quantification techniques often used. Here, we applied confocal microscopy, flow virometry and nanoparticle tracking analysis (NTA) to assess the production process of Gag virus-like particles (VLPs) in the Sf9 cell/baculovirus expression vector system (BEVS). These novel techniques were implemented in an optimization workflow based on Design of Experiments (DoE) and desirability functions to determine the best production conditions. A higher level of sensitivity was observed for NTA and confocal microscopy but flow virometry proved to be more accurate. Interestingly, extracellular vesicles were detected as an important source of contamination of this system. The synergistic interplay of viable cell concentration at infection (CCI), multiplicity of infection (MOI) and time of harvest (TOH) was assessed on five objective responses: VLP assembly, baculovirus infection, VLP production, cell viability and VLP productivity. Two global optimal conditions were defined, one targeting the maximal yield of VLPs and the other providing a balance between production and assembled VLPs. In both cases, a low MOI proved to be the best condition to achieve the highest VLP production and productivity yields. Cryo-EM analysis of nanoparticles produced in these conditions showed the typical size and morphology of HIV-1 VLPs. This study presents an integrative approach based on the combination of DoE and direct nanoparticle quantification techniques to comprehensively optimize the production of VLPs and other viral-based biotherapeutics.


Shift work and risk of incident dementia: a study of two population-based cohorts.
This study aimed to investigate the association between shift work and incident dementia in two population-based cohorts from the Swedish Twin Registry (STR). The STR-1973 sample included 13,283 participants born 1926-1943 who received a mailed questionnaire in 1973 that asked about status (ever/never) and duration (years) of shift work employment. The Screening Across the Lifespan Twin (SALT) sample included 41,199 participants born 1900-1958 who participated in a telephone interview in 1998-2002 that asked about night work status and duration. Dementia diagnoses came from Swedish patient registers. Cox proportional-hazards regression was used to estimate hazard ratios (HR) with 95% confidence intervals (CI). Potential confounders such as age, sex, education, diabetes, cardiovascular disease and stroke were included in adjusted models. In genotyped subsamples (n = 2977 in STR-1973; n = 10,366 in SALT), APOE ε4 status was considered in models. A total of 983 (7.4%) and 1979 (4.8%) dementia cases were identified after a median of 41.2 and 14.1 years follow-up in the STR-1973 and SALT sample, respectively. Ever shift work (HR 1.36, 95% CI 1.15-1.60) and night work (HR 1.12, 95% CI 1.01-1.23) were associated with higher dementia incidence. Modest dose-response associations were observed, where longer duration shift work and night work predicted increased dementia risk. Among APOE ε4 carriers, individuals exposed to ≥ 20 years of shift work and night work had increased dementia risk compared to day workers. Findings indicate that shift work, including night shift work, compared to non-shift jobs is associated with increased dementia incidence. Confirmation of findings is needed.


Genome and catabolic subproteomes of the marine, nutritionally versatile, sulfate-reducing bacterium Desulfococcus multivorans DSM 2059.
Sulfate-reducing bacteria (SRB) are key players of the carbon- and sulfur-cycles in the sediments of the world's oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO2) a variety of organic acids, including fatty acids up to C14, as well as aromatic compounds. In this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; ~91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) β-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO2. Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed. A highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.


trans Isomeric fatty acids are inversely related to the availability of long-chain PUFAs in the perinatal period.
We summarize data on the potential interaction of trans isomeric fatty acids [trans fatty acids (TFAs)] with the availability of long-chain polyunsaturated fatty acids (LC-PUFAs) in the perinatal period. Today, TFA intakes in pregnant and lactating women can be estimated to be ∼1% of energy in the majority of the population. The significant inverse associations seen between TFAs and LC-PUFAs in pregnant women in 3 different European populations investigated in a recent study raise doubts about the nutritional adequacy of high TFA intakes during pregnancy. In a recent study on the TFA content of human milk in a sizable group of mothers at the sixth week of lactation, both arachidonic and docosahexaenoic acids correlated significantly inversely to 18-carbon TFAs but not to 16-carbon TFAs, and at the sixth month of lactation arachidonic acid correlated significantly inversely to 18-carbon TFAs but not to 16-carbon TFAs. Similarly, significant inverse correlations were seen between 18-carbon TFAs and arachidonic and docosahexaenoic acids in both artery and vein wall lipids in a sizable group of healthy term infants. The TFA data obtained in umbilical blood vessel wall lipids were related to the neurologic condition of healthy children at 18 mo of age: children with minimal neurologic dysfunction at age 18 mo had significantly higher cord blood vein wall trans octadecadienoic acid values than did neurologically normal children. Total TFA values as well as total 18-carbon TFA values in umbilical vein wall lipids were significantly inversely associated with neurologic optimality score. Contradictory data renders it impossible to draw firm conclusions on the role of TFAs in modifying fetal growth; however, TFA exposure may be a confounding parameter in studies that investigate the relation between fetal fatty acid supply and intrauterine growth.


MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.
By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating.


The Effects of Vertical Yoked Prism on Horizontal Heterophoria.
Vertical yoked prisms (VYPs) have been recommended for the remediation of vergence dysfunction, but the evidence base for their use is mostly low level. This study investigates the effect of VYP on horizontal subjective dissociated heterophoria. Phoria measurement was performed in primary gaze with the Modified Thorington technique at 3 m and at 40 cm on 40 nonpresbyopic young adults. Subjects were seated and head position was held constant. Baseline measures (without yoked prism) of distance and near phoria were measured. Phorias were measured again through the following range of VYP, randomly presented: 2 prism diopters (Δ) base up (BU), 2Δ base down (BD), 5Δ BU, and 5Δ BD. Twenty-six subjects also had their phorias measured with control lenses of +0.125 DS OU (because of unavailability of Plano trial lenses) randomly presented along the other conditions. There was no significant difference overall between phoria measured in any of the yoked prism conditions, including the baseline measure, at distance or near. Neither was there evidence of a predictable esophoric or exophoric shift with either BU or BD prism. Vertical yoked prisms did not exert any immediate effect on horizontal phoria in young adults when posture was controlled. This suggests that, if VYPs do indeed improve horizontal vergence problems, they do not do so by a direct or immediate impact on horizontal phoria.


Improving Family Medicine with Thoughtful Research.
This issue is about improving primary health care outcomes, from behavioral health to opioid issues to diagnosing hypertension to providing hope for childhood obesity. It includes hints for integrating behavioral health and care managers into family medicine practices. Opiate prescribing practices vary considerably between Japan and the United States, with helpful insights for our opiate abuse epidemic. Suicidality is high among patients taking opiates. Diagnosing hypertension the recommended way is not easily accomplished. Primary care clinicians are important in infertility and prostate cancer treatment, and in support of men who commit interpersonal violence and people with cognitive impairment who wander. The "July effect" seems to persist. Parents' views on obesity in children can be changed-for the better. Family physicians have less burnout than has been previously reported, and many provide palliative care. Doctors think diseases, patients think about how well they feel. Do we find healthy lifestyles in retirement?


[Hypercalcemia and multiple osteolytic lesions in a child with disseminated paracoccidioidomycosis and pulmonary tuberculosis].
To describe the case of a child with paracoccidioidomycosis who presented hypercalcemia with multiple osteolytic lesions. A 6-year-old boy was admitted with a one-month history of fever and hepatosplenomegaly. On admission, he looked sick, pale, and had disseminated lymphadenopathy and hepatosplenomegaly. The laboratory findings included anemia (hemoglobin = 6.8 g/dl), eosinophilia (1,222/mm3), thrombocytopenia (102,000/mm3), and hypoalbuminemia (serum albumin = 2.2 g/dl). Paracoccidioides brasiliensis was identified in bone marrow examination. In the second week after admission, the patient presented joint pain, poor activity and difficulty in walking. He presented hypercalcemia (maximum value = 14.9 mg%) and reduction in renal function, which lasted for two weeks. On the 42nd day after admission, his chest X-ray showed lytic lesions in clavicle, scapula, ribs, and humerus, with bilateral slipped capital humeral epiphysis. The patient presented nephrocalcinosis and nephrolithiasis, reduction in creatinine clearance and evidence of tubular lesions. At the end of the second month after admission, Mycobacterium tuberculosis was isolated in gastric washing. The child received treatment for paracoccidioidomycosis and tuberculosis and has not had any sequelae for 3 years. The development of symptomatic hypercalcemia leading to renal lesion, associated with multiple osteolytic lesions, had never been described in paracoccidioidomycosis. Although pulmonary tuberculosis was diagnosed and could be related to hypercalcemia, the sudden onset of hypercalcemia and its normalization without specific treatment for tuberculosis suggests that bone lysis was the most important factor in the genesis of hypercalcemia.


O-linked β-N-acetylglucosamine transferase directs cell proliferation in idiopathic pulmonary arterial hypertension.
Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked β-N-acetylglucosamine modification, O-linked β-N-acetylglucosamine transferase (OGT), and O-linked β-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked β-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH.


Immobilization of Nonactivated Unfixed Platelets for Real-Time Single-Cell Analysis.
Existing methods for measuring the response of individual platelets to stimulation are limited. They either measure each platelet at one discrete time-point (flow cytometry) or rely on adhesive ligands to immobilize platelets that concomitantly generate activation signals (microscopy). Such methods of immobilization make it impossible to assess resting platelets, the changes that occur as platelets transition from resting to active states, or the signals generated by soluble agonists, such as ADP and thrombin, or by mechanical stimulus, independently from those generated by the adhesive ligand. Here we describe a microscopy method that allows the immobilization of platelets to a glass cover slip without triggering platelet activation. This method makes use of specific antibodies that bind platelet PECAM-1 without activating it. Platelets can therefore be immobilized to PECAM-1 antibody coated biochips without causing activation and perfused with agonists or inhibitors. Using this method, platelets can be stimulated by an array of soluble agonists at any concentration or combination, in the presence or absence of inhibitors or shear forces. This chapter describes in detail this PECAM-1 mediated immobilized platelet method and its use for measuring changes in Ca2+ signaling in individual platelets under a number of different conditions. While we focus on the measurement of Ca2+ dynamics in this chapter, it is important to consider that the basic method we describe will easily lend its self to other measures of platelet activation (integrin activation, shape change, actin dynamics, degranulation), and may, therefore, be used to measure almost any facet of platelet activation.


Theoretical Studies of IR and NMR Spectral Changes Induced by Sigma-Hole Hydrogen, Halogen, Chalcogen, Pnicogen, and Tetrel Bonds in a Model Protein Environment.
Various types of σ-hole bond complexes were formed with FX, HFY, H2FZ, and H3FT (X = Cl, Br, I; Y = S, Se, Te; Z = P, As, Sb; T = Si, Ge, Sn) as Lewis acid. In order to examine their interactions with a protein, N-methylacetamide (NMA), a model of the peptide linkage was used as the base. These noncovalent bonds were compared by computational means with H-bonds formed by NMA with XH molecules (X = F, Cl, Br, I). In all cases, the A-F bond, which lies opposite the base and is responsible for the σ-hole on the A atom (A refers to the bridging atom), elongates and its stretching frequency undergoes a shift to the red with a band intensification, much as what occurs for the X-H bond in a H-bond (HB). Unlike the NMR shielding decrease seen in the bridging proton of a H-bond, the shielding of the bridging A atom is increased. The spectroscopic changes within NMA are similar for H-bonds and the other noncovalent bonds. The C=O bond of the amide is lengthened and its stretching frequency red-shifted and intensified. The amide II band shifts to higher frequency and undergoes a small band weakening. The NMR shielding of the O atom directly involved in the bond rises, whereas the C and N atoms both undergo a shielding decrease. The frequency shifts of the amide I and II bands of the base as well as the shielding changes of the three pertinent NMA atoms correlate well with the strength of the noncovalent bond.


Genetic Determinants of the Interventricular Septum Are Linked to Ventricular Septal Defects and Hypertrophic Cardiomyopathy.
A large proportion of genetic risk remains unexplained for structural heart disease involving the interventricular septum (IVS) including hypertrophic cardiomyopathy and ventricular septal defects. This study sought to develop a reproducible proxy of IVS structure from standard medical imaging, discover novel genetic determinants of IVS structure, and relate these loci to diseases of the IVS, hypertrophic cardiomyopathy, and ventricular septal defect. We estimated the cross-sectional area of the IVS from the 4-chamber view of cardiac magnetic resonance imaging in 32 219 individuals from the UK Biobank which was used as the basis of genome wide association studies and Mendelian randomization. Measures of IVS cross-sectional area at diastole were a strong proxy for the 3-dimensional volume of the IVS (Pearson r=0.814, P=0.004), and correlated with anthropometric measures, blood pressure, and diagnostic codes related to cardiovascular physiology. Seven loci with clear genomic consequence and relevance to cardiovascular biology were uncovered by genome wide association studies, most notably a single nucleotide polymorphism in an intron of CDKN1A (rs2376620; β, 7.7 mm2 [95% CI, 5.8-11.0]; P=6.0×10-10), and a common inversion incorporating KANSL1 predicted to disrupt local chromatin structure (β, 8.4 mm2 [95% CI, 6.3-10.9]; P=4.2×10-14). Mendelian randomization suggested that inheritance of larger IVS cross-sectional area at diastole was strongly associated with hypertrophic cardiomyopathy risk (pIVW=4.6×10-10) while inheritance of smaller IVS cross-sectional area at diastole was associated with risk for ventricular septal defect (pIVW=0.007). Automated estimates of cross-sectional area of the IVS supports discovery of novel loci related to cardiac development and Mendelian disease. Inheritance of genetic liability for either small or large IVS, appears to confer risk for ventricular septal defect or hypertrophic cardiomyopathy, respectively. These data suggest that a proportion of risk for structural and congenital heart disease can be localized to the common genetic determinants of size and shape of cardiovascular anatomy.


Proposal of novel staging system CNM (Crohn's primary site, nodes, mesentery) to predict postoperative recurrence of Crohn's disease.
After oncologic resection, histological grading and staging of the tumor give important prognostic information about the future risk of recurrence and hence influence the subsequent management plan. Several studies and their meta-analysis have shown that various histological features (e.g., microscopic positive resection margins, plexitis, granuloma, mesenteric inflammatory activity) can predict postoperative clinical/endoscopic/surgical recurrence after resection in Crohn's disease (CD). Inclusion of mesentery in surgical resection specimens has been shown to reduce surgical recurrence after ileocolonic resection in CD. However, there is no uniform histopathological staging system for risk stratification in postoperative CD to systematically predict postoperative recurrence. This is because the prediction to date is based on clinical characteristics (smoking status, disease phenotype, surgical history). Histopathological predictors are still not adopted in routine clinical practice due to the lack of a uniform staging system, heterogeneity of published studies and lack of standardized definition of histological features. In this article, we attempted to incorporate all such histological features in a single histological staging system CNM (Crohn's primary site [resection margin positivity, plexitis, granuloma, depth of infiltration], nodes [presence of granuloma], mesentery [involved or not]) in surgical resection specimen in CD. The proposed CNM classification would help to enable systematic reporting, design future clinical trials, stratify postoperative recurrence risk and choose appropriate postoperative prophylaxis.


To treat or not to treat: comparison of different criteria used to determine whether weight loss is to be recommended.
Excess body fat is a major risk factor for disease primarily due to its endocrine activity. In recent years several criteria have been introduced to evaluate this factor. Nevertheless, treatment need is currently assessed only on the basis of an individual's Body Mass Index (BMI), calculated as body weight (in kg) divided by height in m2. The aim of our study was to determine whether application of the BMI, compared to adiposity-based criteria, results in underestimation of the number of subjects needing lifestyle intervention. We compared treatment need based on BMI classification with four adiposity-based criteria: percentage body fat (%BF), considered both alone and in relation to metabolic syndrome risk (MS), waist circumference (WC), as an index of abdominal fat, and Body Fat Mass Index (BFMI, calculated as fat mass in kg divided by height in m2) in 63 volunteers (23 men and 40 women, aged 20 - 65 years). According to the classification based on BMI, 6.3% of subjects were underweight, 52.4% were normal weight, 30.2% were overweight, and 11.1% were obese. Agreement between the BMI categories and the other classification criteria categories varied; the most notable discrepancy emerged in the underweight and overweight categories. BMI compared to almost all of the other adiposity-based criteria, identified a lower percentage of subjects for whom treatment would be recommended. In particular, the proportion of subjects for whom clinicians would strongly recommend weight loss on the basis of their BMI (11.1%) was significantly lower than those identified according to WC (25.4%, p = 0.004), %BF (28.6%, p = 0.003), and MS (33.9%, p = 0.002). The use of the BMI alone, as opposed to an assessment based on body composition, to identify individuals needing lifestyle intervention may lead to unfortunate misclassifications. Population-specific data on the relationships between body composition, morbidity, and mortality are needed to improve the diagnosis and treatment of at-risk individuals.


Closed-loop power and focus control of laser welding for full-penetration monitoring.
We describe a closed-loop control system ensuring full penetration in welding by controlling the focus position and power of a 4-kW Nd:YAG laser. A focus position monitoring system was developed based on the chromatic aberration of the focusing optics. With the laser power control system we can determine the degree of penetration by analyzing the keyhole image intensity profile. We demonstrate performance in bead-on-plate welding of Inconel 718 and titanium. The focus control system maintained a focal position on tilted and nonflat workpieces, and the penetration monitoring technique successfully controlled the laser power to maintain the full-penetration regime in the presence of linear and step changes of thickness. Finally we discuss the performances and the limits of the systems when applied to a realistic complex aerospace component.


Craniopharyngiomas: intratumoral chemotherapy with interferon-alpha: a multicenter preliminary study with 60 cases.
The authors assessed the efficacy of intratumoral interferon-alpha (IFNalpha)-based chemotherapy in pediatric patients with cystic craniopharyngiomas. In a prospective multicenter study of 60 pediatric patients, the authors assessed the efficacy of intratumoral INFalpha2A-based chemotherapy. The study was conducted between 2000 and 2009 at 3 locations: the Medical School of the Federal University of São Paulo, Catholic University of Rome, and the Neurosurgery Institute of Santiago, Chile. The assessment included clinical and radiological control examinations, side effects observed, and total dose used. Sixty cases of cystic craniopharyngioma were analyzed. The cohort consisted of 35 male and 25 female children (mean age 11 years). Clinical and radiological improvement was achieved in 76% of the cases. New endocrinological deficits were observed in 13% of the cases. In approximately 30% of the patients, the evolution included some light side effects, the most common being headache (33%) and eyelid edema (28%). The number of cycles varied from 1 to 9 (mean 5 cycles), and the total dose applied per cycle was 36,000,000 IU. This has been the largest documented series of intratumoral chemotherapy using INFalpha for the control of cystic craniopharyngiomas. The treatment has proved efficacious; there was no mortality, and morbidity rates were low.


DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages.
We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs.


Renal blood flow in acute cerebral ischemia in spontaneously hypertensive rats: effects of alpha- and beta-adrenergic blockade.
The influences of acute cerebral ischemia on renal hemodynamics were examined in spontaneously hypertensive rats in which cerebral ischemia was induced by bilateral carotid artery occlusion. Renal and cerebral blood flow were measured with a hydrogen clearance technique. Either phenoxybenzamine (0.5 mg/kg body wt) or propranolol (2 mg/kg) was given i.v. immediately after ischemia was induced to examine the drugs' effects on cerebral and renal hemodynamics. One hour after ischemia, cerebral blood flow was markedly reduced to 5, 3, and almost 0% of the preischemic value in the untreated, phenoxybenzamine-treated, and propranolol-treated rats, respectively. In contrast, renal blood flow at that time was decreased to 65, 88, and 67%, respectively. The calculated renal vascular resistance was similarly increased to 151% in the untreated and 136% in the propranolol-treated rats, but decreased to 82% in the phenoxybenzamine-treated rats. The present results indicate that in acute cerebral ischemia renal blood flow was considerably decreased with concomitant increased renal vascular resistance, and that such reduction in renal blood flow was minimized by alpha-adrenergic blockade but not by beta-blockade. It is concluded that activation of the alpha-adrenergic system in acute cerebral ischemia causes renal vasoconstriction.


A Novel Hybrid Plasmid Carrying Multiple Antimicrobial Resistance and Virulence Genes in Salmonella enterica Serovar Dublin.
Virulence plasmids and antibiotic resistance plasmids are usually maintained separately in Salmonella spp.; however, we report an instance of a hybrid plasmid (pN13-01125) in Salmonella enterica serovar Dublin. Review of the complete sequence of the 172,265-bp plasmid suggests that pN13-01125 is comprised of the previously described pSDVr and pSH696_135 plasmids and that the mechanism of hybridization likely involves IS6 (IS26) insertion sequence elements. The plasmid has a low conjugation frequency, confers resistance to six classes of antimicrobials, and contains a complete spv virulence operon.


LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus.
Myxococcus xanthus, a member of the Proteobacteria delta-class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage-inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock-out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light-induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT-PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non-inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA-regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage-inducible genes in bacteria belonging to different phyla.