Getting specific: making taxonomic and ecological sense of large sequencing data sets.
Eukaryotic microbes comprise a diverse collection of phototrophic and heterotrophic creatures known to play fundamental roles in ecological processes. Some can be identified by light microscopy, generally the largest and with conspicuous shapes, while the smallest can be counted by epifluorescence microscopy or flow cytometry but remain largely unidentified. Microbial diversity studies greatly advanced with the analysis of phylogenetic markers sequenced from natural assemblages. Molecular surveys began in 1990 targeting marine bacterioplankton (Giovannoni et al. ) and first approached microbial eukaryotes in three studies published in 2001 (Díez et al. ; López-García et al. ; Moon-van der Staay et al. ). These seminal studies, based on cloning and Sanger sequencing the complete 18S rDNA, were critical for obtaining broad pictures of microbial diversity in contrasted habitats and for describing novel lineages by robust phylogenies, but were limited by the number of sequences obtained. So, inventories of species richness in a given sample and community comparisons through environmental gradients were very incomplete. These limitations have been overcome with the advent of high-throughput sequencing (HTS) methods, initially 454-pyrosequencing, today Illumina and soon others to come. In this issue of Molecular Ecology, Egge et al. () show a nice example of the use of HTS to study the biodiversity and seasonal succession of a particularly important group of marine microbial eukaryotes, the haptophytes. Temporal changes were analysed first at the community level, then at the clade level, and finally at the lowest rank comparable to species. Interesting and useful ecological insights were obtained at each taxonomic scale. Haptophyte diversity differed along seasons in a systematic manner, with some species showing seasonal preferences and others being always present. Many of these species had no correspondence with known species, pointing out the high level of novelty in microbial assemblages, only accessible by molecular tools. Moreover, the number of species detected was limited, agreeing with a putative scenario of constrained evolutionary diversification in free-living small eukaryotes. This study illustrates the potential of HTS to address ecological relevant questions in an accessible way by processing large data sets that, nonetheless, need to be treated with a fair understanding of their limitations.


Physico-chemical studies on the 11S globulin in soybean seeds: size and shape determination of the molecule.
The molecular shape of the 11S globulin, the major storage protein of soybean seeds, was estimated to be an oblate ellipsoid with a revolution axial ratio of 8.11-8.38 with or without scarce hydration according to the procedure of Simha & Perrin by measuring the partial specific volume, diffusion coefficient, molecular weight and volume fraction intrinsic viscosity. The length of the major axis of the molecule is 178-180 A and that of the minor axis 22 A. Subsequently, the shape factor beta, and the hydrodynamically effective volume, Ve, of the protein were calculated by the procedure of Scheraga & Mandelkern. Consequently, the 11S globulin molecule was also an oblate ellipsoid from beta. Ve was found to be equal to MV/N, the anhydrous volume of protein. It was therefore concluded that the protein existed in a rigid and nearly anhydrous state in solution.


Temporal dynamics in microbial soil communities at anthrax carcass sites.
Anthrax is a globally distributed disease affecting primarily herbivorous mammals. It is caused by the soil-dwelling and spore-forming bacterium Bacillus anthracis. The dormant B. anthracis spores become vegetative after ingestion by grazing mammals. After killing the host, B. anthracis cells return to the soil where they sporulate, completing the lifecycle of the bacterium. Here we present the first study describing temporal microbial soil community changes in Etosha National Park, Namibia, after decomposition of two plains zebra (Equus quagga) anthrax carcasses. To circumvent state-associated-challenges (i.e. vegetative cells/spores) we monitored B. anthracis throughout the period using cultivation, qPCR and shotgun metagenomic sequencing. The combined results suggest that abundance estimation of spore-forming bacteria in their natural habitat by DNA-based approaches alone is insufficient due to poor recovery of DNA from spores. However, our combined approached allowed us to follow B. anthracis population dynamics (vegetative cells and spores) in the soil, along with closely related organisms from the B. cereus group, despite their high sequence similarity. Vegetative B. anthracis abundance peaked early in the time-series and then dropped when cells either sporulated or died. The time-series revealed that after carcass deposition, the typical semi-arid soil community (e.g. Frankiales and Rhizobiales species) becomes temporarily dominated by the orders Bacillales and Pseudomonadales, known to contain plant growth-promoting species. Our work indicates that complementing DNA based approaches with cultivation may give a more complete picture of the ecology of spore forming pathogens. Furthermore, the results suggests that the increased vegetation biomass production found at carcass sites is due to both added nutrients and the proliferation of microbial taxa that can be beneficial for plant growth. Thus, future B. anthracis transmission events at carcass sites may be indirectly facilitated by the recruitment of plant-beneficial bacteria.


[Breast biopsy performed by the helicoid biopsy technique: an experimental study].
to assess the helicoid biopsy performance when carrying out breast biopsies. thirty patients with breast cancer submitted to mastectomy were selected at random. Women with a tumor of petreous consistency, nonpalpable, submitted to previous surgical manipulation or containing fluid were excluded. The helicoid biopsy kit and a core biopsy device with a cannula and a 14-gauge-needle, respectively, were used to collect a fragment each from a healthy area and from the tumor of each surgical specimen, for a total of 120 fragments for histological study. Data were analyzed statistically by the parametric Student's t-test and by the Kappa concordance index at the 95% confidence level, using the SPSS software, version 13. the mean patient's age was 51.6 (± 11.1) years old. The core biopsy showed 93.3% sensitivity, 100% specificity and 96.7% accuracy, and the helicoid biopsy showed 96.7% sensitivity, 100% specificity, and 98.3% accuracy. The comparison of tumor histology and biopsy fragments revealed a high degree of concordance in the diagnoses (Kappa equal to 0.9, with p<0.05). both methods provided a highly accurate histological diagnosis of the lesions. The results of the present study demonstrate that the helicoid biopsy is a reliable alternative for the preoperative diagnosis of breast lesions.


Time trends in the adjustment of Swedish teenage girls: a 26-year comparison of 15-year-olds.
The aim of this study was to investigate stability and change over 26 years in self-reported adjustment of Swedish teenage girls. Data were collected with the same questionnaire from two school-cohorts in a middle-sized Swedish community: 522 girls attending Grade 8 (approximately at age 15) in 1970, and 529 girls attending Grade 8 in 1996. The first cohort was part of the longitudinal research programme Individual Development and Adaptation (IDA). In most domains, adjustment problems were approximately as common in 1996 as in 1970, with two exceptions: more girls reported problems with self-esteem and antisocial problems in 1996. In the antisocial domain, a polarization process was indicated, with an increase also in the number of girls without adjustment problems. In the relational domains, especially peer relations, there was an increase in positive adjustment. The results are discussed in relation to earlier findings and to social changes during the period.


Pharmaceutical and nutraceutical approaches for preventing liver carcinogenesis: chemoprevention of hepatocellular carcinoma using acyclic retinoid and branched-chain amino acids.
The poor prognosis for patients with hepatocellular carcinoma (HCC) is associated with its high rate of recurrence in the cirrhotic liver. Therefore, more effective strategies need to be urgently developed for the chemoprevention of this malignancy. The malfunction of retinoid X receptor α, a retinoid receptor, due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and may be a promising target for HCC chemoprevention. Acyclic retinoid (ACR), a synthetic retinoid, can prevent HCC development by inhibiting retinoid X receptor α phosphorylation and improve the prognosis for this malignancy. Supplementation with branched-chain amino acids (BCAA), which are used to improve protein malnutrition in patients with liver cirrhosis, can also reduce the risk of HCC in obese cirrhotic patients. In experimental studies, both ACR and BCAA exert suppressive effects on HCC development and the growth of HCC cells. In particular, combined treatment with ACR and BCAA cooperatively inhibits the growth of HCC cells. Furthermore, ACR and BCAA inhibit liver tumorigenesis associated with obesity and diabetes, both of which are critical risk factors for HCC development. These findings suggest that pharmaceutical and nutraceutical approaches using ACR and BCAA may be promising strategies for preventing HCC and improving the prognosis of this malignancy.


Ten-year evaluation of conservative and surgical treatment of gingival recession. A case series study.
In the last years the treatment of non-inflammatory periodontal diseases has greatly changed. Apico-coronal dimension of gingival tissue is not considered to be of utmost importance, but significance of tissue thickness over each tooth is stressed. Purpose of this study was to show results of conservative and surgical treatment of gingival recession. Sample consisted of two groups of subjects, which have been treated in one of stated ways during ten years. The data was obtained on the beginning and after ten years of recall. Both groups showed increased dimension of keratinized gingiva during observed time with decrease of gingival recession, plaque- and gingival index. It is considered that treatment should start with conservative measures with necessary motivation of the patients. If it does not show improvement, one should consider best surgical method available for each patient. Clinical results show success in both groups, meaning that treatment was properly decided on.


Usefulness of myocardial parametric imaging to evaluate myocardial viability in experimental and in clinical studies.
To evaluate whether myocardial parametric imaging (MPI) is superior to visual assessment for the evaluation of myocardial viability. Myocardial contrast echocardiography (MCE) was assessed in 11 pigs before, during, and after left anterior descending coronary artery occlusion and in 32 patients with ischaemic heart disease by using intravenous SonoVue administration. In experimental studies perfusion defect area assessment by MPI was compared with visually guided perfusion defect planimetry. Histological assessment of necrotic tissue was the standard reference. In clinical studies viability was assessed on a segmental level by (1) visual analysis of myocardial opacification; (2) quantitative estimation of myocardial blood flow in regions of interest; and (3) MPI. Functional recovery between three and six months after revascularisation was the standard reference. In experimental studies, compared with visually guided perfusion defect planimetry, planimetric assessment of infarct size by MPI correlated more significantly with histology (r2 = 0.92 versus r2 = 0.56) and had a lower intraobserver variability (4% v 15%, p < 0.05). In clinical studies, MPI had higher specificity (66% v 43%, p < 0.05) than visual MCE and good accuracy (81%) for viability detection. It was less time consuming (3.4 (1.6) v 9.2 (2.4) minutes per image, p < 0.05) than quantitative blood flow estimation by regions of interest and increased the agreement between observers interpreting myocardial perfusion (kappa = 0.87 v kappa = 0.75, p < 0.05). MPI is useful for the evaluation of myocardial viability both in animals and in patients. It is less time consuming than quantification analysis by regions of interest and less observer dependent than visual analysis. Thus, strategies incorporating this technique may be valuable for the evaluation of myocardial viability in clinical routine.


Placentation in the fallopian tube.
The processes of implantation and placentation have been studied in 105 tubal pregnancies. Successful placentation occurred only with plicomural or mural implantations and in these the early development of the placenta occurred in the tube in exactly the same way as it does in the uterus, although tubal placentation was usually membranaceous in nature, possibly because of an inadequate blood supply. Invasion of the placental bed by interstitial extravillous cytotrophoblast occurred in the tube in the same manner as it does in the uterus whereas extravillous trophoblast invaded tubal vessels in the same fashion as it does the spiral arteries in the uterus. Subsequent development of the placenta in the tube differed from that in the uterus in so far as villous invasion of the tubal tissues, with consequent development of the placenta in the subserosa or on the serosa, was common. The ability of the blastocyst to implant in the tube with subsequent normal placentation indicates that these processes are purely a function of trophoblastic tissue, with maternal tissues playing only a passive role.


[Giant esophageal fibrovascular polyp. Apropos of a case].
Fibrovascular polyps, together with leiomyomas, constitute the great majority of rare benign tumors of the oesophagus. Their size and their frequently high level of attachment sometimes give them a spectacular clinical presentation. The hazards involved in the interpretation of radiological and endoscopic data account for frequent mistakes in their diagnosis. The determination of the pedicle site defines the choice of the surgical approach. A diagnostic error and the choice of an incorrect approach are the two main causes of therapeutic difficulties.


Influence of decompression sickness on vasomotion of isolated rat vessels.
Several studies have demonstrated that endothelial function is impaired following a dive even without decompression sickness. During this study we determined the effect of decompression sickness on endothelium-dependent and independent vasoreactivity. For this purpose twenty-seven male Sprague-Dawley rats were submitted to a simulated dive up to 1,000 kPa absolute pressure and divided into 3 groups: safe diving without decompression sickness or dives provoking mild or severe sickness. A fourth control group remained at atmospheric pressure. Endothelium-dependent and independent vasomotion was assessed ex vivo by measuring isometric tension in rings of abdominal aorta and mesenteric arteries. Dose-response curves were obtained with phenylephrine, acetylcholine and sodium nitroprusside. Acetylcholine-induced relaxation was measured in the presence of L-NAME, indometacin or both of them at once.Contraction was significantly decreased after each protocol compared with the control rats. Additionally, the response in animals from the severe group was significantly different from that of the safe and mild groups. Dose response curves for acetylcholine alone and in the presence of inhibitors remained unchanged. We did not observe differences in endothelium-dependent vasodilation after diving or in the presence of decompression sickness. Contractile response to phenylephrine was progressively impaired with increased decompression stress. These results may indicate smooth muscle injury.


Short-term effects of oral administration of Pistacia lentiscus oil on tissue-specific toxicity and drug metabolizing enzymes in mice.
Pistacia lentiscus (Anacardiaceae) is a flowering plant traditionally used in the treatment of various skin, respiratory, and gastrointestinal disorders. The aim of this study was to assess whether Pistacia lentiscus oil has any short term toxic effects in vivo and in vitro. Pistacia lentiscus oil (100µl) was administered orally into mice for 5 days. Measurements of body weight did not show any weight loss. Serum concentration of LDH did not show any significant statistical difference when compared to control mice. Similarly, blood, kidney or liver function tests showed no toxicity with Pistacia lentiscus oil when compared to the control group. Examination of gastrointestinal tissues sections revealed similar structural features with no difference in cell proliferation. In this context, pharmacological dilutions of Pistacia lentiscus oil (10(-6) - 10(-3)) did not affect the viability (cell death and proliferation) of mouse gastric stem cells, human colorectal cancer cells HT29, human hepatoma cells HepG2. However, it appears that at the dose and time point studied, Pistacia lentiscus oil treatment has targeted various cytochrome P450s and has specifically inhibited the activities and the expression of CYP2E1, CYP3A4, CYP1A1 and CYP1A2 differentially in different tissues. Our results also demonstrate that there is no appreciable effect of Pistacia lentiscus oil on the GSH-dependent redox homoeostasis and detoxification mechanism in the tissues. These data suggest a good safety profile of short term oral use of Pistacia lentiscus oil as a monotherapy in the treatment of various skin, respiratory, and gastrointestinal disorders. However, due to its inhibitory effect of various cytochrome P450s and mainly CYP3A4, this might have implications on the bioavailability and metabolism of drugs taken in combination with Pistacia lentiscus oil. More attention is needed when Pistacia lentiscus oil is intended to be uses in combination with other pharmacological agents in order to avoid potential drug-drug interaction leading to toxicity. This study will help in safer use of Pistacia lentiscus oil for therapeutic purpose.


Microcircuitry of agranular frontal cortex: contrasting laminar connectivity between occipital and frontal areas.
Neocortex is striking in its laminar architecture. Tracer studies have uncovered anatomical connectivity among laminae, but the functional connectivity between laminar compartments is still largely unknown. Such functional connectivity can be discerned through spontaneous neural correlations during rest. Previous work demonstrated a robust pattern of mesoscopic resting-state connectivity in macaque primary visual cortex (V1) through interlaminar cross-frequency coupling. Here we investigated whether this pattern generalizes to other cortical areas by comparing resting-state laminar connectivity between V1 and the supplementary eye field (SEF), a frontal area lacking a granular layer 4 (L4). Local field potentials (LFPs) were recorded with linear microelectrode arrays from all laminae of granular V1 and agranular SEF while monkeys rested in darkness. We found substantial differences in the relationship between the amplitude of gamma-band (>30 Hz) LFP and the phase of alpha-band (7-14 Hz) LFP between these areas. In V1, gamma amplitudes in L2/3 and L5 were coupled with alpha-band LFP phase in L5, as previously described. In contrast, in SEF phase-amplitude coupling was prominent within L3 and much weaker across layers. These results suggest that laminar interactions in agranular SEF are unlike those in granular V1. Thus the intrinsic functional connectivity of the cortical microcircuit does not seem to generalize across cortical areas.


Chronic illness and smoking cessation.
Smoking is among the leading causes of premature mortality and preventable death in the United States. Although smoking contributes to the probability of developing chronic illness, little is known about the relationship between quitting smoking and the presence of chronic illness. The present study investigated the association between diagnoses of one or more chronic diseases (diabetes, hypertension, or high cholesterol) and smoking status (former or current smoker). The data analyzed were a subset of questions from a 155-item telephone-administered community survey that assessed smoking status, demographic characteristics, and presence of chronic disease. The study sample consisted of 3,802 randomly selected participants. Participants with diabetes were more likely to report being former smokers, after adjusting for sociodemographic characteristics, whereas having hypertension or high cholesterol was not associated significantly with smoking status. The likelihood of being a former smoker did not increase as number of diagnosed chronic diseases increased. Participants who were women, older (aged 65+), or single were significantly less likely to be former smokers. Participants with at least a college degree, those with incomes of 50,000+ US dollars, and those who were underweight or obese were more likely to be former smokers. These findings were inconsistent with research that has suggested that having a chronic illness or experiencing a serious medical event increases the odds of smoking cessation. Supporting prior research, we found that being male, having a higher income, and being obese were associated with greater likelihood of being a former smoker.


Effects of delayed gastric emptying on postprandial glucose kinetics, insulin sensitivity, and β-cell function.
Controlling meal-related glucose excursions continues to be a therapeutic challenge in diabetes mellitus. Mechanistic reasons for this need to be understood better to develop appropriate therapies. To investigate delayed gastric emptying effects on postprandial glucose turnover, insulin sensitivity, and β-cell responsivity and function, as a feasibility study prior to studying patients with type 1 diabetes, we used the triple tracer technique C-peptide and oral minimal model approach in healthy subjects. A single dose of 30 μg of pramlintide administered at the start of a mixed meal was used to delay gastric emptying rates. With delayed gastric emptying rates, peak rate of meal glucose appearance was delayed, and rate of endogenous glucose production (EGP) was lower. C-peptide and oral minimal models enabled the assessments of β-cell function, insulin sensitivity, and β-cell responsivity simultaneously. Delayed gastric emptying induced by pramlintide improved total insulin sensitivity and decreased total β-cell responsivity. However, β-cell function as measured by total disposition index did not change. The improved whole body insulin sensitivity coupled with lower rate of appearance of EGP with delayed gastric emptying provides experimental proof of the importance of evaluating pramlintide in artificial endocrine pancreas approaches to reduce postprandial blood glucose variability in patients with type 1 diabetes.


Multitarget Strategies to Reduce Myocardial Ischemia/Reperfusion Injury: JACC Review Topic of the Week.
Many treatments have been identified that confer robust cardioprotection in experimental animal models of acute ischemia and reperfusion injury. However, translation of these cardioprotective therapies into the clinical setting of acute myocardial infarction (AMI) for patient benefit has been disappointing. One important reason might be that AMI is multifactorial, causing cardiomyocyte death via multiple mechanisms, as well as affecting other cell types, including platelets, fibroblasts, endothelial and smooth muscle cells, and immune cells. Many cardioprotective strategies act through common end-effectors and may be suboptimal in patients with comorbidities. In this regard, emerging data suggest that optimal cardioprotection may require the combination of additive or synergistic multitarget therapies. This review will present an overview of the state of cardioprotection today and provide a roadmap for how we might progress towards successful clinical use of cardioprotective therapies following AMI, focusing on the rational combination of judiciously selected, multitarget therapies. This paper emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.


Trophic divergence despite morphological convergence in a continental radiation of snakes.
Ecological and phenotypic convergence is a potential outcome of adaptive radiation in response to ecological opportunity. However, a number of factors may limit convergence during evolutionary radiations, including interregional differences in biogeographic history and clade-specific constraints on form and function. Here, we demonstrate that a single clade of terrestrial snakes from Australia--the oxyuranine elapids--exhibits widespread morphological convergence with a phylogenetically diverse and distantly related assemblage of snakes from North America. Australian elapids have evolved nearly the full spectrum of phenotypic modalities that occurs among North American snakes. Much of the convergence appears to involve the recurrent evolution of stereotyped morphologies associated with foraging mode, locomotion and habitat use. By contrast, analysis of snake diets indicates striking divergence in feeding ecology between these faunas, partially reflecting regional differences in ecological allometry between Australia and North America. Widespread phenotypic convergence with the North American snake fauna coupled with divergence in feeding ecology are clear examples of how independent continental radiations may converge along some ecological axes yet differ profoundly along others.


Transcriptional regulation of GATA-3 by an intronic regulatory region and fetal liver zinc finger protein 1.
GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. The transcriptional regulation of GATA-3, however, remains elusive. In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene. The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro. By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes. More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro. Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.


Nutritional value of commercial and traditional lettuce (Lactuca sativa L.) and wild relatives: Vitamin C and anthocyanin content.
Lettuce is the most consumed leafy vegetable though the most popular varieties have a low nutritional value. Our objective was to accurately quantify vitamin C and anthocyanins in wild relatives, and commercial and traditional varieties. Wild species and traditional varieties contained more total ascorbic acid (TAA) than commercial varieties (21% and 8%, respectively). In contrast, commercial varieties had significantly higher content of anthocyanins than traditional varieties and wild species (6 and 8 times more, respectively). TAA was significantly higher in green than in red lettuces (18%). TAA was also significantly higher in the leaves of two wild species than in stems. Cyanidin 3-O-(6'-O-malonylglucoside) was the most abundant anthocyanin (97%), present in most samples. The rankings of accessions by vitamin C and anthocyanin contents can be useful for consumers worried about the impacts of food on their wellbeing and for breeders aiming to improve lettuce by biofortification with health-promoting compounds.


N-Eicosapentaenoyl Dopamine, A Conjugate of Dopamine and Eicosapentaenoic Acid (EPA), Exerts Anti-inflammatory Properties in Mouse and Human Macrophages.
A large body of evidence suggests that dietary n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), contribute to a reduced inflammatory tone thereby lowering the risk for several chronic and degenerative diseases. Different mechanisms have been proposed to explain these anti-inflammatory effects, including those involving endocannabinoids and endocannabinoid-like molecules. In this context, fatty acid amides (FAAs), conjugates of fatty acids with amines or amino acids, are an emerging class of compounds. Dopamine conjugates of DHA (N-docosahexaenoyl dopamine, DHDA) and EPA (N-eicosapentaenoyl dopamine, EPDA) have previously been shown to induce autophagy, apoptosis, and cell death in different tumor lines. Additionally, DHDA has displayed anti-inflammatory properties in vitro. Here, we tested the immune-modulatory properties of EPDA in mouse RAW 264.7 and human THP-1 macrophages stimulated with lipopolysaccharide (LPS). EPDA suppressed the production of monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in both cell lines, and nitric oxide (NO), and macrophage-inflammatory protein-3α (MIP3A) in RAW 264.7 macrophages. At a transcriptional level, EPDA attenuated cyclooxygenase-2 (COX-2) expression in both cell lines and that of MCP-1, IL-6, and interleukin-1β (IL-1β) in THP-1 macrophages. Although further research is needed to reveal whether EPDA is an endogenous metabolite, our data suggest that this EPA-derived conjugate possesses interesting immune-modulating properties.


Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.
The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).


The INSAR Community Collaborator Request: Using community-academic partnerships to enhance outcomes of participatory autism research.
Participatory approaches, in which researchers work together with members of the autism community (e.g., autistic people, family members, caregivers, or other stakeholders) to design, conduct, and disseminate research, have become increasingly prominent within the field of autism research over the past decade. Despite growing academic and community interest in conducting participatory studies, stakeholder collaboration remains infrequent in autism research, at least partially due to systemic barriers. To help reduce barriers to engaging in participatory autism research, the International Society for Autism Research (INSAR) Autistic Researchers Committee has launched the INSAR Community Collaborator Request (ICCR; https://www.autism-insar.org/page/iccr), a platform on the INSAR website that allows autism researchers conducting participatory research to seek out stakeholder collaborators from the autism community (including both autistic people and their family members/caregivers, as relevant to a given research project). Interested stakeholders also have the opportunity to subscribe to ICCR posts, allowing them to be alerted of new opportunities for collaboration and potentially increasing their involvement in autism research. Overall, the ICCR provides a venue to connect autism researchers with potential community collaborators, reducing barriers to participatory autism research and increasing the frequency of successful community-academic partnerships within the field. We are hopeful that in the long term, such changes will lead to greater alignment between research outputs and the goals of the greater autism community, and consequently an increase in the overall quality and relevance of autism research.


Anti-obesity effects of capsaicin and the underlying mechanisms: a review.
Obesity and overweight have become serious health problems in the world and are linked to a variety of metabolic disorders. Phytochemicals with a weight-loss effect have been widely studied for the past few decades. Capsaicin is the major bioactive component in red chili peppers with many beneficial functions. Its anti-obesity effects have been evaluated extensively using different model systems, including cell models, animal models and human subjects. In this paper, anti-obesity effects of capsaicin are reviewed and the underlying mechanisms are characterized.


Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.
Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.


Representative subsampling of sedimenting blood.
It is often necessary to extract a small amount of a suspension, such as blood, from a larger sample of the same material for the purposes of diagnostics, testing or imaging. A practical challenge is that the cells in blood sediment noticeably on the time scale of a few minutes, making a representative subsampling of the original sample challenging. Guided by experimental data, we develop a Kynch sedimentation model to discuss design considerations that ensure a representative subsampling of blood, from a container of constant cross-sectional area, for the entire range of physiologically relevant hematocrit over a specified time of interest. Additionally, we show that this design may be modified to exploit the sedimentation and perform subsampling to achieve either higher or lower hematocrit relative to that of the original sample. Thus, our method provides a simple tool to either concentrate or dilute small quantities of blood or other sedimenting suspensions.


Respiratory irritation associated with inhalation of boron trifluoride and fluorosulfonic acid.
The objectives of this study were to examine the respiratory irritancy of boron trifluoride (BF(3)) and fluorosulfonic acid (FSA) following acute inhalation exposure. Testing was conducted using groups of 10 male and 10 female rats (BF(3)) or groups of 6 male rats (FSA). Rats were exposed for a single 4-h period (BF(3)) or a single 1-h period (FSA) and necropsied 1 or 14 days after exposure (BF(3)) or 14 days after exposure (FSA). Measurements consisted of clinical signs, body weight, kidney and lung weight, histopathology (BF(3)), and breathing parameters (FSA) and were used to evaluate the possible irritating effects of these compounds. The results indicated treatment-related findings in the larynx and trachea in the rats exposed to 74.4 mg/m(3) BF(3), consisting of ventral cartilage necrosis, hemorrhage, and an increase in ventral epithelial hyperplasia and ventral inflammatory cell inflammation 24 h postexposure. In the animals sacrificed 14 days postexposure, the only notable observation was ventral cartilage necrosis, present in 2 animals. The next lower level tested, 24.6 mg/m(3) BF, was considered a no-observed-adverse-effects level (NOAEL). A concentration of 4125 mg/m(3) FSA resulted in a clearly decreased breathing rate during and shortly after exposure with 67% (4/6) mortality on days 5-9 after exposure. A concentration of 845 mg/m(3) FSA resulted in only minor signs of irritation, consisting of slight changes in breathing pattern shorlty after exposure. The results of the present 4-h inhalation study with BF(3) indicated that respiratory irritation was present at a level of 74.4 mg/m(3) whereas 24.6 mg/m(3) was a NOAEL. A single 1-h exposure to 845 mg/m(3) FSA resulted in only minor signs of respiratory irritation, indicating that on a mass basis FSA is no more toxic or irritating than hydrogen fluoride (HF) or sulfuric acid.


ANGPLT3: A novel modulator of lipid metabolism.
Angiopoietin-like proteins (ANGPTLs) have emerged as an important regulator of lipid and glucose metabolism as well as insulin sensitivity. ANGPTL3 plays a key role in regulating circulating triglycerides (TG) and cholesterol levels through reversible inhibition of lipoprotein lipase (LPL) and endothelial lipase enzymes activity. Loss of function mutation of ANGPTL3 gene has been identified in many subjects with familial combined hypolipidemia. ANGPTL4 produces irreversible inhibition of LPL activity, while ANGPTL8 enhances the activity of ANGPTL3, which highlight the interplay between the different ANGPTLs in a coordinated manner to regulate lipid metabolism during different nutritional states. This new class of lipid modulators may serve as potential novel therapeutic target for reducing plasma lipoprotein and treatment of metabolic syndrome.


Postnatal growth, neurobehavioral and neurophysiologic changes of prenatal low-dose beta-radiation from tritiated water in mice.
Pregnant adult C57BL/6J mice were randomly assigned to four groups: one sham and three irradiated groups that were exposed to -irradiation from tritiated water (HTO) by a single intraperitoneal injection on Day 12.5 of gestation. The offspring received cumulative doses of 0.036, 0.071, and 0.213 Gy, respectively. The litters were observed for postnatal growth (body weight, brain weight), the development of four physiologic milestones (pinna detachment, eye opening, testes decent, vaginal opening), the acquisition age of several reflexes (cliff avoidance, air righting) and sensory functions (auditory startle, thermal reflex), movement and coordination functions and activity (pivoting, foot splay, continuous corridor activity), learning and memory performance (shock avoidance, conditioning reflex), and the density of CA1-CA4 hippocampal pyramidal neurons. Modest but significant dose-dependent neuronal death and functional impairment were seen in both 0.071 and 0.213 Gy groups. In conclusion, even prenatal low-dose beta-radiation may impair murine central nervous system (CNS) development suggesting the potential importance of minimizing environmental exposure during human pregnancy.


AID and caspase 8 shape the germinal center response through apoptosis.
Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.


Entanglement Dynamics of Coupled Quantum Oscillators in Independent NonMarkovian Baths.
This work strives to better understand how the entanglement in an open quantum system, here represented by two coupled Brownian oscillators, is affected by a nonMarkovian environment (with memories), here represented by two independent baths each oscillator separately interacts with. We consider two settings, a 'symmetric' configuration wherein the parameters of both oscillators and their baths are identical, and an 'asymmetric' configuration wherein they are different, in particular, a 'hybrid' configuration, where one of the two coupled oscillators interacts with a nonMarkovian bath and the other with a Markovian bath. Upon finding the solutions to the Langevin equations governing the system dynamics and the evolution of the covariance matrix elements entering into its entanglement dynamics, we ask two groups of questions: (Q1) Which time regime does the bath's nonMarkovianity benefit the system's entanglement most? The answers we get from detailed numerical studies suggest that (A1) For an initially entangled pair of oscillators, we see that in the intermediate time range, the duration of entanglement is proportional to the memory time, and it lasts a fraction of the relaxation time, but at late times when the dynamics reaches a steady state, the value of the symplectic eigenvalue of the partially transposed covariance matrix barely benefit from the bath nonMarkovianity. For the second group of questions: (Q2) Can the memory of one nonMarkovian bath be passed on to another Markovian bath? And if so, does this memory transfer help to sustain the system's entanglement dynamics? Our results from numerical studies of the asymmetric hybrid configuration indicate that (A2) A system with a short memory time can acquire improvement when it is coupled to another system with a long memory time, but, at a cost of the latter. The sustainability of the bipartite entanglement is determined by the party which breaks off entanglement most easily.


Structure analysis and comparative characterization of the cytochrome c' and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum.
The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.


Clostridium herbivorans sp. nov., a cellulolytic anaerobe from the pig intestine.
A new cellulolytic anaerobic clostridium was isolated from the intestinal tract of pigs. The single isolate was a gram-positive, motile rod, formed terminal to subterminal swollen sporangia, and required a fermentable carbohydrate for growth. Cellulose, cellobiose, maltose, starch, and glycogen supported growth, but glucose and fructose did not. The major end products from the fermentation of cellobiose were butyrate and formate; minor amounts of hydrogen and ethanol were also formed. Ruminal fluid (15%) or yeast extract (1%) was required for good growth. The optimum temperature for growth was 39 to 42 degrees C, and the optimum pH was 6.8 to 7.2. Cell lysis occurred rapidly once stationary growth was reached. A 16S rRNA sequence analysis showed that the strain was related to a group of gram-positive anaerobes that includes Clostridium oroticum and the cellulolytic species Clostridium polysaccharolyticum and Clostridium populeti. The DNA base composition of the isolate is 38 mol% G + C. We propose the name Clostridium herbivorans for this organism; strain 54408 (= ATCC 49925) is the type strain.


Involvement of the pituitary-specific transcription factor pit-1 in somatolactotrope cell growth and death: an approach using dominant-negative pit-1 mutants.
The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1Delta1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.


Using graphs to link data across the product lifecycle for enabling smart manufacturing digital threads.
Smart manufacturing promises to provide significant increases in productivity and effectiveness of manufacturing systems by better connecting the data from people, processes, and things. However, there is no uniform, generalized method for deploying linked-data concepts to the manufacturing domain. The literature describes and commercial vendors offer centralized data repository solutions, but these types of approaches quickly breakdown under the intense burden of managing and reconciling all the data flowing in and out of the various repositories across the product lifecycle. In this paper, we introduce a method for linking and tracing data throughout the product lifecycle using graphs to form digital threads. We describe a prototype implementation of the method and a case study to demonstrate an information round-trip for a product assembly between the design, manufacturing, and quality domains of the product lifecycle. The expected impact from this novel, standards-based, linked-data method is the ability to use digital threads to provide data, system, and viewpoint interoperability in the deployment of smart manufacturing to realize industry's $30 Billion annual opportunity.


The association between serum vitamin B12 deficiency and tension-type headache in Turkish children.
This study aimed to determine the relationship between serum vitamin B12 level and tension-type headache. The study groups consisted of 75 patients (40 females, 35 males) with headache and a control group of 49 healthy children (25 females, 24 males). Serum vitamin B12 level < 200 pg/ml was defined as deficient, and < 160 pg/ml as severely deficient. The serum vitamin B12 level was measured by the electrochemiluminescence (ECLIA) method. The serum vitamin B12 levels in the headache and control groups were 273.01 ± 76.77 and 316.22 ± 74.53 pg/ml, with the difference determined as statistically significant (p = 0.003). In the case group, 18/75 patients (24%) had a serum vitamin B12 level below the normal of 200 pg/ml, and in the control group 4/49 (8%) patients were also below the normal range (p = 0.021). The serum vitamin B12 level in the children with tension-type headache was significantly lower than that in the control group. From the results of the study, it was concluded that there may be an association between vitamin B12 level and tension-type headache. However, further clinical studies are needed.


RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise.
The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.


Adult inclusion conjunctivitis. Clinical characteristics and corneal changes.
Twenty-five consecutive cases of adult inclusion conjunctivitis were studied. Diagnosis was based on the finding of typical Halberstaedter-Prowazek inclusion bodies on conjunctival scrapings. Corneal involvement was common (20/25 patients); most frequent was superficial epithelial keratitis (15/25). Other changes seen included subepithelial nummular keratitis (6/25), marginal keratitis (2/25), (4/25). There seemed to be a predilection for involvement of the upper half of the cornea. Conjunctival scarring occurred in one patient only. Associated genitourinary symptoms were spontaneously reported in three patients only; however, on careful questioning, an additional 12 patients were found to have a history of urethritis or cervicitis. A comparison with viral follicular conjunctivitis is made. The location and pattern of keratopathy, associated genitourinary complaints, mucopurulent nature of discharge, and lack of response to standard topical therapy would seem to suggest chlamydial cause. Conjunctival scrapings are very helpful in differential diagnosis between viral and chlamydial conjunctivitis.


Perspectives about policy implementation: A learning opportunity from the 2003-2013 Malawi HIV/AIDS Policy.
Malawi published its first ever HIV and AIDS policy in 2003. The implementation of the policy provided a very necessary and historic step in Malawi's organized response towards HIV and AIDS. Many achievements were registered in the period this policy was implemented. However, some components of the policy were not well-implemented. Our study explored barriers to implementation of provider initiated HIV testing and counseling (PITC) for sexually transmitted infections (STI) within general outpatient settings. Malawi also launched a revised HIV and AIDS Policy in December 2013. Although not part of this policy analysis, future years of implementation may face related issues observed during the implementation of the 2003-2013 policy. This is a non-experimental, descriptive study using a case study design. We examined the implementation of provider initiated HIV testing and counseling component of the Malawi HIV and AIDS policy from 2003-2013 focusing on STI and outpatient clinic settings. We sought to understand perspectives of various stakeholders and users of the policy. We conducted in-depth interviews with policy makers, health care worker supervisors, health care workers and health rights activists. Major problems which affected the implementation of the 2003-2013 HIV policy were: selective prioritization of policies by government, lack of involvement of implementers in the policy making process, non-awareness of health workers about the existence of the policy, lack of healthcare worker training, unsatisfactory supervision of policy implementation, poor harmonization of policies, lack of clarity about guidance to those directly implementing, unclear roles and reporting authority among the main national coordinating units. Good leadership, effective coordination, involvement of key players in the policy making process, dissemination to primary users and decentralization or empowerment of local supervisors is key to successful policy implementation.


Method of alkalization and monitoring of urinary pH for prevention of recurrent uric acid urolithiasis: a systematic review.
Uric acid (UA) urolithiasis comprises around 5-10% of all stones and can frequently recur. Due to the fact that UA stones form in acidic urine with a pH <5.5, these patients require special attention compared to other stone patients. The international guidelines suggest treatment and metaphylaxis by urinary alkalization. The objective of this review is to critically asses the available evidence concerning the method and efficacy of this treatment modality. A systematic review on the methods of metaphylactic therapy using oral alkalization of UA urolithiasis was conducted by two authors. Evidence was sought using a predefined search strategy in seven different databases. The provided evidence was critically evaluated using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Cochrane collaboration tool for assessing the risk of bias. Twelve manuscripts were included of which one was a randomised trial. They focussed on ways to alkalize urine and its effect on stone recurrence. Because of their methodology and heterogeneity, the evidence is presented in a narrative review. There were differences in medication used for alkalizing urine, ways of monitoring urine pH and evaluating its efficacy. The reported outcomes also differed between studies. There is currently a lack of clear evidence for the method of alkalization of urine and the method of pH measurement. Besides this, for an established treatment modality, there is lack of long term results for the alkalization therapy. In conclusion, urine alkalization is an established treatment modality for the metaphylaxis of UA urolithiasis despite the lack of evidence from high quality studies on the methods of alkalization and its treatment efficacy. The studies published on this topic are scarce and contain notable risks of bias which should be kept in mind when interpreting the stated results.


Total laparoscopic hysterectomy for large uterus.
In this review, we assessed the feasibility of total laparoscopic hysterectomy (TLH) in cases of very large uteri weighing more than 500 grams. We have analyzed whether it is possible for an experienced laparoscopic surgeon to perform efficient total laparoscopic hysterectomy for large myomatous uteri regardless of the size, number and location of the myomas. Retrospective review (Canadian Task Force Classification II-1) Dedicated high volume Gynecological laparoscopy centre. 173 women with symptomatic myomas who underwent total laparoscopic hysterectomy at our center. There were no exclusion criteria based on the size number or location of myomas. TLH and modifications of performing the surgery by ligating the uterine arteries prior, myomectomy followed by hysterectomy, direct morcellation after uterine artery ligation. 72% of patients had previous normal vaginal delivery and 28% had previous cesarean section. Average clinical size of the uterus was 18 weeks (10, 32). The average weight of the specimen was 700 grams (500, 2240). The average duration of surgery was 107 min (40, 300) and the average blood loss was 228 ml (10, 3200). Total laparoscopic hysterectomy is a technically feasible procedure. It can be performed by experienced surgeons for large uteri regardless of the size, number or location of the myomas.


United States Valuation of EQ-5D-5L Health States Using an International Protocol.
To derive a US-based value set for the EQ-5D-5L questionnaire using an international, standardized protocol developed by the EuroQol Group. Respondents from the US adult population were quota-sampled on the basis of age, sex, ethnicity, and race. Trained interviewers guided participants in completing composite time trade-off (cTTO) and discrete choice experiment (DCE) tasks using the EuroQol Valuation Technology software and routine quality control measures. Data were modeled using a Tobit model for cTTO data, a mixed logit model for DCE data, and a hybrid model that combined cTTO and DCE data. Model performance was compared on the basis of logical ordering of coefficients, statistical significance, parsimony, and theoretical considerations. Of 1134 respondents, 1062, 1099, and 1102 respondents provided useable cTTO, DCE, and cTTO or DCE responses, respectively, on the basis of quality control criteria and interviewer judgment. Respondent demographic characteristics and health status were similar to the 2015 US Census. The Tobit model was selected as the preferred model to generate the value set. Values ranged from -0.573 (55 555) to 1 (11 111), with 20% of all predicted health states scores less than 0 (ie, worse than dead). A societal value set for the EQ-5D-5L was developed that can be used for economic evaluations and decision making in US health systems. The internationally established, standardized protocol used to develop this US-based value set was recommended by the EuroQol Group and can facilitate cross-country comparisons.


The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids.
The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.


Follow-up of stable chromosomal aberrations in gamma-ray irradiated non-human primates.
The purpose of this study was to examine a new approach to retrospective biological dosimetry, by using a long-term animal model to determine the stability of translocation frequency after in vivo irradiation. While the frequency of dicentrics is known to decrease over time, the persistence of more stable chromosomal aberrations such as translocations could be useful if their stability were definitively proved. Four monkeys (Macaca fascicularis) were exposed to two different doses of ionizing radiation: 2 Gy whole body irradiation for two and 4 Gy for two others. Blood samples were obtained at various times after irradiation. Both total and two-way translocations were detected by fluorescence in situ hybridization. Translocations were scored in stable cells, that is, those without dicentrics, rings or fragments. The course of translocation frequency was analysed at four time-points: one hour (H1), 2 months (M2), 10 months (M10) and 31 months (M31) after irradiation. We observed two separate trends in translocation frequency: Total translocation frequency decreased slightly in animals irradiated with a dose of 2 Gy, while two-way translocation frequency was relatively stable in all irradiated animals. We confirmed the long-term stability of translocations and found that it seems to depend on the type of the translocation recorded. Overall translocations were stable for up to 31 months regardless of dose, but two-way translocations were more stable than those that were non-reciprocal, especially in stable cells.


The importance of solid-phase distribution on the oral bioaccessibility of Ni and Cr in soils overlying Palaeogene basalt lavas, Northern Ireland.
Potentially toxic elements (PTEs) including nickel and chromium are often present in soils overlying basalt at concentrations above regulatory guidance values due to the presence of these elements in underlying geology. Oral bioaccessibility testing allows the risk posed by PTEs to human health to be assessed; however, bioaccessibility is controlled by factors including mineralogy, particle size, solid-phase speciation and encapsulation. X-ray diffraction was used to characterise the mineralogy of 12 soil samples overlying Palaeogene basalt lavas in Northern Ireland, and non-specific sequential extraction coupled with chemometric analysis was used to determine the distribution of elements amongst soil components in 3 of these samples. The data obtained were related to total concentration and oral bioaccessible concentration to determine whether a relationship exists between the overall concentrations of PTEs, their bioaccessibility and the soils mineralogy and geochemistry. Gastric phase bioaccessible fraction (BAF %) ranged from 0.4 to 5.4 % for chromium in soils overlying basalt and bioaccessible and total chromium concentrations are positively correlated. In contrast, the range of gastric phase BAF for nickel was greater (1.4-43.8 %), while no significant correlation was observed between bioaccessible and total nickel concentrations. However, nickel BAF was inversely correlated with total concentration. Solid-phase fractionation information showed that bioaccessible nickel was associated with calcium carbonate, aluminium oxide, iron oxide and clay-related components, while bioaccessible chromium was associated with clay-related components. This suggests that weathering significantly affects nickel bioaccessibility, but does not have the same effect on the bioaccessibility of chromium.


Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans.
Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro. Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-α, receptor-interacting protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen.


Genome-wide expression analysis of LBD genes in tomato (Solanum lycopersicum L.) under different light conditions.
Lateral organ boundaries (LOB) domain (LBD) genes, a gene family that encodes the transcription factors (TFs) of plants, plays crucial functions in the development and growth of plants. Currently, genome-wide studies of the LBD family are still limited to tomato (Solanum lycopersicum L.), which is considered an important economic crop. In this study, we performed a genome-wide analysis of LBD in tomato. In total, 56 LBDs were found in the tomato genome. Protein alignment and phylogenetic classification showed that LBDs were conserved with other species. Since light emitting diodes (LEDs) light have promising applications for tomato growth. To better understand the potential function of LBDs in response to LED light in tomato, we conducted a genome-wide expression analysis of LBD genes under different light conditions. As expected, different LED lights affected the tomato growth (e.g. hypocotyl length). RNA-seq data showed that eight LBDs in tomato seedlings were differentially expressed under different light treatments, including white, blue, red, and far-red light, compared to the dark-grown condition. It indicates that these LBDs might regulate plant development in different LED light conditions. Interestingly, two LBD genes (SlLBD1 and SlLBD2) were found to be differentially expressed in four distinct lights, which might be involved in regulating the plant architecture via a complicated TF network, which can be taken into consideration in further investigation.


Clinical and research implications of revising psychological tests.
This article integrates those of other contributors to this special section, "Methods and Implications of Revising Assessment Instruments," to underscore important conceptual factors to consider when undertaking test revisions. These considerations include determination of when test measures have become sufficiently understood to be incorporated in a test revision, cohort effects, revision of administration formats and test instructions, and comparisons of performance levels across test versions. The discussion of these factors also takes into consideration clinical practice and educational implications of making a transition to revised test versions.


Efficacy of trastuzumab in unselected patients with HER2-positive metastatic breast cancer: a retrospective analysis.
The addition of trastuzumab to chemotherapy for HER2-positive metastatic breast cancer has significantly improved progression-free survival and overall survival, although most patients develop resistance or have a primarily resistant disease. The aim of the study was to describe the efficacy and safety of a first-line treatment in unselected metastatic HER2-positive breast cancer patients, treated according to clinical practice. From 2000 to 2009, we conducted a retrospective multi-institutional analysis of 182 consecutive patients with HER2-positive metastatic breast cancer who underwent first-line treatment with trastuzumab. The primary end points were progression-free survival and overall survival; the secondary end points were survival after progression in patients treated with second-line chemotherapy with or without trastuzumab and safety. A total of 172 patients were analyzed. Median progression-free survival and overall survival were 1.2 (95% CI, 1.1-1.4) and 4.4 years (95% CI, 3.6-5.4), respectively. For 100 patients who received second-line chemotherapy, median survival after progression was significantly longer in those who also received trastuzumab: 2.8 (95% CI, 2.1-4.0) versus 1.2 years (95% CI, 0.6-1.9). Although based on retrospective data, the study confirms the role of trastuzumab as first-line treatment in metastatic breast cancer outside of a controlled trial. Moreover, information obtained on the use of trastuzumab beyond disease progression supports its use in this setting.


From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU.
In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell-based corneal therapy.


Cutaneous leishmaniasis: recognition and treatment.
Cutaneous leishmaniasis is a parasitic disease occurring throughout the Americas from Texas to Argentina, and in the Old World, particularly the Middle East and North Africa. It is spread by the female sandfly. The condition is diagnosed every year in travelers, immigrants, and military personnel. Physicians in the United States must be alert to the diagnosis of leishmaniasis in travelers returning from endemic areas. Physicians working for short periods in endemic areas often must make the diagnosis and should be aware of local disease patterns. When faced with a possible leishmanial skin lesion, a skin scraping with microscopic analysis is the best test. Punch biopsies with tissue-impression smears also can be diagnostic. Needle aspiration of tissue fluid from the margin of a lesion can yield fluid for culture to isolate the organism and identify the species. Immunologic tests are being developed, including a highly sensitive polymerase chain reaction test. The treatment mainstay is pentavalent antimony (e.g., sodium stibogluconate). Not all patients require treatment; many lesions heal spontaneously. Antimonials have a high incidence of reversible adverse effects. Other medications used for treatment include amphotericin B, pentamidine isethionate, paromomycin, and antifungals. This disease must be considered in at-risk patients, and family physicians should know the basics of diagnosis and where to go for more help.


Norepinephrine, left ventricular disorders and volume excess in ESRD.
Sympathetic over-activity is a hallmark of end stage renal disease (ESRD). Left ventricular (LV) disorders and volume overload are pervasive in ESRD and sympathetic over-activity may be a relevant mediator of the cardiovascular (CV) risk by these alterations in this population. We investigated the relationship between a combined biomarker of LV disorders and volume excess, atrial natriuretic peptide (ANP), and the plasma concentration of nor-epinephrine (NE) in 227 ESRD patients without heart failure at baseline and modelled the risk for incident CV events by these biomarkers over a 3.5 years follow-up. Plasma NE was strongly and independently related to ANP (β = 0.31, P < 0.001). In a multivariate Cox's regression analysis, ANP was an independent predictor of these events [HR (1-SD) 1.25, 95 % CI 1.01-1.54]. However, when NE was introduced into the multivariate model, HR by ANP reduced substantially (1.14, 95% CI 0.91-1.42) and was no longer significant (P = 0.25) while the CV risk signalled by NE was clinically relevant (HR 1.29, 95% CI 1.05-1.59) and statistically significant (P = 0.02). In ESRD patients without heart failure, NE is strongly and independently related to ANP. The predictive power of ANP for CV events is largely captured by NE in a statistical model including both biomarkers. These data suggest that sympathetic over-activity may be a relevant mediator of the high risk of CV events triggered by LV disorders and volume excess in this population. However, further mechanistic and intervention studies are needed to prove the nature (causal/non causal) of these findings.


Connective tissue activating peptide III expression disappears progressively with increased dysplasia in human cervical epithelium.
The aim of this study was to explore the possible involvement of CTAP-III in the development of cervical cancer as it progresses through several cervical intraepithelial neoplasia (CIN) stages. Twenty-four cervical specimens were obtained by direct punch biopsy, conization, or hysterectomy. Diagnosis of CIN I to CIN III was based on standard morphological criteria in 12 specimens. Tissue specimens were also obtained from 4 normal uteri and 8 cases of invasive squamous cell cervical carcinoma. RT-PCR, using CTAP-III-specific primers, was used to identify CTAP-III mRNA and polyclonal antibodies, directed against the N-terminus of CTAP-III, for immunostaining of the CTAP-III protein. RT-PCR yielded amplified fragments in RNA derived from normal cervical tissue, while no PCR product was detected in the invasive cervical carcinoma tissue. The PCR product corresponded to a CTAP-III plasmid PCR product. Both tissues expressed the same amounts of GAPDH mRNA as the control for the integrity and equal amounts of the isolated RNA. In each of the 16 specimens of normal cervices and of CIN tissues, epithelial cells were stained with the anti-CTAP-III antibodies. In normal epithelium, CTAP-III staining was homogeneously distributed in all epithelial layers, except in the highly active and proliferating basal cells. CTAP-III was localized at the epithelial cell membrane or between adjacent epithelial cells in a granular, chain-like pattern of staining. In the CIN specimens, CTAP-III staining was no longer seen in the deep epithelial layers, consistent with the dysplastic appearance of the cells, and remained in the seemingly normal superficial epithelial layers. Cells of invasive cervical carcinoma did not stain for CTAP-III and were detected in endothelial cells of capillary blood vessels. The specific localization of CTAP-III between adjacent epithelial cells suggests a possible role of this chemokine in maintaining the normal architecture of epithelial tissues. Its progressive disappearance in increasingly severe CIN may be applied to distinguish between specific stages in the progression of cervical carcinoma.


Unity and diversity of executive functions in creativity.
Increasing evidence suggests that executive functions (EFs) - a set of general-purpose control processes that regulate thoughts and behaviors - are relevant for creativity. However, EF is not a unitary process, and it remains unclear which specific EFs are involved. The present study examined the association between the three EFs, both uniquely (EF-Specific) and together (Common EF), and three measures of creativity. Participants (N = 47) completed a divergent thinking test, and self-reported their real-life creative accomplishments. A subset of participants indicated their involvement in the artistic or information technology (IT) professions. Results indicated that fluency (but not originality) of divergent thinking was uniquely predicted by working memory Updating. Better response Inhibition predicted higher number of real-world artistic creative achievements. Involvement in the artistic (versus IT) professions was associated with better Common EF, and with enhanced mental set Shifting abilities. Results demonstrate that different EFs predict creativity depending on its operational definition.


Advances in developing noncovalent small molecules targeting Keap1.
Kelch-like ECH-associated protein 1 (Keap1) is a drug target for diseases involving oxidative stress and inflammation. There are three covalent Keap1-binding drugs on the market, but noncovalent compounds that inhibit the interaction between Keap1 and nuclear factor erythroid 2-related factor 2 (Nrf2) represent an attractive alternative. Both compound types prevent degradation of Nrf2, leading to the expression of antioxidant and antiinflammatory proteins. However, their off-target profiles differ as do their exact pharmacodynamic effects. Here, we discuss the opportunities and challenges of targeting Keap1 with covalent versus noncovalent inhibitors. We then provide a comprehensive overview of current noncovalent Keap1-Nrf2 inhibitors, with a focus on their pharmacological effects, to examine the therapeutic potential for this compound class.


Oophorectomy, hormone therapy, and subclinical coronary artery disease in women with hysterectomy: the Women's Health Initiative coronary artery calcium study.
Surgical menopause has been associated with an increased risk of coronary heart disease events. In this study, we aimed to determine the associations between coronary artery calcium (CAC) and hysterectomy, oophorectomy, and hormone therapy use with a focus on the duration of menopause for which there was no hormone therapy use. In a substudy of the Women's Health Initiative placebo-controlled trial of conjugated equine estrogens (0.625 mg/d), we measured CAC by computed tomography 1.3 years after the trial was stopped. Participants included 1,064 women with previous hysterectomy, aged 50 to 59 years at baseline. The mean trial period was 7.4 years. Imaging was performed at a mean of 1.3 years after the trial was stopped. Mean age was 55.1 years at randomization and 64.8 years at CAC measurement. In the overall cohort, there were no significant associations between bilateral oophorectomy, years since hysterectomy, years since hysterectomy without taking hormone therapy (HT), years since bilateral oophorectomy, and years of HT use before Women's Health Initiative enrollment and the presence of CAC. However, there was a significant interaction between bilateral oophorectomy and prerandomization HT use for the presence of any CAC (P = 0.05). When multivariable analyses were restricted to women who reported no previous HT use, those with bilateral oophorectomy had an odds ratio of 2.0 (95% CI: 1.2-3.4) for any CAC compared with women with no history of oophorectomy, whereas among women with unilateral or partial oophorectomy, the odds of any CAC was 1.7 (95% CI: 1.0-2.8). Among women with bilateral oophorectomy, HT use within 5 years of oophorectomy was associated with a lower prevalence of CAC. Among women with previous hysterectomy, subclinical coronary artery disease was more prevalent among those with oophorectomy and no prerandomization HT use, independent of traditional cardiovascular disease risk factors. The results suggest that factors related to oophorectomy and the absence of estrogen treatment in oophorectomized women may be related to coronary heart disease.


[Intergeneric conjugational crossing of Escherichia coli with Salmonella typhimurium. II. Transfer of a polA1 mutation from Escherichia coli to Salmonella typhimurium and its phenotypic expression in the salmonella genome].
The Escherichia coli structural gene for DNA polymerase I was inserted into Salmonella typhimurium chromosome by conjugal transfer. The genetic analysis of P1-mediated transduction of obtained hybrid showed that polA gene is located in it between metE and rha loci and is cotransduced with metE (about 50%) and rha (12%). The phenotypic properties of polA1 hybrid E. coliXS. typhimurium concerning UV-MMS-NG and gamma-ray sensitivity are similar to the polA1 mutants of E. coli.


Comparison effects and electron spin resonance studies of α-Fe2O4 spinel type ferrite nanoparticles.
α-Fe2O4 spinel type ferrite nanoparticles have been synthesized by cetyltrimethylammonium bromide (CTAB) and ethylenediaminetetraacetic acid (EDTA) assisted hydrothermal route by using NaOH solution. Electron spin resonance (ESR/EPR) measurements of α-Fe2O4 nanoparticles have been performed by a conventional x-band spectrometer at room temperature. The comparison effect of nanoparticles prepared by using CTAB and EDTA in different α-doping on the structural and morphological properties have been investigated in detail. The effect of EDTA-assisted synthesis for α-Fe2O4 nanoparticles are refined, and thus the spectroscopic g-factor are detected by using ESR signals. These samples can be considered as great benefits for magnetic recording media, electromagnetic and drug delivery applications.


Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.
To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.


OBSERVATIONS ON HEATING HAY IN THE FLOODED REGIONS OF NORTHERN VERMONT.
The waters of the recent floods in Vermont and Massachusetts reached the haymows of hundreds of barns. Excessive heating set in almost immediately after the flood waters receded, endangering the farm buildings. Observations were made at 13 different farms in the valleys of the Winooski, Lamoille and Mississquoi Rivers. These observations are summarized in the following statements: 1. From half a foot to seventeen feet of the piles were under water. 2. In every pile of wet hay observed some "heating" had taken place, frequently to the point of being considered dangerous. 3. Heat was generated in the bottom layers of the piles and, escaping up through the hay, led to the production of draughts of hot gases or "flues" rising to the surface. 4. The large quantity of moisture carried with the hot gases from the lower layers was condensed on the upper, cooler hay, or in the air above. Many hay piles had been soaked throughout by the falling condensed moisture. 5. While the hot hay was being removed from the barns only one farmer had observed any charred materials. 6. The maximum temperature found (besides one case of fire) was 74 degrees C., though temperatures above 70 degrees were recorded in other places. 7. The most marked evidence of excessive heating was observed (by the farmers) on the second and third days after the recession of the flood waters. 8. One authentic case of "spontaneous" combustion of hay caused by the flood was reported. The outstanding features were: a. The lower two feet of the pile consisted of old hay from the preceding season (1926). b. Covering this lower 2-foot section of old hay was a 6-inch layer of first cutting alfalfa. c. Two feet beneath the top surface of the 42-foot pile was another 6-inch layer of alfalfa (second cutting). d. This hay pile which fired "spontaneously" was the only one containing even a small quantity of alfalfa. The urgent need for extensive research upon the problem of the "spontaneous" heating of farm products was emphasized by the lack of scientific knowledge with which to meet the situation.


The Pathogenesis of Systemic Sclerosis: An Understanding Based on a Common Pathologic Cascade across Multiple Organs and Additional Organ-Specific Pathologies.
Systemic sclerosis (SSc) is a multisystem autoimmune and vascular disease resulting in fibrosis of various organs with unknown etiology. Accumulating evidence suggests that a common pathologic cascade across multiple organs and additional organ-specific pathologies underpin SSc development. The common pathologic cascade starts with vascular injury due to autoimmune attacks and unknown environmental factors. After that, dysregulated angiogenesis and defective vasculogenesis promote vascular structural abnormalities, such as capillary loss and arteriolar stenosis, while aberrantly activated endothelial cells facilitate the infiltration of circulating immune cells into perivascular areas of various organs. Arteriolar stenosis directly causes pulmonary arterial hypertension, scleroderma renal crisis and digital ulcers. Chronic inflammation persistently activates interstitial fibroblasts, leading to the irreversible fibrosis of multiple organs. The common pathologic cascade interacts with a variety of modifying factors in each organ, such as keratinocytes and adipocytes in the skin, esophageal stratified squamous epithelia and myenteric nerve system in gastrointestinal tract, vasospasm of arterioles in the heart and kidney, and microaspiration of gastric content in the lung. To better understand SSc pathogenesis and develop new disease-modifying therapies, it is quite important to understand the complex pathogenesis of SSc from the two distinct perspectives, namely the common pathologic cascade and additional organ-specific pathologies.


Domoic acid toxicity in Californian sea lions (Zalophus californianus): clinical signs, treatment and survival.
Eighty-one Californian sea lions (Zalophus californianus) with signs of domoic acid toxicity stranded along the coast of California in 1998 when there were blooms of the domoic acid-producing alga Pseudonitzschia australis off-shore. In 2000, a further 184 sea lions stranded with similar clinical signs, but the strandings occurred both during detectable algal blooms and after the blooms had subsided. The clinical signs in these 265 Californian sea lions included seizures, ataxia, head weaving, decreased responsiveness to stimuli and scratching behaviour. Affected animals had high haematocrits, and eosinophil counts, and high activities of serum creatine kinase. They were treated supportively by using fluid therapy, diazepam, lorazepam and phenobarbitone. Fifty-five of the 81 sea lions (68 per cent) affected in 1998 and 81 of the 184 (44 per cent) affected in 2000 died despite the treatment. Three of the 23 sea lions which survived in 1998 were tracked with satellite and radiotransmitters; they travelled as far south as San Miguel Island, California, and survived for at least three months. Eleven of the 129 animals which were released stranded within four months of being released.


[HIV-related dementia].
HIV-Dementia (HIV-D) is an important cause of dementia worldwide. Since the introduction of highly active retroviral therapy, the prevalence of HIV-D has decreased significantly but the proportion of those patients with CD4 at the higher range is increasing. HIV-associated minor cognitive impairment precedes the dementia. The latter is characterized by cognitive, behavioral and motor deficits. Neurological assessment, brain imaging, cerebrospinal fluid analysis and immunologic parameters are important diagnostic tools. Neuropathological data show inflammation and atrophy. The virus attacks brain astrocytes and neurons directly or indirectly by activation of macrophages. HIV-D is therefore an immunologic, inflammatory, and neurodegenerative disease. In the future, drugs will block cell-cell signaling or apoptosis.


Development and Validation of a Nomogram for Early Detection of Malignant Gallbladder Lesions.
Preoperative decision-making for differentiating malignant from benign lesions in the gallbladder remains challenging. We aimed to create a diagnostic nomogram to identify gallbladder cancer (GBC), especially for incidental GBC (IGBC), before surgical resection. A total of 587 consecutive patients with pathologically confirmed gallbladder lesions from a hospital were randomly assigned to a training cohort (70%) and an internal validation cohort (30%), with 287 patients from other centers as an external validation cohort. Radiological features were developed by the least absolute shrinkage and selection operator logistic regression model. Significant radiological features and independent clinical factors, identified by multivariate analyses, were used to construct a nomogram. A diagnostic nomogram was established by age, CA19.9, and 6 radiological features. The values of area under the curve in the internal and external validation cohorts were up to 0.91 and 0.89, respectively. The calibration curves for probability of GBC showed optimal agreement between nomogram prediction and actual observation. Compared with previous methods, it demonstrated superior sensitivity (91.5%) and accuracy (85.1%) in the diagnosis of GBC. The accuracy using the nomogram was significantly higher in GBC groups compared with that by radiologists in the training cohort (P < 0.001) and similarly in each cohort. Notably, most of the IGBC, which were misdiagnosed as benign lesions, were successfully identified using this nomogram. A novel nomogram provides a powerful tool for detecting the presence of cancer in gallbladder masses, with an increase in accuracy and sensitivity. It demonstrates an unprecedented potential for IGBC identification.


Bacteriophages and genetic mobilization in sewage and faecally polluted environments.
Bacteriophages are one of the most abundant entities on the planet and are present in high concentrations within humans and animals, mostly in the gut. Phages that infect intestinal bacteria are released by defecation and remain free in extra-intestinal environments, where they usually persist for longer than their bacterial hosts. Recent studies indicate that a large amount of the genetic information in bacterial genomes and in natural environments is of phage origin. In addition, metagenomic analysis reveals that a substantial number of bacterial genes are present in viral DNA in different environments. These facts support the belief that phages can play a significant role in horizontal gene transfer between bacteria. Bacteriophages are known to transfer genes by generalized and specialized transduction and indeed there are some examples of phages found in the environment carrying and transducing genes of bacterial origin. A successful transduction in the environment requires certain conditions, e.g. phage and bacterial numbers need to exceed certain threshold concentrations, the bacteria need to exist in an infection-competent physiological state, and lastly, the physical conditions in the environment (pH, temperature, etc. of the supporting matrix) have to be suitable for phage infection. All three factors are reviewed here, and the available information suggests: (i) that the number of intestinal bacteria and phages in faecally contaminated environments guarantees bacteria-phage encounters, (ii) that transduction to intestinal bacteria in the environment is probable, and (iii) that transduction is more frequent than previously thought. Therefore, we suggest that phage-mediated horizontal transfer between intestinal bacteria, or between intestinal and autochthonous bacteria in extra-intestinal environments, might take place and that its relevance for the emergence of new bacterial strains and potential pathogens should not be ignored.


Metastatic uveal melanoma: biology and emerging treatments.
Uveal melanoma is the most common primary intraocular cancer in adults. Nearly half of primary uveal melanoma tumors metastasize, but there are currently no effective therapies for metastatic uveal melanoma. The recent discovery of mutations that underlie uveal melanoma metastasis, growth, and survival provide a key to the molecular understanding of this disease. Much work is now underway to leverage this knowledge to develop effective therapies. This review summarizes recently discovered molecular features of uveal melanoma and therapies being explored to capitalize on this knowledge.


[Constitutions and generalities in traditional Tibetan medicine].
The present work is the result of a preliminary study promoted by C.I.S.ME.T. (the International Tibetan Medicine Study Centre) and aims to unify the diagnostic and therapeutic language of various medical cultures. In line with the spirit of the W.H.O. aimed at safeguarding the cultural heritage represented by popular and traditional medicine, encouraged by the Tibetan Medical Centre and under the auspices of His Holiness the Dalai Lama a terminological and conceptual integration of the basic elements of traditional Tibetan medicine is proposed. The Rlung, Bad Kan, Mkris Pa constitution is correlated with embryological anatomy. The 5 exhalations, 5 biles and 5 phlegbs are analysed from a tissue viewpoint with a search for parallels with embryological tissues.


Evolutionary changes affecting rapid identification of 2008 Newcastle disease viruses isolated from double-crested cormorants.
A morbidity-mortality event involving virulent Newcastle disease virus (NDV) in wild double-crested cormorants (Phalacrocorax auritus) occurred in North America in the summer of 2008. All 22 viruses isolated from cormorants were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene. However, the USDA-validated reverse transcription-PCR assay targeting the fusion gene that is specific for virulent isolates identified only 1 of these 22 isolates. Additionally, several of these isolates have been sequenced, and this information was used to identify genomic changes that caused the failure of the test and to revisit the evolution of NDV in cormorants. The forward primer and fusion probe were redesigned from the 2008 cormorant isolate sequence, and the revised fusion gene test successfully identified all 22 isolates. Phylogenetic analyses using both the full fusion sequence and the partial 374-nucleotide sequence identified these isolates as genotype V, with their nearest ancestor being an earlier isolate collected from Nevada in 2005. Histopathological analysis of this ancestral strain revealed morphological changes in the brain consistent with that of the traditional mesogenic pathotypes in cormorants. Intracerebral pathogenicity assays indicated that each of these isolates is virulent with values of >0.7 but not more virulent than earlier isolates reported from Canada.