Simulating the ghost: quantum dynamics of the solvated electron.
The nature of the bulk hydrated electron has been a challenge for both experiment and theory due to its short lifetime and high reactivity, and the need for a high-level of electronic structure theory to achieve predictive accuracy. The lack of a classical atomistic structural formula makes it exceedingly difficult to model the solvated electron using conventional empirical force fields, which describe the system in terms of interactions between point particles associated with atomic nuclei. Here we overcome this problem using a machine-learning model, that is sufficiently flexible to describe the effect of the excess electron on the structure of the surrounding water, without including the electron in the model explicitly. The resulting potential is not only able to reproduce the stable cavity structure but also recovers the correct localization dynamics that follow the injection of an electron in neat water. The machine learning model achieves the accuracy of the state-of-the-art correlated wave function method it is trained on. It is sufficiently inexpensive to afford a full quantum statistical and dynamical description and allows us to achieve accurate determination of the structure, diffusion mechanisms, and vibrational spectroscopy of the solvated electron.


Gorillas with spondyloarthropathies express an MHC class I molecule with only limited sequence similarity to HLA-B27 that binds peptides with arginine at P2.
The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the alpha1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 demonstrate that, like HLA-B27, this gorilla MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27.


Functional significance of concomitant inactivation of hMLH1 and hMSH6 in tumor cells of the microsatellite mutator phenotype.
Genetic or epigenetic inactivation of one of the DNA mismatch repair (MMR) genes in tumor precursor cells causes a profound mutator phenotype, known as the microsatellite mutator phenotype (MMP). This mutator phenotype induces mutations not only in cancer genes that drive tumorigenesis but also in other DNA repair genes. The functional significance of these successive DNA repair gene mutations, however, has not been substantiated. Here we show that the concomitant inactivation of two DNA MMR genes (hMLH1 and hMSH6) increases the mutator phenotype. We isolated cell clones of the SW48 MMP-positive cell line with either active or inactive hMSH6. All of these clones lacked expression of hMLH1 because of promoter hypermethylation. Compared with inactivation of hMLH1 alone, the additional inactivation of hMSH6 produced a higher mutation rate and a different spectrum of mutations in the endogenous hprt gene. These results confirm our model that the mutator phenotype can increase during tumorigenesis by the consecutive inactivation of different members of the DNA MMR system. Thus, a stronger mutator phenotype accelerates the accumulation of mutations in target cancer genes, which, in turn, speeds up tumor progression. The results of this study also have significant impact on our understanding of the mechanism of DNA MMR.


Placental telomere length decreases with gestational age and is influenced by parity: a study of third trimester live-born twins.
In contrast to the postnatal period, little is known about telomere length (TL) during prenatal life. The decrease in placental TL remains unknown, although intra uterine growth retardation and preeclampsia are associated with shorter placental TL. The aim of this study is to assess the decrease of placental TL during the third trimester of gestation and to explore the role of potential "growth influencing factors". The study sample consisted of 329 live-born twins from the East Flanders Prospective Twin Survey. TL was determined using a multiplex quantitative PCR method. Gestational age, sex, birth order, placental characteristics, parity, maternal and paternal age, diabetes, hypertension, smoking, alcohol use, and socio economic status (SES) were considered "growth influencing factors". Bivariable multilevel regression analysis with "growth influencing factors" was performed. Placental TL ranged from 4.3 kbp to 84.4 kbp with a median of 10.8 kbp. Ln(TL) decreased in a linear fashion with an estimated TL decreasing from 13.98 kbp at 28 weeks to 10.56 kbp at 42 weeks. The regression coefficient of gestational age became smaller if considered together with SES (b = -0.017; p = 0.08) or diabetes (b = -0.018; p = 0.07) and bigger if considered together with parity (b = -0.022; p = 0.02), indicating that part of the association between gestational age and telomere length is explained by these three confounding factors. Placental TL decreases during the third trimester of gestation of live-born twins with approximately 25% indicating that telomere shortening may play a role in aging of the placenta.


Effect of Soils from Six Management Systems on Root-knot Nematodes and Plant Growth in Greenhouse Assays.
The effects of soil management systems on root-knot nematode (Meloidogyne incognita) eggs and gall incidence on tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) following tomato were evaluated. Soil was collected from a replicated field experiment in which six management systems were being assessed for vegetable production. Soil management systems were conventional production, organic production, bahiagrass (Paspalum notatum) pasture, bahiagrass: Stylosanthes (Stylosanthes guianensis) pasture, bare ground fallow, and weed fallow. Soil was collected from field plots and used in greenhouse experiments. Identification of egg-parasitic fungi and the incidence of root-knot nematode galling were assessed both on tomato and cucumber planted in the same pots following the removal of tomato plants. Organic, bare ground fallow and conventional production treatments reduced galling both on tomato and on cucumber following tomato. Although no treatment consistently enhanced egg-parasitic fungi, management system did affect egg viability and the types of fungi isolated from parasitized eggs.


Before-After Study of an Electronic Order Set for Reversal of Vitamin K Antagonist-Associated Intracerebral Hemorrhage.
Vitamin K antagonist (VKA)-associated intracerebral hemorrhages (ICHs) are more likely to expand and are associated with higher mortality than primary ICH. Prompt reversal of anticoagulant effect with prothrombin complex concentrate (PCC) may promote hemostasis and decrease hematoma expansion. The aim of this study was to evaluate the impact of an electronic order set designed to standardize and facilitate more timely reversal of coagulopathy in VKA-associated ICH. We identified all adults who received PCC for VKA-associated ICH from June 2012 to June 2015 at University of California San Francisco Medical Center, which included a period before and after an electronic order set became available in 2014. We abstracted baseline demographics and clinical data from electronic health records. The primary outcome was time from radiographic identification of ICH to administration of PCC. Thirty-one patients received PCC for VKA-associated ICH, including 17 patients before and 14 patients after the order set became available. Baseline demographics and clinical features were similar. Order set use was associated with a significant decrease in the time from identification of ICH on imaging to the administration of PCC (median 83 vs 45 minutes; P = .02), more accurate dosing (29.4% vs 92.9%; P < .01), and a shorter time from the PCC order to follow-up international normalized ratio (INR) testing (median 164 vs 85 minutes, P = .001). An electronic order set for administering PCC for VKA-associated ICH was associated with significantly faster time to PCC administration and increased dosing accuracy.


Profiling the Responses of Soccer Substitutes: A Review of Current Literature.
Depending upon competition regulations, the laws of soccer allow between three and an unlimited number of substitutions that can be made on either a permanent or rolling basis. Substitutes are typically introduced to minimise/offset the effects of fatigue, alter tactics, replace players deemed as underperforming or injured, and/or give playing time to youth players or to squad members returning from injury. While the match-day practices of substitutes include participation in the pre-match warm-up, and sporadic periods of rewarm-up activity, it is currently unclear as to whether these pre-entry preparations facilitate optimal match performance thereafter. Acknowledging the contextual factors that possibly influence substitutes' performance, this review summarises the presently available literature on soccer substitutes, and makes recommendations for future research. Literature searching and screening yielded 13 studies, which have typically focused on characterising: (1) the patterns, including timing, of substitutes' introduction; (2) indices of match-performance; and (3) the emotional experiences of soccer substitutes. The majority of substitutions occur after the first-half has ended (i.e. at half-time or during the second-half), with introduced players exceeding the second-half physical performances of those who started the match. Observations of progressive improvements in running performance as playing time increases, and findings that substitutes mostly experience negative emotions, highlight the potential inadequacies of pre-match preparations, and present future research opportunities. Additional work is therefore needed to confirm these findings and to determine the efficacy of current preparation strategies, thereby providing opportunities to assess then address substitutes' pre-pitch entry preparations, on-field performance and emotional responses.


Physical activity profiles and perceived environmental determinants in New Zealand: a national cross-sectional study.
A minority of adults in developed countries engage in sufficient physical activity (PA) to achieve health benefits. This study aims to identify modifiable perceived resources and barriers to PA among New Zealand adults. Secondary analysis of a 2003 nationally representative cross-sectional mail survey, stratified by region, age, and ethnicity, and analyzed utilizing ordinal logistic regression. Overall, n = 8038 adults responded to the survey, of whom 49% met updated guidelines for sufficient PA. Perceived accessibility of local resources was associated with PA; however, for some resources there was more awareness among individuals whose predominant activity was not commonly associated with that resource (eg, health clubs and walkers). Perceived local environmental barriers demonstrated negative (steep hills, crime, dogs) and positive (unmaintained footpaths) associations. The absence of perceived environmental barriers was strongly associated with increased activity, suggesting the number of barriers may be a critical factor. Complex relationships between perceptions of local environments and activity patterns among adults were found. Although complex, these results demonstrate positive associations between awareness of resources and perceived lack of barriers with being sufficiently physically active for health. Therefore, investments in provision and/or promotion of local resources have the potential to enable active healthy communities.


Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.
Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.


Indicators for healthy ageing--a debate.
Definitions of healthy ageing include survival to a specific age, being free of chronic diseases, autonomy in activities of daily living, wellbeing, good quality of life, high social participation, only mild cognitive or functional impairment, and little or no disability. The working group Epidemiology of Ageing of the German Association of Epidemiology organized a workshop in 2012 with the aim to present different indicators used in German studies and to discuss their impact on health for an ageing middle-European population. Workshop presentations focused on prevalence of chronic diseases and multimorbidity, development of healthy life expectancy at the transition to oldest-age, physical activity, assessment of cognitive capability, and functioning and disability in old age. The communication describes the results regarding specific indicators for Germany, and hereby contributes to the further development of a set of indicators for the assessment of healthy ageing.


Stemness genes expression in naïve vs. osteodifferentiated human dental-derived stem cells.
Mesenchymal stem cells (MSCs) have been deeply investigated in regenerative medicine because of their crucial role in tissue healing, such as tissue regeneration. Dental-derived stem cells (d-DSCs) are easily available from dental tissues, which can be isolated from all age patients with minimal discomfort. Normal unerupted third molars tooth buds were collected from adolescents' patients underwent to extractions for orthodontic reasons. The expression of the genes Kruppel-like factor 4 (Klf-4), octamer-binding transcription factor 4 (Oct-4), homeobox transcription factor Nanog (NANOG) was investigated in d-DSCs obtained from dental bud (DBSCs), differentiated toward osteoblastic phenotype and not. Our results showed that DBSCs expressed Oct-4, Nanog, and Klf-4 in undifferentiated conditions and interestingly the expression of such genes increased when the cells were kept in osteogenic medium. These attractive stemness properties, together with the effortlessly isolation, during common oral and maxillofacial surgical procedures, from undifferentiated tissues such as dental bud, make this kind of d-DSCs a promising tool in regenerative medicine, having the potential for clinical applications, and reinforcing the present challenge to develop new preventive and healing strategies in tissue regeneration.


Cryptic sexual populations account for genetic diversity and ecological success in a widely distributed, asexual fungus-growing ant.
Sex and recombination are central processes in life generating genetic diversity. Organisms that rely on asexual propagation risk extinction due to the loss of genetic diversity and the inability to adapt to changing environmental conditions. The fungus-growing ant species Mycocepurus smithii was thought to be obligately asexual because only parthenogenetic populations have been collected from widely separated geographic localities. Nonetheless, M. smithii is ecologically successful, with the most extensive distribution and the highest population densities of any fungus-growing ant. Here we report that M. smithii actually consists of a mosaic of asexual and sexual populations that are nonrandomly distributed geographically. The sexual populations cluster along the Rio Amazonas and the Rio Negro and appear to be the source of independently evolved and widely distributed asexual lineages, or clones. Either apomixis or automixis with central fusion and low recombination rates is inferred to be the cytogenetic mechanism underlying parthenogenesis in M. smithii. Males appear to be entirely absent from asexual populations, but their existence in sexual populations is indicated by the presence of sperm in the reproductive tracts of queens. A phylogenetic analysis of the genus suggests that M. smithii is monophyletic, rendering a hybrid origin of asexuality unlikely. Instead, a mitochondrial phylogeny of sexual and asexual populations suggests multiple independent origins of asexual reproduction, and a divergence-dating analysis indicates that M. smithii evolved 0.5-1.65 million years ago. Understanding the evolutionary origin and maintenance of asexual reproduction in this species contributes to a general understanding of the adaptive significance of sex.


Wilms tumor imaging: patient costs and protocol compliance.
To evaluate the patient costs for imaging and compliance with imaging protocols in pediatric patients in the National Wilms Tumor Study (NWTS) IV. The medical and imaging records of 60 patients (28 male, 32 female; aged 3 days to 12.6 years) in NWTS IV were reviewed. Initial imaging and follow-up imaging were evaluated separately. Three levels of follow-up compliance were evaluated. The total patient cost for imaging was $442,180: $94,212 for initial and $347,968 for follow-up studies. Many areas of potential cost savings were identified. Protocol compliance was variable. Seventy-five percent of patients underwent studies in full compliance with the initial protocol requirements. For follow-up, compliance was 0%-80% for different studies at different compliance levels. For no study was compliance 100%. Imaging costs in pediatric patients with Wilms tumor are substantial. Protocol compliance was not optimal.


Comparing Outcomes with Bone Marrow or Peripheral Blood Stem Cells as Graft Source for Matched Sibling Transplants in Severe Aplastic Anemia across Different Economic Regions.
Bone marrow (BM) is the preferred graft source for hematopoietic stem cell transplantation (HSCT) in severe aplastic anemia (SAA) compared with mobilized peripheral blood stem cells (PBSCs). We hypothesized that this recommendation may not apply to those regions where patients present later in their disease course, with heavier transfusion load and with higher graft failure rates. Patients with SAA who received HSCT from an HLA-matched sibling donor from 1995 to 2009 and reported to the Center for International Blood and Marrow Transplant Research or the Japan Society for Hematopoietic Cell Transplantation were analyzed. The study population was categorized by gross national income per capita and region/countries into 4 groups. Groups analyzed were high-income countries (HIC), which were further divided into United States-Canada (n = 486) and other HIC (n = 1264); upper middle income (UMIC) (n = 482); and combined lower-middle, low-income countries (LM-LIC) (n = 142). In multivariate analysis, overall survival (OS) was highest with BM as graft source in HIC compared with PBSCs in all countries or BM in UMIC or LM-LIC (P < .001). There was no significant difference in OS between BM and PBSCs in UMIC (P = .32) or LM-LIC (P = .23). In LM-LIC the 28-day neutrophil engraftment was higher with PBSCs compared with BM (97% versus 77%, P = .002). Chronic graft-versus-host disease was significantly higher with PBSCs in all groups. Whereas BM should definitely be the preferred graft source for HLA-matched sibling HSCT in SAA, PBSCs may be an acceptable alternative in countries with limited resources when treating patients at high risk of graft failure and infective complications.


Cytoskeleton-dependent transport of cytoplasmic particles in previtellogenic to mid-vitellogenic ovarian follicles of Drosophila: time-lapse analysis using video-enhanced contrast microscopy.
In Drosophila oogenesis, several morphogenetic determinants and other developmental factors synthesized in the nurse cells have been shown to accumulate in the oocyte during pre- to mid-vitellogenic stages. However, the mechanisms of the involved intercellular transport processes that seem to be rather selective have not been revealed so far. We have investigated in vitro, by means of video-enhanced contrast time-lapse microscopy, the transport of cytoplasmic particles from the nurse cells through ring canals into the oocyte during oogenesis stages 6-10A. At stage 7, we first observed single particles moving into the previtellogenic oocyte. The particle transfer was strictly unidirectional and seemed to be selective, since only some individual particles moved whereas other particles lying in the vicinity of the ring canals were not transported. The observed transport processes were inhibitable with 2,4-dinitrophenol, cytochalasin B or N-ethylmaleimide, but not with microtubule inhibitors. At the beginning of vitellogenesis (stage 8), the selective translocation of particles through the ring canals became faster (up to 130 nm/second) and more frequent (about 1 particle/minute), whereas during mid-vitellogenesis (stages 9-10A) the velocity and the frequency of particle transport decreased again. Following their more or less rectilinear passage through the ring canals, the particles joined a circular stream of cytoplasmic particles in the oocyte. This ooplasmic particle streaming started at stage 6/7 with velocities of about 80 nm/second and some reversals of direction at the beginning. The particle stream in the oocyte was sensitive to colchicine and vinblastine, but not to cytochalasin B, and we presume that it reflects the rearrangement of ooplasmic microtubules described recently by other authors. We propose that during stages 7-10A, a selective transport of particles into the oocyte occurs through the ring canal along a polarized scaffold of cytoskeletal elements in which microfilaments are involved. This transport might be driven by a myosin-like motor molecule. Either attached to, or organized into, such larger particles or organelles, specific mRNAs and proteins might become selectively transported into the oocyte.


Rho-ROCK signal pathway regulates microtubule-based process formation of cultured podocytes--inhibition of ROCK promoted process elongation.
Podocytes, renal glomerular visceral epithelial cells, have two kinds of processes, namely major processes containing microtubules (MTs) and foot processes with actin filaments (AFs). The present study investigated how MTs are organized by the Rho-ROCK signal transduction pathway during process formation of podocytes. After induction of differentiation, podocytes of the conditionally immortalized mouse cell line were treated with Y-27632, a specific inhibitor of ROCK, and exoenzyme C3, an inhibitor of RhoA, as well as with forskolin whose effects include inhibition of RhoA, in order to inhibit the Rho-ROCK pathway. Inhibition of ROCK significantly enhanced the formation of thick processes containing MT bundles. Y-27632 promoted process formation even in the presence of latrunculin A which disrupts AFs, strongly suggesting that ROCK directly regulates MT assembly. Treatment with Y-27632 increased MT stability, and stabilized MTs preferentially localized in podocyte processes. Moreover, when treated with a combination of Y-27632 and forskolin, and with Y-27632 and C3 as well, podocytes developed not only MT-based thick processes but also AF-based thin projections. These data indicate a contribution of ROCK in MT organization to promote podocyte process formation, although it was originally thought to regulate AF assembly. AF-based thin projections seem to be induced mainly by inhibition of RhoA and ROCK. The present study reveals a significant role of the Rho-ROCK signal pathway in the reorganization of both MTs and AFs during process formation of podocytes.


Association of the Affordable Care Act's Medicaid Expansion With Care Quality and Outcomes for Low-Income Patients Hospitalized With Heart Failure.
Heart failure (HF) is the leading cause of morbidity and mortality in the United States. Despite advancement in the management of HF, outcomes remain suboptimal, particularly among the uninsured. In 2014, the Affordable Care Act expanded Medicaid eligibility, and millions of low-income adults gained insurance. Little is known about Medicaid expansion's effect on inpatient HF care. We used the American Heart Association's Get With The Guidelines-Heart Failure registry to assess changes in inpatient care quality and outcomes among low-income patients (<65 years old) hospitalized for HF after Medicaid expansion, in expansion, and nonexpansion states. Patients were classified as low-income if covered by Medicaid, uninsured, or missing insurance. Expansion states were those that implemented expansion in 2014. Piecewise logistic multivariable regression models were constructed to track quarterly trends of quality and outcome measures in the pre (January 1, 2010-December 31, 2013) and postexpansion (January 1, 2014-June 30, 2017) periods. These measures were compared between expansion versus nonexpansion states during the postexpansion period. The cohort included 58 804 patients hospitalized across 391 sites. In states that expanded Medicaid, uninsured HF hospitalizations declined from 7.9% to 4.4%, and Medicaid HF hospitalizations increased from 18.3% to 34.6%. Defect-free HF care was increasing during the preexpansion period (adjusted odds ratio/quarter, 1.06; 95% confidence interval, 1.03-1.08) but did not change after expansion (adjusted odds ratio, 0.99; 95% confidence interval, 0.97-1.02). Patterns were similar for other quality measures. There were no quality measures for which the rate of improvement sped up after expansion. In-hospital mortality rates remained similar during the preexpansion (adjusted odds ratio, 0.99; 95% confidence interval, 0.96-1.02) and postexpansion periods (adjusted odds ratio, 1.00; 95% confidence interval, 0.97-1.03). Among nonexpansion states, uninsured HF hospitalizations increased (11.6% to 16.7%) as did Medicaid HF hospitalizations (17.9% to 26.6%), and no quarterly improvement was observed for most quality measures in the post compared with preexpansion period. During the postexpansion period, defect-free care and mortality did not differ between expansion and nonexpansion states. Medicaid expansion was associated with a significant decline in uninsured HF hospitalizations but not improvements in quality of care or in-hospital mortality among sites participating in a national quality improvement initiative. Efforts beyond insurance expansion are needed to improve in-hospital outcomes for low-income patients with HF.


Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress.
It has been well established that the tumor microenvironment can promote tumor cell adaptation and survival. However, the mechanisms that influence malignant progression have not been clearly elucidated. We have previously demonstrated that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR). Here, we show that tumors derived from K-Ras-transformed Perk(-/-) mouse embryonic fibroblasts (MEFs) are smaller and exhibit less angiogenesis than tumors with an intact ISR. Furthermore, Perk promotes a tumor microenvironment that favors the formation of functional microvessels. These observations were corroborated by a microarray analysis of polysome-bound RNA in aerobic and hypoxic Perk(+/+) and Perk(-/-) MEFs. This analysis revealed that a subset of proangiogenic transcripts is preferentially translated in a Perk-dependent manner; these transcripts include VCIP, an adhesion molecule that promotes cellular adhesion, integrin binding, and capillary morphogenesis. Taken with the concomitant Perk-dependent translational induction of additional proangiogenic genes identified by our microarray analysis, this study suggests that Perk plays a role in tumor cell adaptation to hypoxic stress by regulating the translation of angiogenic factors necessary for the development of functional microvessels and further supports the contention that the Perk pathway could be an attractive target for novel antitumor modalities.


Steroid control of acute middle ear inflammation in a mouse model.
To investigate steroids for their potential for therapeutic approaches to control otitis media. Glucocorticoids and mineralocorticoids have differential effects on inflammation and fluid absorption, but little is known of their control of middle and inner ear manifestations of acute otitis media. Both glucocorticoid (prednisolone and dexamethasone) and mineralocorticoid (aldosterone and fludrocortisone) steroids were investigated for their ability to reduce inflammatory symptoms in a mouse otitis media model. Academic medical center. Acute inflammation was induced by transtympanic injection of heat killed Streptococcus pneumoniae to 100 BALB/c mice. Twenty mice in each experimental group (prednisolone, dexamethasone, aldosterone, and fludrocortisone) were given a steroid in their drinking water the day before inoculation, and these treatments were continued until the mice were killed for histologic examination. Twenty control mice were treated with water only. Histologic measure of inflammation: middle ear fluid, inflammatory cell number, and tympanic membrane thickness. Histologic middle ear morphometrics showed significant steroid effects at both 3 and 5 days in reduction of fluid area, cell number, and tympanic membrane thickness. Glucocorticoids were most effective in controlling inflammation. Interestingly, the mineralocorticoids were also effective in reducing the inflammatory response at 5 days, suggesting that their fluid transport function helped clear disease. Thus, steroid control of middle ear disease may be useful in alleviating symptoms faster and reducing the risk to the inner ear.


Psychological variables in hypertension: relationship to casual or ambulatory blood pressure in men.
The evidence linking hypertension with personality or psychological characteristics, such as anger, anxiety, or depression, remains equivocal. This may be due in part to limitations of personality theory, confounding by awareness of hypertension, and/or inherent difficulties in measuring blood pressure. This study was designed to investigate the association between mild hypertension as defined by both ambulatory and casual (clinic) blood pressure measurements and various measures of personality and psychological characteristics. We examined this association in a population-based sample of 283 men between the ages of 30 and 60 years from eight work sites in New York City, using an ambulatory blood pressure monitor and controlling for age, race/ethnicity, and body mass index. We found no consistent difference between participants with mild hypertension and those with normal blood pressure on any of the psychological variables assessed, including Type A behavior pattern, state and trait anger, anger expression, anxiety, symptoms of psychological distress, locus of control, or attributional style. Results were not due to the use of antihypertensive medication by some of the participants with hypertension nor to the dichotomization of blood pressure into those with and without mild hypertension. This contrasts with previous findings from this study showing a sizable association of ambulatory blood pressure and hypertension with job strain (a situational measure), age, and body mass index. These null results suggest that situational, biological, and perhaps behavioral factors are the primary determinants of mild hypertension and that the predictive significance of psychological or dispositional factors is low or negligible in those without overt cardiovascular disease.


It's NOT rocket science: rethinking our metaphors for research in health professions education.
The health professional education community is struggling with a number of issues regarding the place and value of research in the field, including: the role of theory-building versus applied research; the relative value of generalisable versus contextually rich, localised solutions, and the relative value of local versus multi-institutional research. In part, these debates are limited by the fact that the health professional education community has become deeply entrenched in the notion of the physical sciences as presenting a model for 'ideal' research. The resulting emphasis on an 'imperative of proof' in our dominant research approaches has translated poorly to the domain of education, with a resulting denigration of the domain as 'soft' and 'unscientific' and a devaluing of knowledge acquired to date. Similarly, our adoption of the physical sciences''imperative of generalisable simplicity' has created difficulties for our ability to represent well the complexity of the social interactions that shape education and learning at a local level. Using references to the scientific paradigms associated with the physical sciences, this paper will reconsider the place of our current goals for education research in the production and evolution of knowledge within our community, and will explore the implications for enhancing the value of research in health professional education. Reorienting education research from its alignment with the imperative of proof to one with an imperative of understanding, and from the imperative of simplicity to an imperative of representing complexity well may enable a shift in research focus away from a problematic search for proofs of simple generalisable solutions to our collective problems, towards the generation of rich understandings of the complex environments in which our collective problems are uniquely embedded.


National Survey Indicates that Individual Vaccination Decisions Respond Positively to Community Vaccination Rates.
Some models of vaccination behavior imply that an individual's willingness to vaccinate could be negatively correlated with the vaccination rate in her community. The rationale is that a higher community vaccination rate reduces the risk of contracting the vaccine-preventable disease and thus reduces the individual's incentive to vaccinate. At the same time, as for many health-related behaviors, individuals may want to conform to the vaccination behavior of peers, counteracting a reduced incentive to vaccinate due to herd immunity. Currently there is limited empirical evidence on how individual vaccination decisions respond to the vaccination decisions of peers. In the fall of 2014, we used a rapid survey technology to ask a large sample of U.S. adults about their willingness to use a vaccine for Ebola. Respondents expressed a greater inclination to use the vaccine in a hypothetical scenario with a high community vaccination rate. In particular, an increase in the community vaccination rate from 10% to 90% had the same impact on reported utilization as a nearly 50% reduction in out-of-pocket cost. These findings are consistent with a tendency to conform with vaccination among peers, and suggest that policies promoting vaccination could be more effective than has been recognized.


Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise.
The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min(-1) ; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg(-1) ) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg(-1) ) of l-[ring-(13) C6 ]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation.


Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model.
As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.


The role of the physician and the medical profession in the prevention of international torture and in the treatment of its survivors. American College of Physicians.
The prevention of torture and the treatment of survivors are issues that concern an increasing number of physicians in their daily work. Every day, thousands of men, women, and children are subjected to violence and are forced to flee their homelands. There are more than 18 million refugees in the world and hundreds of thousands of persons seeking asylum, many of them in the United States. Physicians are often the first to interview these victims of abuse. Torture has serious and long-lasting health consequences. Thus, physicians can play a key role in documenting and preventing many forms of abuse and in treating survivors. In some areas, physicians may become the targets of arrest because of their work as clinicians or as influential members of their communities. They may also face disturbing ethical dilemmas as they witness torture or its results. As members of the medical profession, physicians have an obligation to their peers around the world. This report reviews the current state of physicians' involvement in the prevention of international torture and in the treatment of its victims. We propose ways in which physicians can become involved by caring for survivors of torture and by providing expert testimony on behalf of victims who seek asylum. We discuss how the medical profession complements the efforts of individual physicians by providing an infrastructure to support and guide their work. Medical organizations can adopt and disseminate ethical principles that specifically address human rights and their violation. They can coordinate letter-writing networks for human rights, organize or sponsor fact-finding missions, and develop continuing medical education courses on topics such as the identification and treatment of victims of torture. We conclude that physicians can make a difference, both as clinicians and as advocates for the health of the public and the protection of the human rights. The American College of Physicians will continue to advocate for the rights of persons and communities to live in dignity and peace, free of the fear of unjust imprisonment or torture.


Should Health Systems Share Genetic Findings With At-Risk Relatives When the Proband Is Deceased? Interviews With Individuals Diagnosed With Lynch Syndrome.
Genetic information has health implications for patients and their biological relatives. Death of a patient before sharing a genetic diagnosis with at-risk relatives is a missed opportunity to provide important information that could guide interventions to minimize cancer-related morbidity and mortality in relatives. We performed semi-structured interviews with individuals diagnosed with Lynch syndrome at 1 of 4 health systems to explore their perspectives on whether health systems should share genetic risk information with relatives following a patient's death. An inductive, open-coding approach was used to analyze audio-recorded content, with software-generated code reports undergoing iterative comparative analysis by a qualitative research team to identify broad themes and representative participant quotes. Among 23 participating interviewees, 19 supported health systems informing relatives about their Lynch syndrome risk while the remaining 4 were conflicted about patient privacy. Most (n=22) wanted their Lynch syndrome diagnosis shared with relatives if they were unable to share and to be informed of their own risk if a diagnosed relative was unable to share. The most common issues noted regarding information-sharing with relatives included patient privacy and privacy laws (n=8), potential anxiety (n=5), and lack of contact information for relatives (n=3). Interviewee perspectives on how health systems could communicate genetic findings generated a consensus: When - a few months after but within a year of the patient's death; How - explanatory letter and follow-up phone call; and Who - a knowledgeable professional. Interviews demonstrated strong and consistent perspectives from individuals diagnosed with Lynch syndrome that health systems have a role and responsibility to inform relatives of genetic findings following a patient's death.


Effects of A(3) adenosine receptor activation and gene knock-out in ischemic-reperfused mouse heart.
To characterize effects of A(3) adenosine receptor (A(3)AR) activation and gene knock-out on responses to ischemia-reperfusion in mouse heart. Perfused hearts from wild-type and A(3)AR gene knock-out (A(3)AR KO) mice were subjected to 20 min ischemia and 30 min reperfusion. Functional responses were assessed and changes in energy metabolism and cytosolic pH monitored via 31P-NMR spectroscopy. Selective A(3)AR agonism with 100 nM 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (chloro-IB-MECA) enhanced post-ischemic contractile recovery without altering contracture development in wild-type hearts, an effect unrelated to non-selective activation of A(1) or A(2) adenosine receptors. Chloro-IB-MECA also improved recovery in hearts overexpressing A(1)ARs. Paradoxically, post-ischemic recovery was enhanced by A(3)AR KO. Developed pressure, +dP/dt, and -dP/dt all recovered to higher levels in A(3)AR KO (70-80% of pre-ischemia) vs. wild-type hearts (45-50% of pre-ischemia) (P<0.05). Enhanced recovery was unrelated to recoveries of ATP, phosphocreatine (PCr), inorganic phosphate (P(i)), energy state ([ATP]/[ADP] x [P(i)], DeltaG(ATP)) or cytosolic pH. Selective A(3)AR activation is cardioprotective in wild-type hearts and hearts overexpressing A(1)ARs, yet A(3)AR gene deletion generates an ischemia-tolerant phenotype without altering energy metabolism or pH. This may be due to compensatory changes or undefined genotypic differences in A(3)AR KO vs. wild-type hearts.


"Under Pressure" - How fungi evade, exploit, and modulate cells of the innate immune system.
The human immune system uses an arsenal of effector mechanisms to prevent and counteract infections. Yet, some fungal species are extremely successful as human pathogens, which can be attributed to a wide variety of strategies by which these fungi evade, exploit, and modulate the immune system. These fungal pathogens normally are either harmless commensals or environmental fungi. In this review we discuss how commensalism, but also life in an environmental niche without human contact, can drive the evolution of diverse and specialized immune evasion mechanisms. Correspondingly, we discuss the mechanisms contributing to the ability of these fungi to cause superficial to life-threatening infections.


Cavitation-enhanced extravasation for drug delivery.
A flow-through tissue-mimicking phantom composed of a biocompatible hydro-gel with embedded tumour cells was used to assess and optimize the role of ultrasound-induced cavitation on the extravasation of a macromolecular compound from a channel mimicking vessel in the gel, namely a non-replicating luciferase-expressing adenovirus (Ad-Luc). Using a 500 KHz therapeutic ultrasound transducer confocally aligned with a focussed passive cavitation detector, different exposure conditions and burst mode timings were selected by performing time and frequency domain analysis of passively recorded acoustic emissions, in the absence and in the presence of ultrasound contrast agents acting as cavitation nuclei. In the presence of Sonovue, maximum ultraharmonic emissions were detected for peak rarefactional pressures of 360 kPa, and maximum broadband emissions occurred at 1250 kPa. The energy of the recorded acoustic emissions was used to optimise the pulse repetition frequency and duty cycle in order to maximize either ultraharmonic or broadband emissions while keeping the acoustic energy delivered to the focus constant. Cell viability measurements indicated that none of the insonation conditions investigated induces cell death in the absence of a therapeutic agent (i.e. virus). Phase contrast images of the tissue-mimicking phantom showed that short range vessel disruption can occur when ultra-harmonic emissions (nf0/2) are maximised whereas formation of a micro-channel perpendicular to the flow can be obtained in the presence of broadband acoustic emissions. Following Ad-Luc delivery, luciferase expression measurements showed that a 60-fold increase in its bioavailability can be achieved when broadband noise emissions are present during insonation, even for modest contrast agent concentrations. The findings of the present study suggest that drug delivery systems based on acoustic cavitation may help enhance the extravasation of anticancer agents, thus increasing their penetration distance to hypoxic regions and poorly vascularised tumour regions.


Gathering "tea"--from necessity to connectedness with nature. Local knowledge about wild plant gathering in the Biosphere Reserve Grosses Walsertal (Austria).
Wild plant gathering is an essential element in livelihood strategies all over the world. However due to changing circumstances in Europe, the reason for gathering has altered from one of necessity in the past to a pleasurable activity today. Wild plant gathering has therefore also received renewed attention as a form of intangible cultural heritage expressing local preferences, habits and man's relationship with nature. In the Biosphere Reserve Grosses Walsertal (Austria), local people's knowledge of the gathering of wild plants and their perception of their own gathering activities are being documented. The focus of this paper is on the uses of herbal teas and the informal guidelines for gathering plants that have been issued by the Bergtee (mountain tea) association. Thirty-six free-list interviews were conducted with subsequent semi-structured interviews and three focus group meetings held with members of the Bergtee association. Participatory observation (gathering and processing plants, mixing and marketing tea) also allowed for greater understanding of what had been reported. In total, 140 different gathered plant species were listed by respondents. Herbal tea is the most frequently mentioned use. The Bergtee association, founded by a young man and two middle-aged women in the valley, is a good example of the link between biological and cultural diversity, with the aim of sharing the biosphere reserve's natural treasures as well as local plant-related knowledge in the form of herbal tea products. The association's informal guidelines for gathering reflect people's attitude to nature: monetary income does not play a major role in gathering plants; instead people's appreciation of the value of the nature around them is to the fore. Gathering wild plants can be seen as an expression of people's regional identity. The conscious appreciation of nature and related local knowledge is crucial for the sustainable conservation and use of the Biosphere Reserve's resources.


Supporting continuous learning in a large organization: the role of group and organizational perspectives.
Many organizations recognize the need to continuously adapt and learn in order to survive and remain competitive. Learning and therefore change in organizations is driven in two ways. First, there is strategically driven learning, motivated by high-level factors such as market changes, company mergers and newly emerging approaches to organizational management and workplace learning. These changes reveal themselves in the introduction of new training programmes, recruitment strategies and knowledge management methodologies. Second, there is local, continuous learning occurring from the ground up. This is revealed as workers become more adept at their job through experience and collaboration with colleagues. Continuous learning is more gradual and requires local autonomy. This paper describes an experiment in supporting local, continuous learning, and its dissemination, but driven by a strategic initiative of the organization. This work raised many issues concerning the difficulty of integrating local and global organizational influences on learning. We outline lessons learned and suggestions as to the extent to which it is possible to align continuous learning with a company-wide perspective.


Cyclohexene ring and Fjord region twist inversion in stereoisomeric DNA adducts of enantiomeric benzo[c]phenanthrene diol epoxides.
The sterically hindered, nonplanar fjord region polycyclic aromatic hydrocarbons (PAHs) have been of great interest because of the exceptionally high mutagenic and tumorigenic activity of certain of their metabolically activated diol epoxides. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region PAH, is metabolically activated to a pair of enantiomers, 1S,2R,3R,4S-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene, (+)-anti-B[c]PhDE, and the corresponding 1R,2S,3S,4R enantiomer, (-)-anti-B[c]PhDE. Both of these can bind covalently to the amino group of purines in DNA via trans addition. In the present work we carry out an extensive computational investigation of the 1R(+) and 1S(-)-trans-anti-B[c]Ph adducts to the base guanine, with the goal of delineating the conformational possibilities for the fjord region and the adjacent cyclohexene-type benzylic ring and their relevance to DNA duplexes. We created 10 369 starting structures for each adduct and minimized the energy using AMBER 5.0. A limited set of conformational families is computed, in which the R isomer structures are near mirror images of the S isomer. The benzylic rings are essentially all half-chair-type. Cyclohexene-type ring inversion as well as fjord region twist inversion are possible for each isomer and are correlated. DNA duplexes modified by fjord region adducts select conformers from the allowed families that optimize stacking interactions, which contributes to the stability of the carcinogen-intercalated DNA duplex structures [Cosman et al. (1993) Biochemistry 32, 12488-12497; Cosman et al. (1995) Biochemistry 34, 1295-1307; Suri et al. (1999) J. Mol. Biol. 292, 289-307; Lin et al. (2001) J. Mol. Biol. 306, 1059-1080]. In turn, this stability could contribute to the resistance to repair by the human nucleotide excision system observed in fjord region adducts [Buterin et al. (2000) Cancer Res. 60, 1849-1856].


[Stability of diurnal blood pressure rhythm during 48-hour ambulatory blood pressure monitoring].
Evaluation of nocturnal blood pressure (BP) fall is important for prognosis and selection of antihypertensive therapy. We assessed stability of BP diurnal rhythm in 56 hospitalized hypertensive patients by means of 48-hour ambulatory BP monitoring. According to the degree of congruence of BP fall during two nights of monitoring the following groups of patients were distinguished: with similar diurnal BP rhythm in both nights (72% of all patients, including 42% with sufficient and 30% with insufficient nocturnal BP decline), with different diurnal BP rhythms - with sufficient fall of BP in one night and insufficient in another (28% of all patients). BP variability in all patients did not change and could be considered very stable. Thus when insufficient nocturnal BP fall is detected its stability should be confirmed by repeat BP monitoring.


Stratification of patient risk based on prostate-specific antigen doubling time after radical retropubic prostatectomy.
To assess the risk of local recurrence, systemic progression, and death from cancer among patients who experience biochemical relapse after radical retropubic prostatectomy and to stratify those patients by prostate-specific antigen (PSA) doubling time (DT). We identified patients who experienced biochemical recurrence (defined as a PSA level < or =0.4 ng/mL) after radical prostatectomy from January 1, 1990, to December 31, 1999, for prostate adenocarcinoma. The PSA-DT was calculated by log linear regression using all PSA values within 2 years of biochemical recurrence. Local recurrence- and systemic progression- free survival and cancer-specific survival were estimated using the Kaplan-Meier method and analyzed by the log-rank test and Cox models. Biochemical recurrence was noted in 1521 (27%) of 5533 men during the follow-up period. Of the 1064 patients with a calculable PSA-DT, 322 (30%) had a PSA-DT of less than 1 year, 357 (34%) had a PSA-DT of 1 to 9.9 years, and 385 (36%) had a PSA-DT of 10 years or more. Patients with a PSA-DT of 10 years or more were less likely to have a higher preoperative PSA level, Gleason score, advanced pathologic stage, and seminal vesicle invasion. Patients with a PSA-DT of 10 years or more were at low risk of local recurrence (hazard ratio [HR], 0.09; 95% confidence interval [CI], 0.06-0.14; compared with patients with a PSA-DT of <1 year), systemic progression (HR, 0.05; 95% CI, 0.02-0.13), or death from cancer (HR, 0.15; 95% CI, 0.05-0.43). Prostate-specific antigen DT is an independent predictor of clinical disease recurrence and mortality after surgical biochemical failure. Risk stratification into high-, intermediate-, and low-risk categories based on the PSA-DT provides helpful clinical information and assists in the development of salvage therapy trials.


Changes in Mitochondrial Morphology and Bioenergetics in Human Lymphoblastoid Cells With Four Novel OPA1 Mutations.
Mutations in the optic atrophy 1 gene (OPA1) have been reported in patients with autosomal dominant optic atrophy (ADOA). OPA1 plays important roles in mitochondrial dynamics and cell apoptosis. The link between OPA1 mutations and changes in bioenergetics is still not fully resolved. The aim of this study was to investigate the effects of OPA1 mutations on the mitochondrial tubular network and bioenergetics. We established lymphoblastoid cell lines from four ADOA families harboring different OPA1 mutations, unaffected relatives (internal control cell lines), and unrelated normal controls (normal control cell lines). OPA1 splice variants and mRNA were analyzed by reverse transcription-PCR and quantitative real-time PCR. Protein isoforms were examined by Western blotting. The mitochondrial network was visualized by confocal microscopy. Mitochondrial bioenergetics were assessed using a Seahorse XF24 flux analyzer. Mitochondrial membrane potential and oxidative damage were analyzed by flow cytometry. OPA1 mutant cell lines showed significant decreases in OPA1 mRNA and protein expression, mitochondrial membrane potential, and ATP synthesis. A marked deficiency of the long isoform of OPA1 was observed in cells with OPA1 mutations in the middle domain and GTPase effector domain. Confocal microscopy revealed increased mitochondrial fragmentation in OPA1 mutant cells. OPA1 mutant cells also displayed reduced oxygen consumption and underwent glycolysis to produce ATP. Moreover, OPA1 mutations caused the accumulation of oxidative damage. Our experiments demonstrated that OPA1 mutations induced mitochondrial fragmentation, uncoupled mitochondrial respiration, and elicited dysfunctional bioenergetics. However, there were no significant differences among the various OPA1 mutations.


Late-phase contrast-enhanced ultrasound reflects biological features of instability in human carotid atherosclerosis.
Development of translational functional imaging modalities for atherosclerosis risk stratification is sought for stroke prediction. Our group has developed late-phase contrast-enhanced ultrasound (LP-CEUS) to quantify microbubble contrast retention within carotid atherosclerosis and shown it to separate asymptomatic plaques from those responsible for recent cerebrovascular events. We hypothesized that microbubbles are retained in areas of plaque inflammation, aiming to examine whether LP-CEUS signal reflects plaque biology. Subjects awaiting carotid endarterectomy (n=31) underwent axial LP-CEUS and diseased intimal segments were symmetrically divided in the long axis. Half-specimens underwent quantitative immunohistochemical analysis for CD68 (macrophages) and CD31 (angiogenesis). Half-specimens were processed for atheroma cell culture and supernatant collected at 24 hours for multianalyte profiling for 34 analytes. Percentage area immunopositivity was significantly higher in subjects in which normalized plaque late-phase intensity was ≥0 versus <0 (CD68 mean 11.8 versus 6.68, P=0.004; CD31 mean 9.45 versus 4.82, P=0.025). Interleukin-6, matrix metalloproteinase-1, and matrix metalloproteinase-3 were significantly higher by multianalyte profiling when LP-CEUS was ≥0. LP-CEUS reflects biological features of inflammation and angiogenesis, key features predisposing to plaque rupture. Further investigation of LP-CEUS as a tissue-specific marker of inflammation for risk stratification of carotid atherosclerosis is warranted.


Structural and Functional Interrogation of Selected Biological Nitrogen Removal Systems in the United States, Denmark, and Singapore Using Shotgun Metagenomics.
Conventional biological nitrogen removal (BNR), comprised of nitrification and denitrification, is traditionally employed in wastewater treatment plants (WWTPs) to prevent eutrophication in receiving water bodies. More recently, the combination of selective ammonia to nitrite oxidation (nitritation) and autotrophic anaerobic ammonia oxidation (anammox), collectively termed deammonification, has also emerged as a possible energy- and cost-effective BNR alternative. Herein, we analyzed microbial diversity and functional potential within 13 BNR processes in the United States, Denmark, and Singapore operated with varying reactor configuration, design, and operational parameters. Using next-generation sequencing and metagenomics, gene-coding regions were aligned against a custom protein database expanded to include all published aerobic ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), anaerobic ammonia oxidizing bacteria (AMX), and complete ammonia oxidizing bacteria (CMX). Overall contributions of these N-cycle bacteria to the total functional potential of each reactor was determined, as well as that of several organisms associated with denitrification and/or structural integrity of microbial aggregates (biofilm or granules). The potential for these engineered processes to foster a broad spectrum of microbial catabolic, anabolic, and carbon assimilation transformations was elucidated. Seeded sidestream DEMON® deammonification systems and single-stage nitritation-anammox moving bed biofilm reactors (MBBRs) and a mainstream Cleargreen reactor designed to enrich in AOB and AMX showed lower enrichment in AMX functionality than an enriched two-stage nitritation-anammox MBBR system treating mainstream wastewater. Conventional BNR systems in Singapore and the United States had distinct metagenomes, especially relating to AOB. A hydrocyclone process designed to recycle biomass granules for mainstream BNR contained almost identical structural and functional characteristics in the overflow, underflow, and inflow of mixed liquor (ALT) rather than the expected selective enrichment of specific nitrifying or AMX organisms. Inoculum used to seed a sidestream deammonification process unexpectedly contained <10% of total coding regions assigned to AMX. These results suggest the operating conditions of engineered bioprocesses shape the resident microbial structure and function far more than the bioprocess configuration itself. We also highlight the advantage of a systems- and metagenomics-based interrogation of both the microbial structure and potential function therein over targeting of individual populations or specific genes.


A microwell array platform for picoliter membrane protein assays.
A novel microwell chip is developed that can be used to detect protein binding in a liquid environment, together with a liquid handling system that allows the performance of assays with picoliter volumes. A PDMS well structure is cast on a planar optical waveguide, providing reaction containers combined with a high-sensitivity fluorescence readout system. Individual wells of the array can be addressed, filled, and rinsed using a contact-mode pin and ring spotter. This allows for immunoassays in a heavily multiplexed way, as all steps of the assay can be individually chosen per well. An array density of over 1000 wells cm(-2) is used for the current experiments. The wells provide a protected liquid environment in which the handling of proteins in their natural state is possible, thus maintaining their activity. The membrane protein annexin V is chosen as a model protein to demonstrate the current possibilities. Annexin V binds to phosphatidylserine (PS) head groups of lipids in a Ca(2+)-dependent manner and is often chosen as a marker for cell apoptosis. Lipid vesicles with and without PS are spotted in individual wells and spontaneously formed a planar lipid bilayer on the bottom of the buffer-filled wells. Annexin V can be used to distinguish between wells containing PS groups previously incorporated in the membrane patches and reference wells without PS head groups. Also, the dependence on the calcium concentration can be shown. Fluorescence readout of the assays is performed using a highly sensitive system based on a planar optical waveguide.


Radical hysterectomy and pelvic lymphadenectomy for stage IB2 cervical cancer.
We wished to evaluate survival and adverse outcomes of patients with stage IB2 cervical cancer treated primarily with radical hysterectomy and lymphadenectomy. A review was performed of all patients undergoing primary radical hysterectomy for stage IB2 cervical cancer at two institutions from 1987 to 2002. Patients were stratified into low, intermediate (Gynecologic Oncology Group protocol 92 criteria), and high-risk (positive nodes, margins, or parametria) groups. Survival and progression-free interval were analyzed using the Kaplan-Meier method and multivariate analysis. Seventy-two patients underwent primary type III radical hysterectomy and lymphadenectomy (72 pelvic, 58 pelvic and paraaortic). Patients were classified as low (n = 6), intermediate (n = 49), or high (n = 17) risk for recurrence. Adjuvant therapy was administered to 94%, 12%, and 0% of the high-, intermediate-, and low-risk groups, respectively. Five-year survival was 72%, while 5-year progression-free survival was 63%. Five-year overall and progression-free survival by risk group were 47% and 40% (high-risk), 80% and 66% (intermediate-risk), 100% and 100% (low-risk). Predictors of survival in multivariate analysis were Caucasian race (P = 0.001), older age (P = 0.017), inner 2/3 cervical wall invasion (P = 0.045), and absence of lymph-vascular invasion (P < 0.001). Major complications were experienced by 10/72 (13.9%) patients. Among 34 patients who received radiation therapy, two (5.9%) experienced complications attributable to radiation. Radical hysterectomy and lymphadenectomy followed by tailored adjuvant therapy is a reasonable alternative to primary radiotherapy for stage IB2 cervical cancer. Patients with low- and intermediate-risk factors have satisfactory results after primary surgical management. A prospective randomized trial will clarify the optimal mode of initial therapy for patients with stage IB2 disease.


Triclosan Impairs Hippocampal Synaptic Plasticity and Spatial Memory in Male Rats.
Triclosan, a widely used industrial and household agent, is present as an antiseptic ingredient in numerous products of everyday use, such as toothpaste, cosmetics, kitchenware, and toys. Previous studies have shown that human brain and animal tissues contain triclosan, which has been found also as a contaminant of water and soil. Triclosan disrupts heart and skeletal muscle Ca2+ signaling, damages liver function, alters gut microbiota, causes colonic inflammation, and promotes apoptosis in cultured neocortical neurons and neural stem cells. Information, however, on the possible effects of triclosan on the function of the hippocampus, a key brain region for spatial learning and memory, is lacking. Here, we report that triclosan addition at low concentrations to hippocampal slices from male rats inhibited long-term potentiation but did not affect basal synaptic transmission or paired-pulse facilitation and modified the content or phosphorylation levels of synaptic plasticity-related proteins. Additionally, incubation of primary hippocampal cultures with triclosan prevented both the dendritic spine remodeling induced by brain-derived neurotrophic factor and the emergence of spontaneous oscillatory Ca2+ signals. Furthermore, intra-hippocampal injection of triclosan significantly disrupted rat navigation in the Oasis maze spatial memory task, an indication that triclosan impairs hippocampus-dependent spatial memory performance. Based on these combined results, we conclude that triclosan exerts highly damaging effects on hippocampal neuronal function in vitro and impairs spatial memory processes in vivo.


[Use of asthma controller drugs at admission to a pediatric pulmonology outpatient clinic].
Inhaled corticosteroids are widely used in developed countries. On the other hand, due to its high cost, this kind of drug is underused in asthmatic patients living in developing countries. The present study aims at verifying the use of controller medication in children with moderate or severe persistent asthma at admission to a specialized outpatient facility. Descriptive study with 560 children aged 4 to 14, with diagnosed asthma, who were admitted between April 1996 and December 2000. The patients were randomly selected. Of 560 patients, 61.8% were male; 69.5% were between 4 and 9 years old. The first acute attack occurred before the first year of life in 55.8% of the cases, and 70.5% had physician-diagnosed persistent moderate asthma. In the previous 12 months, 42.7% of the children had been admitted to the hospital and 92.7% were assisted in emergency rooms. Considering those living in the metropolitan area of Belo Horizonte, the average use of some kind of controller drug was 27.3%, and 17.1% of inhaled corticosteroids. The use of oral corticosteroids decreased from 14.3% in 1996 to 4.2% in 2000. the rate of inhaled corticosteroid therapy was higher than that found in Brazilian studies and it is comparable to the results of some international studies. A decrease in the use of oral corticosteroids was also observed. Both findings may be related to the implementation of an asthma program in the public health system of Belo Horizonte, which began in 1996.


Structure and Management of an Engineering Senior Design Course.
The design of products and processes is an important area in engineering. Students in engineering schools learn fundamental principles in their courses but often lack an opportunity to apply these methods to real-world problems until their senior year. This article describes important elements that should be incorporated into a senior capstone design course. It includes a description of the general principles used in engineering design and a discussion of why students often have difficulty with application and revert to trial and error methods. The structure of a properly designed capstone course is dissected and its individual components are evaluated. Major components include assessing resources, identifying projects, establishing teams, understanding requirements, developing conceptual designs, creating detailed designs, building prototypes, testing performance, and final presentations. In addition to the course design, team management and effective mentoring are critical to success. This article includes suggested guidelines and tips for effective design team leadership, attention to detail, investment of time, and managing project scope. Furthermore, the importance of understanding business culture, displaying professionalism, and considerations of different types of senior projects is discussed. Through a well-designed course and proper mentoring, students will learn to apply their engineering skills and gain basic business knowledge that will prepare them for entry-level positions in industry.


Developing the Capacity for Rapid-Cycle Improvement at a Large Freestanding Children's Hospital.
To develop the capacity for rapid-cycle improvement at the unit level, a large freestanding children's hospital designated 2 inpatient units with normal patient loads and workforce as pilot "Innovation Units" where frontline staff was trained to lead rigorous improvement portfolios. Frontline staff received improvement training, and interdisciplinary teams brainstormed ideas for tests of change. Ideas were prioritized using an impact-effort evaluation and an assessment of how they aligned with high-level goals. A template for each test summarized the following: the opportunity for improvement, the test being conducted, dates for the tests, driver diagrams, metrics to measure effects, baseline data, results, findings, and next steps. Successful interventions were implemented and disseminated to other units. Multidisciplinary staff generated 150 improvement ideas and Innovation Units collectively ran >40 plan-do-study-act cycles. Of the 10 distinct improvement projects, elements of all 10 were deemed "successful" and fully implemented on the unit, and elements from 8 were spread to other units. More than 3 years later, elements of all of the successful improvements are still in practice in some form on the units, and each unit has tested >20 additional improvement ideas, using multiple plan-do-study-act cycles to refine them. The Innovation Unit model successfully engaged frontline staff in improvement work and established a sustainable system and framework for managing rigorous improvement portfolios at the unit level. Other hospitals and health care delivery settings may find our quality improvement approach helpful, especially because it is rooted in the microsystem of care delivery.


High prevalence of significant histology in asymptomatic chronic hepatitis B patients with genotype C and high serum HBV DNA levels.
Current treatment guidelines suggest that antiviral therapy be considered for chronic hepatitis B (CHB) patients with high viral load if a biopsy shows significant liver disease despite alanine aminotransferase (ALT) levels two times or less than the upper limit of normal (ULN). We evaluated the histological findings in CHB patients with high viral load and persistently normal or slightly elevated serum ALT levels. Between January 2003 and June 2006, 105 consecutive treatment-naive patients with CHB who underwent ultrasonography-guided percutaneous liver biopsy, had detectable serum HBV DNA (>10(5) copies/mL) in a direct hybridization assay and normal or slightly elevated serum ALT levels (≤2 × ULN) for at least 12 months were included in a prospective study. Histological assessment was based on the METAVIR scoring system. Significant histology was defined as fibrosis stage ≥F2 or necroinflammation grade ≥A2. Among the 105 CHB patients with high viral load and persistently normal or slightly elevated serum ALT levels for at least 12 months, significant fibrosis (F2-F4 fibrosis) was observed in 63 patients (60.0%) and the actual significant histology was found in 65 patients (61.9%). On multivariate analysis, serum ALT levels and age at which they entered the study were independent factors associated with significant histology. Odds ratios for significant histology increased progressively according to serum ALT levels and age. In conclusion, a large proportion of CHB patients with genotype C, high viral load and ALT ≤2 × ULN had significant liver disease on liver biopsy and should be considered for antiviral therapy.


Minimally Invasive Surgery for Axillary Osmidrosis Using a Combination of Subcutaneous Tissue Removal and a 1,444-nm Nd:YAG Laser.
Many treatment modalities have been developed for axillary osmidrosis. It is well known that the surgical treatment has the best results. However, there is a high possibility of side effects. The 1,444-nm lipolysis laser has been recently introduced to remove the apocrine glands. So far, subdermal coagulation treatment with a 1,444-nm Nd:YAG laser may be the least invasive and most effective therapy for axillary osmidrosis. However, according to our previous experience, the recurrence rate was 20%~30%. This emphasizes the need for combination of surgical method and non-surgical method and we combined subcutaneous tissue removal and photothermocoagulation with a 1,444-nm Nd:YAG laser. Three patients for bilateral axillary osmidrosis were enrolled. After an incision of about one-third the length of the widest transverse diameter, the apocrine glands were separated from the skin. And then apocrine glands within the marked area were destroyed by irradiation with a 1,444-nm Nd:YAG laser thereafter. All patients exhibited no relapse of axillary osmidrosis and were satisfied with the treatment results. A combination of subcutaneous tissue removal and Interstitial laser photothermocoagulation with a 1,444-nm Nd:YAG laser could be an effective treatment for mild to moderate axillary osmidrosis.


Hamilton-Jacobi equation in momentum space.
The application of the Hamilton-Jacobi equation to isotropic optical materials leads to the well-known eikonal equation which provides the surfaces normal to the ray trajectories. The symmetry between the coordinates x=(x(1),x(2),x(3)) and the momenta p=(p(1),p(2),p(3)) in the Hamiltonian formulation of Geometrical Optics establishes a dual Hamilton-Jacobi equation for "wavefronts" in the momentum space. This equation is also an eikonal equation when the refractive index distribution has spherical symmetry. In this case, another spherical symmetric refractive index distribution may exist such that the ray trajectories in the coordinates and momentum space are exchanged (examples of this case are given: Maxwell fish-eye, Eaton lens and Luneburg lens). The relationship between the wavefronts in the coordinate and momentum space is also analyzed. Curved orthogonal coordinates are considered as well.


HelitronScanner uncovers a large overlooked cache of Helitron transposons in many plant genomes.
Transposons make up the bulk of eukaryotic genomes, but are difficult to annotate because they evolve rapidly. Most of the unannotated portion of sequenced genomes is probably made up of various divergent transposons that have yet to be categorized. Helitrons are unusual rolling circle eukaryotic transposons that often capture gene sequences, making them of considerable evolutionary importance. Unlike other DNA transposons, Helitrons do not end in inverted repeats or create target site duplications, so they are particularly challenging to identify. Here we present HelitronScanner, a two-layered local combinational variable (LCV) tool for generalized Helitron identification that represents a major improvement over previous identification programs based on DNA sequence or structure. HelitronScanner identified 64,654 Helitrons from a wide range of plant genomes in a highly automated way. We tested HelitronScanner's predictive ability in maize, a species with highly heterogeneous Helitron elements. LCV scores for the 5' and 3' termini of the predicted Helitrons provide a primary confidence level and element copy number provides a secondary one. Newly identified Helitrons were validated by PCR assays or by in silico comparative analysis of insertion site polymorphism among multiple accessions. Many new Helitrons were identified in model species, such as maize, rice, and Arabidopsis, and in a variety of organisms where Helitrons had not been reported previously to our knowledge, leading to a major upward reassessment of their abundance in plant genomes. HelitronScanner promises to be a valuable tool in future comparative and evolutionary studies of this major transposon superfamily.


Exploratory spatial data analysis for the identification of risk factors to birth defects.
Birth defects, which are the major cause of infant mortality and a leading cause of disability, refer to "Any anomaly, functional or structural, that presents in infancy or later in life and is caused by events preceding birth, whether inherited, or acquired (ICBDMS)". However, the risk factors associated with heredity and/or environment are very difficult to filter out accurately. This study selected an area with the highest ratio of neural-tube birth defect (NTBD) occurrences worldwide to identify the scale of environmental risk factors for birth defects using exploratory spatial data analysis methods. By birth defect registers based on hospital records and investigation in villages, the number of birth defects cases within a four-year period was acquired and classified by organ system. The neural-tube birth defect ratio was calculated according to the number of births planned for each village in the study area, as the family planning policy is strictly adhered to in China. The Bayesian modeling method was used to estimate the ratio in order to remove the dependence of variance caused by different populations in each village. A recently developed statistical spatial method for detecting hotspots, Getis's 7, was used to detect the high-risk regions for neural-tube birth defects in the study area. After the Bayesian modeling method was used to calculate the ratio of neural-tube birth defects occurrences, Getis's statistics method was used in different distance scales. Two typical clustering phenomena were present in the study area. One was related to socioeconomic activities, and the other was related to soil type distributions. The fact that there were two typical hotspot clustering phenomena provides evidence that the risk for neural-tube birth defect exists on two different scales (a socioeconomic scale at 6.84 km and a soil type scale at 22.8 km) for the area studied. Although our study has limited spatial exploratory data for the analysis of the neural-tube birth defect occurrence ratio and for finding clues to risk factors, this result provides effective clues for further physical, chemical and even more molecular laboratory testing according to these two spatial scales.


Body composition and morphological assessment of nutritional status in adults: a review of anthropometric variables.
Evaluation of body composition is an important part of assessing nutritional status and provides prognostically useful data and an opportunity to monitor the effects of nutrition-related disease progression and nutritional intervention. The aim of this narrative review is to critically evaluate body composition methodology in adults, focusing on anthropometric variables. The variables considered include height, weight, body mass index and alternative indices, trunk measurements (waist and hip circumferences and sagittal abdominal diameter) and limb measurements (mid-upper arm and calf circumferences) and skinfold thickness. The importance of adhering to a defined measurement protocol, checking measurement error and the need to interpret measurements using appropriate population-specific cut-off values to identify health risks were highlighted. Selecting the optimum method for assessing body composition using anthropometry depends on the purpose (i.e. evaluating obesity or undernutrition) and requires practitioners to have a good understanding of both practical and theoretical limitations and to be able to interpret the results wisely.


Host choice and penetration by Schistosoma haematobium miracidia.
Schistosome parasites commonly show specificity to their intermediate mollusc hosts and the degree of specificity can vary between parasite strains and geographical location. Here the role of miracidial behaviour in host specificity of Schistosoma haematobium on the islands of Zanzibar is investigated. In choice-chamber experiments, S. haematobium miracidia moved towards Bulinus globosus snail hosts in preference to empty chambers. In addition, miracidia preferred uninfected over patent B. globosus. This preference should benefit the parasite as patent snails are likely to have mounted an immune response to S. haematobium as well as providing poorer resources than uninfected snails. Miracidia also discriminated between the host B. globosus and the sympatric, non-host species Cleopatra ferruginea. In contrast, S. haematobium did not discriminate against the allopatric Bulinus nasutus. Penetration of the host by miracidia was investigated by screening snails 24 h after exposure using polymerase chain reaction (PCR) with S. haematobium specific DraI repeat primers. There was no difference in the frequency of penetration of B. globosus versus B. nasutus. These responses to different snail species may reflect selection pressure to avoid sympatric non-hosts which represent a transmission dead end. The distribution of B. nasutus on Unguja is outside the endemic zone and so there is less chance of exposure to S. haematobium, hence there will be little selection pressure to avoid this non-host snail.


Asymmetric Centromeres Differentially Coordinate with Mitotic Machinery to Ensure Biased Sister Chromatid Segregation in Germline Stem Cells.
Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. How non-genic information could be inherited differentially to establish distinct cell fates is not well understood. Here, we report a series of spatiotemporally regulated asymmetric components, which ensure biased sister chromatid attachment and segregation during ACD of Drosophila male germline stem cells (GSCs). First, sister centromeres are differentially enriched with proteins involved in centromere specification and kinetochore function. Second, temporally asymmetric microtubule activities and polarized nuclear envelope breakdown allow for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. Abolishment of either the asymmetric sister centromeres or the asymmetric microtubule activities results in randomized sister chromatid segregation. Together, these results provide the cellular basis for partitioning epigenetically distinct sister chromatids during stem cell ACDs, which opens new directions to study these mechanisms in other biological contexts.


NPC1 in human white adipose tissue and obesity.
Genetic studies have implicated the NPC1 gene (Niemann Pick type C1) in susceptibility to obesity. To assess the potential function of NPC1 in obesity, we determined its expression in abdominal white adipose tissue (WAT) in relation to obesity. NPC1 mRNA was measured by RT-qPCR in lean and obese individuals, paired samples of subcutaneous (sc) and omental (om) WAT, before and after weight loss, in isolated adipocytes and intact adipose pieces, and in primary adipocyte cultures during adipocyte differentiation. NPC1 protein was examined in isolated adipocytes. NPC1 mRNA was significantly increased in obese individuals in scWAT and omWAT and downregulated by weight loss. NPC1 mRNA was enriched in isolated fat cells of WAT, in scWAT versus omWAT but not modified during adipocyte differentiation. NPC1 protein mirrored expression of mRNA in lean and obese individuals. NPC1 is highly expressed in human WAT adipocytes with increased levels in obese. These results suggest that NPC1 may play a role in adipocyte processes underlying obesity.


Folate deficiency induces dysfunctional long and short telomeres; both states are associated with hypomethylation and DNA damage in human WIL2-NS cells.
The essential role of dietary micronutrients for genome stability is well documented, yet the effect of folate deficiency or excess on telomeres is not known. Accordingly, human WIL2-NS cells were maintained in medium containing 30, 300, or 3,000 nmol/L folic acid (FA) for 42 days to test the hypothesis that chronic folate deficiency would cause telomere shortening and dysfunction. After 14 days, telomere length (TL) in FA-deficient (30 nmol/L) cultures was 26% longer than that of 3,000 nmol/L FA cultures; however, this was followed by rapid telomere attrition over the subsequent 28 days (P trend, P < 0.0001); both long and short telomere status was positively correlated with biomarkers of chromosome instability (P ≤ 0.003) and mitotic dysfunction (P = 0.01), measured by the cytokinesis-block micronucleus cytome (CBMN-cyt) assay. The early increase in TL was associated with FA-deficiency-induced global DNA hypomethylation (P = 0.05), with an effect size similar to that induced by the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. Quantitative PCR analysis indicated a negative association between FA concentration and uracil incorporation into telomeric DNA (r = -0.47, P = 0.1), suggesting a possible plausible mechanism for uracil as a cause of folate deficiency-induced telomere dysfunction or deletion. Peptide nucleic acid-FISH (PNA-FISH) analysis showed that FA deficiency resulted in 60% of micronuclei containing acentric terminal fragments, an observation consistent with the 3-fold increase in terminal deletions (P = 0.0001). Together, these results demonstrate the impact of folate deficiency on biomarkers of telomere maintenance and integrity, and provide evidence that dysfunctional long telomeres may be as important as critically short telomeres as a cause of chromosomal instability.


Reforming Medicare provider payment.
The basic thesis of this paper is that, in the short to medium term (that is to say, the politically relevant term), fee-for-service payment will be the dominant form of Medicare transaction--and that is not such a bad thing. Capitated arrangements will grow, and should be encouraged, especially for long-term-care services, but for reasons to be discussed, it would be a mistake to put too many policy eggs into that basket. As the nascent Prospective Payment System (PPS) for hospital care reveals, fee-for-service payment can take almost an infinite variety of forms, and the connection between the choice of form and the cost impact is highly variable and somewhat uncertain. PPS needs considerable improvement; so, unquestionably, does the current nonsystem of physician payment. An analysis of potential improvements in both arenas will constitute the bulk of this discussion. That emphasis reflects not only the personal predilections of the author, but also the contention that those are the most sensible things to talk about.


Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation.
Three human tumor cell lines of widely differing radiosensitivity were used to examine the characteristics of the 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetradium bromide (MTT) assay and to select suitable conditions for its use in assessing the response of cells to ionizing radiation. The optimal concentration of MTT and the time of incubation of the cells with MTT were individualized for each cell line. The relationship between absorbance and cell number was not linear over the wide range of cell numbers that were used. A calibration curve of absorbance against cell number for each cell line was therefore used. Using the assay to quantify metabolically viable cells, growth curves of irradiated and unirradiated cells were constructed on days 0-14 after irradiation. Accurate surviving fractions could be calculated only when cells were in exponential growth. Using this modification to its interpretation, the MTT assay was able to provide a reproducible measure of survival, which compared well with clonogenic cell survival measurements. However, the necessity to optimize conditions of the MTT assay for each cell line severely limits its usefulness in determining the radiosensitivity of cells in primary human tumor cultures.


Complete genome sequence and biological characterization of Moroccan pepper virus (MPV) and reclassification of Lettuce necrotic stunt virus as MPV.
Moroccan pepper virus (MPV) and Lettuce necrotic stunt virus (LNSV) have been steadily increasing in prevalence in central Asia and western North America, respectively, over the past decade. Recent sequence analysis of LNSV demonstrated a close relationship between the coat proteins of LNSV and MPV. To determine the full extent of the relationship between LNSV and MPV, the genomes of three MPV isolates were sequenced and compared with that of LNSV. Sequence analysis demonstrated that genomic nucleotide sequences as well as virus-encoded proteins of the three MPV isolates and LNSV shared 97% or greater identity. A full-length clone of a California LNSV isolate was developed and virus derived from infectious transcripts was used to evaluate host plant reactions under controlled conditions. Symptoms of LNSV matched those described previously for MPV on most of a select series of host plants, although some differences were observed. Collectively, these molecular and biological results demonstrate that LNSV should be classified as MPV within the family Tombusviridae, genus Tombusvirus, and confirm the presence of MPV in North America.


Zirconocene-catalyzed propene polymerization: a quenched-flow kinetic study.
The kinetics of propene polymerization catalyzed by ansa-metallocenes were studied using quenched-flow techniques. Two catalyst systems were investigated, (SBI)ZrMe2/Al(i)Bu3/[Ph3C][CN[B(C6F5)3]2] (1:100:1) at 25.0 degrees C and (SBI)ZrCl(2)/methylalumoxane at 40.0 degrees C (Al:Zr = 2400:1) (SBI = rac-Me(2)Si(1-Indenyl)2). The aims of the study were to address fundamental mechanistic aspects of metallocene-catalyzed alkene polymerizations, catalyst initiation, the quantitative correlation between catalyst structure and the rate of chain propagation, and the nature of dormant states. One of the most important but largely unknown factors in metallocene catalysis is the distribution of the catalyst between dormant states and species actively involved in polymer chain growth. Measurements of polymer yield Y versus reaction time t for propene concentrations [M] = 0.15-0.59 mol L(-1) and zirconocene concentrations in the range [Zr] = (2.38-9.52) x 10(-5) mol L(-1) for the borate system showed first-order dependence on [M] and [Zr]. Up to t approximately 1 s, the half-life of catalyst initiation is comparable to the half-life of chain growth; that is, this phase is governed by non-steady-state kinetics. We propose a rate law which takes account of this and accurately describes the initial rates. Curve fitting of Y(t) data provides an apparent chain growth rate constant k(p)(app) on the order of 10(3) L mol(-1) s(-1). By contrast, the evolution with time of the number-average polymer molecular weight, which is independent of the concentration of catalyst involved, leads to a k(p) which is an order of magnitude larger, (17.2 +/- 1.4) x 10(3) L mol(-1) s(-1). The ratio k(p)(app)/k(p) = 0.08 indicates that under the given conditions only about 8% of the total catalyst is actively engaged in chain growth at any one time. The system (SBI)ZrCl(2)/methylalumoxane is significantly less active, k(p)(app) = 48.4 +/- 2.7 and k(p) = (6 +/- 2) x 10(2) L mol(-1) s(-1), while, surprisingly, the mole fraction of active species is essentially identical, 8%. Evidently, the energetics of the chain growth sequence are strongly modulated by the nature of the counteranion. Increasing the counteranion/zirconium ratio from 1:1 to 20:1 has no influence on catalyst activity. These findings are consistent with a model of closely associated ion pairs throughout the chain growth sequence. For the borate system, propagation is approximately 6000 times faster than initiation, while for the MAO catalyst, k(p)/k(i) approximately 800. Polymers obtained at 25 degrees C show 0.1-0.2 mol % 2,1-regioerrors, and end-group analysis identifies 2,1-misinsertions as the main cause for chain termination (66%), as compared to 34% for the vinylidene end groups. The results suggest that 2,1-regioerrors are a major contributor to the formation of dormant species, even at short reaction times.


Comprehensive analysis of stop codon usage in bacteria and its correlation with release factor abundance.
We present a comprehensive analysis of stop codon usage in bacteria by analyzing over eight million coding sequences of 4684 bacterial sequences. Using a newly developed program called "stop codon counter," the frequencies of the three classical stop codons TAA, TAG, and TGA were analyzed, and a publicly available stop codon database was built. Our analysis shows that with increasing genomic GC content the frequency of the TAA codon decreases and that of the TGA codon increases in a reciprocal manner. Interestingly, the release factor 1-specific codon TAG maintains a more or less uniform frequency (∼20%) irrespective of the GC content. The low abundance of TAG is also valid with respect to expression level of the genes ending with different stop codons. In contrast, the highly expressed genes predominantly end with TAA, ensuring termination with either of the two release factors. Using three model bacteria with different stop codon usage (Escherichia coli, Mycobacterium smegmatis, and Bacillus subtilis), we show that the frequency of TAG and TGA codons correlates well with the relative steady state amount of mRNA and protein for release factors RF1 and RF2 during exponential growth. Furthermore, using available microarray data for gene expression, we show that in both fast growing and contrasting biofilm formation conditions, the relative level of RF1 is nicely correlated with the expression level of the genes ending with TAG.