Competitive Repression of the artPIQM Operon for Arginine and Ornithine Transport by Arginine Repressor and Leucine-Responsive Regulatory Protein in Escherichia coli.
Two out of the three major uptake systems for arginine in Escherichia coli are encoded by the artJ-artPIQM gene cluster. ArtJ is the high-affinity periplasmic arginine-specific binding protein (ArgBP-I), whereas artI encodes the arginine and ornithine periplasmic binding protein (AO). Both ArtJ and ArtI are supposed to combine with the inner membrane-associated ArtQMP2 transport complex of the ATP-binding cassette-type (ABC). Transcription of artJ is repressed by arginine repressor (ArgR) and the artPIQM operon is regulated by the transcriptional regulators ArgR and Leucine-responsive regulatory protein (Lrp). Whereas repression by ArgR requires arginine as corepressor, repression of P artP by Lrp is partially counteracted by leucine, its major effector molecule. We demonstrate that binding of dimeric Lrp to the artP control region generates four complexes with a distinct migration velocity, and that leucine has an effect on both global binding affinity and cooperativity in the binding. We identify the binding sites for Lrp in the artP control region, reveal interferences in the binding of ArgR and Lrp in vitro and demonstrate that the two transcription factors act as competitive repressors in vivo, each one being a more potent regulator in the absence of the other. This competitive behavior may be explained by the partial steric overlap of their respective binding sites. Furthermore, we demonstrate ArgR binding to an unusual position in the control region of the lrp gene, downstream of the transcription initiation site. From this unusual position for an ArgR-specific operator, ArgR has little direct effect on lrp expression, but interferes with the negative leucine-sensitive autoregulation exerted by Lrp. Direct arginine and ArgR-dependent repression of lrp could be observed with a 25-bp deletion mutant, in which the ArgR binding site was artificially moved to a position immediately downstream of the lrp transcription initiation site. This finding is reminiscent of a previous observation made for the carAB operon encoding carbamoylphosphate synthase, where ArgR bound in overlap with the downstream promoter P2 does not block transcription initiated 67 bp upstream at the P1 promoter, and further supports the hypothesis that ArgR does not act as an efficient roadblock.


Homeoprotein Six2 promotes breast cancer metastasis via transcriptional and epigenetic control of E-cadherin expression.
Misexpression of developmental transcription factors occurs often in human cancers, where embryonic programs may be reinstated in a context that promotes or sustains malignant development. In this study, we report the involvement of the kidney development transcription factor Six2 in the metastatic progression of human breast cancer. We found that Six2 promoted breast cancer metastasis by a novel mechanism involving both transcriptional and epigenetic regulation of E-cadherin. Downregulation of E-cadherin by Six2 was necessary for its ability to increase soft agar growth and in vivo metastasis in an immunocompetent mouse model of breast cancer. Mechanistic investigations showed that Six2 represses E-cadherin expression by upregulating Zeb2, in part, through a microRNA-mediated mechanism and by stimulating promoter methylation of the E-cadherin gene (Cdh1). Clinically, SIX2 expression correlated inversely with CDH1 expression in human breast cancer specimens, corroborating the disease relevance of their interaction. Our findings establish Six2 as a regulator of metastasis in human breast cancers and demonstrate an epigenetic function for SIX family transcription factors in metastatic progression through the regulation of E-cadherin.


MRI and CSF biomarkers in normal, MCI, and AD subjects: diagnostic discrimination and cognitive correlations.
To assess the correlations of both MRI and CSF biomarkers with clinical diagnosis and with cognitive performance in cognitively normal (CN) subjects and patients with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). This is a cross-sectional study with data from the Alzheimer's Disease Neuroimaging Initiative, which consists of CN subjects, subjects with aMCI, and subjects with AD with both CSF and MRI. Baseline CSF (t-tau, Abeta(1-42), and p-tau(181P)) and MRI scans were obtained in 399 subjects (109 CN, 192 aMCI, 98 AD). Structural Abnormality Index (STAND) scores, which reflect the degree of AD-like anatomic features on MRI, were computed for each subject. We found no significant correlation between CSF biomarkers and cognitive scores in any of the 3 clinical groups individually. Conversely, STAND scores correlated with both Clinical Dementia Rating-sum of boxes and Mini-Mental State Examination in aMCI and AD (p < or = 0.01). While STAND and all CSF biomarkers were predictors of clinical group membership (CN, aMCI, or AD) univariately (p < 0.001), STAND was more predictive than CSF both univariately and in combined models. CSF and MRI biomarkers independently contribute to intergroup diagnostic discrimination and the combination of CSF and MRI provides better prediction than either source of data alone. However, MRI provides greater power to effect cross-sectional groupwise discrimination and better correlation with general cognition and functional status cross-sectionally. We therefore conclude that although MRI and CSF provide complementary information, MRI reflects clinically defined disease stage better than the CSF biomarkers tested.


An open-source tool for analysis and automatic identification of dendritic spines using machine learning.
Synaptic plasticity, the cellular basis for learning and memory, is mediated by a complex biochemical network of signaling proteins. These proteins are compartmentalized in dendritic spines, the tiny, bulbous, post-synaptic structures found on neuronal dendrites. The ability to screen a high number of molecular targets for their effect on dendritic spine structural plasticity will require a high-throughput imaging system capable of stimulating and monitoring hundreds of dendritic spines in various conditions. For this purpose, we present a program capable of automatically identifying dendritic spines in live, fluorescent tissue. Our software relies on a machine learning approach to minimize any need for parameter tuning from the user. Custom thresholding and binarization functions serve to "clean" fluorescent images, and a neural network is trained using features based on the relative shape of the spine perimeter and its corresponding dendritic backbone. Our algorithm is rapid, flexible, has over 90% accuracy in spine detection, and bundled with our user-friendly, open-source, MATLAB-based software package for spine analysis.


Corpus based learning of stochastic context-free grammar combined with hidden Markov models for tRNA modelling.
tRNA molecule has a well-known second structure in which it folds by pairing of far-off nucleotides. This paper shows a syntactic pattern recognition methodology for model tRNA second structure using stochastic context-free grammars. In order to learn models, structural regions (paired nucleotides) have been learned from categorized samples with full labelled tree with a Corpus based estimation algorithm. Nonstructural regions have been modelled by hidden Markov models and transformed to stochastic regular grammars to fusion together the structural regions. Test with positive samples and negative samples in comparison with Sakakibara achieved 1.81% in sequences error rate, 98.43% in precision and 100% in recall and 100% of SER in negative test. Corpus based algorithm is computational time efficient and required less training samples for converge to the correct model of the tRNA second structure.


Upper respiratory tract bacterial carriage in Aboriginal and non-Aboriginal children in a semi-arid area of Western Australia.
Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are associated with otitis media (OM). Indigenous children experience particularly high rates of OM. Few studies worldwide have described upper respiratory tract (URT) carriage in Indigenous and non-Indigenous children living in the same area. The aim of this study was to describe URT bacterial carriage in Aboriginal and non-Aboriginal children in the Kalgoorlie-Boulder area, Western Australia, as part of an investigation into causal pathways to OM. Five hundred four and 1045 nasopharyngeal aspirates were collected from 100 Aboriginal and 180 non-Aboriginal children, respectively, followed from birth to age 2 years. Standard procedures were used to identify bacteria. Overall carriage rates of S. pneumoniae, M. catarrhalis and H. influenzae in Aboriginal children were 49%, 50% and 41%, respectively, and 25%, 25% and 11% in non-Aboriginal children. By age 2 months S. pneumoniae and M. catarrhalis had been isolated from 37% and 36% of Aboriginal children and from 11% and 12% of non-Aboriginal children, respectively. From age 3 months onward, carriage rates in Aboriginal children were 51% to 67% for S. pneumoniae and M. catarrhalis and 42% to 62% for H. influenzae; corresponding figures for non-Aboriginal children were 26% to 37% for S. pneumoniae and M. catarrhalis and 11% to 18% for H. influenzae. Non-Aboriginal children had higher carriage rates in winter than in summer, but season had little effect in Aboriginal children. Staphylococcus aureus carriage was highest under age 1 month (55% Aboriginal, 61% non-Aboriginal) and was always higher in non-Aboriginal than Aboriginal children. Interventions are needed to reduce high transmission and carriage rates, particularly in Aboriginal communities, to avoid the serious consequences of OM.


A prospective study of central venous catheters placed in a tertiary care Emergency Department: indications for use, infectious complications, and natural history.
Despite successful efforts to improve overall central line-associated bloodstream infections (CLABSI) rates, little is known about CLABSI rates or even central venous catheter insertion practices in the Emergency Department. We sought to determine the baseline CLABSI rate for Emergency Department-inserted central venous catheters and to describe indications for placement, duration of use, and the natural history of these devices.


Implications of Zoonoses From Hunting and Use of Wildlife in North American Arctic and Boreal Biomes: Pandemic Potential, Monitoring, and Mitigation.
The COVID-19 pandemic has re-focused attention on mechanisms that lead to zoonotic disease spillover and spread. Commercial wildlife trade, and associated markets, are recognized mechanisms for zoonotic disease emergence, resulting in a growing global conversation around reducing human disease risks from spillover associated with hunting, trade, and consumption of wild animals. These discussions are especially relevant to people who rely on harvesting wildlife to meet nutritional, and cultural needs, including those in Arctic and boreal regions. Global policies around wildlife use and trade can impact food sovereignty and security, especially of Indigenous Peoples. We reviewed known zoonotic pathogens and current risks of transmission from wildlife (including fish) to humans in North American Arctic and boreal biomes, and evaluated the epidemic and pandemic potential of these zoonoses. We discuss future concerns, and consider monitoring and mitigation measures in these changing socio-ecological systems. While multiple zoonotic pathogens circulate in these systems, risks to humans are mostly limited to individual illness or local community outbreaks. These regions are relatively remote, subject to very cold temperatures, have relatively low wildlife, domestic animal, and pathogen diversity, and in many cases low density, including of humans. Hence, favorable conditions for emergence of novel diseases or major amplification of a spillover event are currently not present. The greatest risk to northern communities from pathogens of pandemic potential is via introduction with humans visiting from other areas. However, Arctic and boreal ecosystems are undergoing rapid changes through climate warming, habitat encroachment, and development; all of which can change host and pathogen relationships, thereby affecting the probability of the emergence of new (and re-emergence of old) zoonoses. Indigenous leadership and engagement in disease monitoring, prevention and response, is vital from the outset, and would increase the success of such efforts, as well as ensure the protection of Indigenous rights as outlined in the United Nations Declaration on the Rights of Indigenous Peoples. Partnering with northern communities and including Indigenous Knowledge Systems would improve the timeliness, and likelihood, of detecting emerging zoonotic risks, and contextualize risk assessments to the unique human-wildlife relationships present in northern biomes.


Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.


The combined effect on survival of four main behavioural risk factors for non-communicable diseases.
To quantify and illustrate the combined effects of WHO's four behavioural risk factors for non-communicable diseases (NCDs) on mortality. Participants (n=16,721) were part of two Swiss population studies conducted between 1977 and 1993. Smoking status, alcohol consumption, physical activity and diet were assessed at baseline. With record linkage in 2008, up to 31years of follow-up with 3,533 deaths could be recorded. Mortality was assessed with Cox proportional hazard models for each risk factor and their combinations. Ten-year survival probabilities for 65- and 75-year-olds were estimated with Weibull regression models. Hazard ratios for the combination of all four risk factors compared to none were 2.41 (1.99-2.93) in men and 2.46 (1.88-3.22) in women. For 65-year-olds, the probability of surviving the next 10years was 86% for men with no risk factors and 67% for men with four. In women, the respective numbers were 90% and 77%. In 75-year-olds, probabilities were 67% and 35% in men, and 74% and 47% in women. The combined impact of four behavioural NCD risk factors on survival probability was comparable in size to a 10-year age difference and bigger than the gender effect.


Maternal personality profile of children affected by migraine.
Empirical evidence of the important role of the family in primary pediatric headache has grown significantly in the last few years, although the interconnections between the dysfunctional process and the family interaction are still unclear. Even though the role of parenting in childhood migraine is well known, no studies about the personality of parents of migraine children have been conducted. The aim of the present study was to assess, using an objective measure, the personality profile of mothers of children affected by migraine without aura (MoA). A total of 269 mothers of MoA children (153 male, 116 female, aged between 6 and 12 years; mean 8.93 ± 3.57 years) were compared with the findings obtained from a sample of mothers of 587 healthy children (316 male, 271 female, mean age 8.74 ± 3.57 years) randomly selected from schools in the Campania, Umbria, Calabria, and Sicily regions. Each mother filled out the Minnesota Multiphasic Personality Inventory - second edition (MMPI-2), widely used to diagnose personality and psychological disorders. The t-test was used to compare age and MMPI-2 clinical basic and content scales between mothers of MoA and typical developing children, and Pearson's correlation test was used to evaluate the relation between MMPI-2 scores of mothers of MoA children and frequency, intensity, and duration of migraine attacks of their children. Mothers of MoA children showed significantly higher scores in the paranoia and social introversion clinical basic subscales, and in the anxiety, obsessiveness, depression, health concerns, bizarre mentation, cynicism, type A, low self-esteem, work interference, and negative treatment indicator clinical content subscales (P < 0.001 for all variables). Moreover, Pearson's correlation analysis showed a significant relationship between MoA frequency of children and anxiety (r = 0.4903, P = 0.024) and low self-esteem (r = 0.5130, P = 0.017), while the MoA duration of children was related with hypochondriasis (r = 0.6155, P = 0.003), hysteria (r = 0.6235, P = 0.003), paranoia (r = 0.5102, P = 0.018), psychasthenia (r = 0.4806, P = 0.027), schizophrenia (r = 0.4350, P = 0.049), anxiety (r = 0.4332, P = 0.050), and health concerns (r = 0.7039, P < 0.001) MMPI-2 scores of their mothers. This could be considered a preliminary study that indicates the potential value of maternal personality assessment for better comprehension and clinical management of children affected by migraine, though further studies on the other primary headaches are necessary.


Immunological properties of melanoma tumor-infiltrating lymphocytes before and after IL-2-based biotherapies.
Immunological properties of melanoma TILs before and/or after IL-2-based biotherapies were investigated. TILs harvested before therapies, including those for adoptive transfer, proliferated well in culture with IL-2 and displayed cytotoxicity relatively restricted to autologous tumor cells. In contrast, TILs during or at the end of IL-2 based therapies did not proliferate in culture with IL-2. TILs from tumors even harvested 45 days after the end of IL-2 therapy modestly proliferated in culture with IL-2 and showed MHC-nonrestricted cytotoxicity. The number of live tumor cells that were yielded from melanomas during or at the end of IL-2-based therapies significantly decreased in all nine patients with metastatic melanomas, regardless of their clinical responses (2 PR, 2 MR, 2 SD, and 3 PD). Collectively, these results suggest that current IL-2-based therapies resulted in both transient nonresponsiveness of TILs to IL-2 and transient decrease in the number of live tumour cells in most melanoma patients.


In-hospital outcomes of inflammatory bowel disease in cannabis users: a nationwide propensity-matched analysis in the United States.
Literature suggests the role of cannabis (marijuana) as an anti-inflammatory agent. However, the impact of recreational marijuana usage on in-hospital outcomes of inflammatory bowel disease (IBD) remains indistinct. We assessed the outcomes of Crohn's disease (CD) as well as ulcerative colitis (UC) with vs. without recreational marijuana usage using a nationally illustrative propensity-matched sample. The Nationwide Inpatient Sample datasets (2010-2014) were queried to identify adults with CD and UC hospitalizations with cannabis use and linked complications using ICD-9 CM codes. Categorical and continuous variables were compared between propensity-matched cohorts using Chi-square and Student's t-test, respectively. Primary endpoints were in-hospital complications, whereas secondary endpoints were the discharge disposition, mean length of stay (LOS) and hospital charges. Propensity-matched cohorts included 6,002 CD (2,999 cannabis users & 3,003 non-users) and 1,481 UC (742 cannabis users & 739 non-users) hospitalizations. In CD patients, prevalence of colorectal cancer (0.3% vs. 1.2%, P<0.001), need for parenteral nutrition (3.0% vs. 4.7%, P=0.001) and anemia (25.6% vs. 30.1%, P<0.001) were lower in cannabis users. However, active fistulizing disease or intraabdominal abscess formation (8.6% vs. 5.9%, P<0.001), unspecific lower gastrointestinal (GI) hemorrhage (4.0% vs. 2.7%, P=0.004) and hypovolemia (1.2% vs. 0.5%, P=0.004) were higher with recreational cannabis use. The mean hospital stay was shorter (4.2 vs. 5.0 days) with less hospital charges ($28,956 vs. $35,180, P<0.001) in cannabis users. In patients with UC, cannabis users faced the higher frequency of fluid and electrolyte disorders (45.1% vs. 29.6%, P<0.001), and hypovolemia (2.7% vs. <11) with relatively lower frequency of postoperative infections (<11 vs. 3.4%, P=0.010). No other complications were significant enough for comparison between the cannabis users and non-users in this group. Like CD, UC-cannabis patients had shorter mean hospital stay (LOS) (4.3 vs. 5.7 days, P<0.001) and faced less financial burden ($30,393 vs. $41,308, P<0.001). We found a lower frequency of colorectal cancer, parenteral nutrition, anemia but a higher occurrences of active fistulizing disease or intraabdominal abscess formation, lower GI hemorrhage and hypovolemia in the CD cohort with cannabis usage. In patients with UC, frequency of complications could not be compared between the two cohorts, except a higher frequency of fluid and electrolyte disorders and hypovolemia, and a lower frequency of postoperative infections with cannabis use. A shorter LOS and lesser hospital charges were observed in both groups with recreational marijuana usage.


Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map-based cloning of AvrLm6.
Map-based cloning of avirulence genes of the AvrLml-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLml, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5' and 3' sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.


Ontogeny can provide insight into the roles of natural and sexual selection in cricket cuticular hydrocarbon evolution.
The often complex cocktails of hydrocarbon compounds found on the cuticles of insects can serve both naturally and sexually selected functions, contributing to an individual's ability to withstand water loss and attract mating partners. However, whether natural and sexual selection act synergistically or antagonistically on a species' cuticular hydrocarbon (CHC) profile remains unclear. Here, we examined the ontogeny of the CHC profile in a species of cricket, Teleogryllus oceanicus, while manipulating humidity during development. We predicted that juvenile crickets should produce only those compounds that contribute to desiccation resistance, while those compounds contributing specifically to male attractiveness should be produced only at sexual maturity. Further, if attractive CHCs come at a cost to desiccation resistance as predicted by some models of sexual selection, then males reared under low humidity should be constrained to invest less in attractive CHCs. Crickets reared under low humidity produced more long-chain methyl-branched alkanes, alkenes and alkadienes than did crickets reared under high humidity. The abundance of n-alkanes was unaffected by humidity treatment. Sexual dimorphism in the CHC profile was not apparent until adult emergence and became exaggerated 10 days after emergence, when crickets were sexually mature. Males produced more of the same compounds that were increased in both sexes under low humidity, but the humidity treatment did not interact with sex in determining CHC abundance. The data suggest that CHC profiles which protect crickets from desiccation might have synergistic effects on male attractiveness, as there was no evidence to suggest males trade-off a CHC profile produced in response to low humidity for one associated with sexual signalling.


Neuropeptide Y-stimulated [(35) S]GTPγs functional binding is reduced in the hippocampus after kainate-induced seizures in mice.
Kainate-induced seizures constitute a model of temporal lobe epilepsy where prominent changes are observed in the hippocampal neuropeptide Y (NPY) system. However, little is known about the functional state and signal transduction of the NPY receptor population resulting from kainate exposure. Thus, in this study, we explored functional NPY receptor activity in the mouse hippocampus and neocortex after kainate-induced seizures using NPY-stimulated [(35) S]GTPγS binding. Moreover, we also studied levels of [(125) I]-peptide YY (PYY) binding and NPY, Y1, Y2, and Y5 receptor mRNA in these kainate-treated mice. Functional NPY binding was unchanged up to 12 h post-kainate, but decreased significantly in all hippocampal regions after 24 h and 1 week. Similarly, a decrease in [(125) I]-PYY binding was found in the dentate gyrus (DG) 1 week post-kainate. However, at 2 h, 6 h, and 12 h, [(125) I]-PYY binding was increased in all regions, and in the CA1 also at 24 h post-kainate. NPY mRNA levels were prominently increased in hippocampal regions, reaching maximum at 12 and 24 h. Y1 and Y5 mRNA levels were lowered in the DG at 24 and 2 h, respectively, while Y2 mRNA levels were elevated at 24 h in the DG and CA3. This study confirms rat kainate studies by showing pronounced adaptive changes in the mouse hippocampus both with regard to NPY synthesis and NPY receptor synthesis and binding, which may contribute to regulating neuronal seizure susceptibility after kainate. However, the potential seizure-suppressant effects of increased NPY gene expression at late time points post-kainate could be attenuated by the novel finding of reduced NPY-receptor G-protein activation.


Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli.
Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.


Intraspecific variations of phosphorus absorption and remobilization, P forms, and their internal buffering in Brassica cultivars exposed to a P-stressed environment.
Translocation of absorbed phosphorus (P) from metabolically inactive sites to active sites in plants growing under P deprivation may increase its P utilization efficiency (PUE). Acclimation to phosphate (Pi) starvation may be caused by a differential storage pool of vacuolar P, its release, and the intensity of re-translocation of absorbed P as P starvation inducible environmental cues (PSIEC) from ambient environment. Biomass assay and three P forms, namely inorganic (Pi), organic (Po), and acid-soluble total (Ptas) were estimated in Brassica cultivars exposed to 10 d P deprivation in the culture media. Considering that -delta Pi/delta t denotes the rate of Pi release, Pi release velocity (RSPi) was determined as the tangent to the equations obtained for Pi f(t) at the mean point in the period of greatest Pi decrease, whereas the inverse of the RSPi was an estimate of the internal Pi buffering capacity (IBCPi). Inter cultivar variations in size of the non-metabolic Pi pool, RSPi, re-translocation of Pi from less to more active metabolic sites, and preferential Pi source and sink compartments were evaluated under P starvation. The cultivar 'Brown Raya' showed the highest Pi storage ability under adequate external P supply, and a more intensive release than 'Rain Bow' and 'Dunkled' under P stress. Cultivar 'B.S.A' was inferior to 'Con-1' in its ability to store and use Pi. Roots and upper leaves were the main sink of Pi stored in the lower and middle leaves of all cultivars and showed lower IBCPi and larger RSPi values than lower and middle leaves. In another trial, six cultivars were exposed to P-free nutrition for 29 d after initial feeding on optimum nutrition for 15 d. With variable magnitude, all of the cultivars re-translocated P from the above ground parts to their roots under P starvation, and [P] at 44 d after transplanting was higher in developing leaves compared with developed leaves. Under P deprivation, translocation of absorbed P from metabolically inactive to active sites may have helped the tolerant cultivars to establish a better rooting system, which provided a basis for tolerance against P starvation and increased PUE. A better understanding of the extent to which changes in the flux of P absorption and re-translocation under PSIEC will help to scavenge Pi from bound P reserves and will bring more sparingly soluble P into cropping systems and obtain capitalization of P reserves.


COBRAme: A computational framework for genome-scale models of metabolism and gene expression.
Genome-scale models of metabolism and macromolecular expression (ME-models) explicitly compute the optimal proteome composition of a growing cell. ME-models expand upon the well-established genome-scale models of metabolism (M-models), and they enable a new fundamental understanding of cellular growth. ME-models have increased predictive capabilities and accuracy due to their inclusion of the biosynthetic costs for the machinery of life, but they come with a significant increase in model size and complexity. This challenge results in models which are both difficult to compute and challenging to understand conceptually. As a result, ME-models exist for only two organisms (Escherichia coli and Thermotoga maritima) and are still used by relatively few researchers. To address these challenges, we have developed a new software framework called COBRAme for building and simulating ME-models. It is coded in Python and built on COBRApy, a popular platform for using M-models. COBRAme streamlines computation and analysis of ME-models. It provides tools to simplify constructing and editing ME-models to enable ME-model reconstructions for new organisms. We used COBRAme to reconstruct a condensed E. coli ME-model called iJL1678b-ME. This reformulated model gives functionally identical solutions to previous E. coli ME-models while using 1/6 the number of free variables and solving in less than 10 minutes, a marked improvement over the 6 hour solve time of previous ME-model formulations. Errors in previous ME-models were also corrected leading to 52 additional genes that must be expressed in iJL1678b-ME to grow aerobically in glucose minimal in silico media. This manuscript outlines the architecture of COBRAme and demonstrates how ME-models can be created, modified, and shared most efficiently using the new software framework.


Impacts of stocking density on development and puberty attainment of replacement beef heifers.
In all, 60 Angus×Hereford heifers were ranked by age and BW (210±2 days and 220±2 kg) on day 0, and assigned to: (a) one of three drylot pens (10×14 m pens; 10 heifers/pen) resulting in a stocking density of 14 m2/heifer (HIDENS; n=3), or (b) one of three pastures (25 ha pastures; 10 heifers/pasture), resulting in a stocking density of 25 000 m2/heifer (LOWDENS; n=3). Pastures were harvested for hay before the beginning of this experiment, and negligible forage was available for grazing to LOWDENS heifers during the experiment (days 0 to 182). All heifers received the same limited-fed diet, which averaged (dry matter basis) 4.0 kg/heifer daily of hay and 3.0 kg/heifer daily of a corn-based concentrate. Heifer shrunk BW was recorded after 16 h of feed and water withdrawal on days -3 and 183 for BW gain calculation. On day 0, heifers were fitted with a pedometer behind their right shoulder. Each week, pedometer results were recorded and blood samples were collected for puberty evaluation via plasma progesterone. Plasma samples collected on days 0, 28, 56, 84, 112, 140, 161 and 182 were also analyzed for cortisol concentrations. On days 0, 49, 98, 147 and 182, hair samples were collected from the tail switch for analysis of hair cortisol concentrations. On days 28, 102 and 175, blood samples were collected for whole blood RNA isolation and analysis of heat shock protein (HSP) 70 and HSP72 mRNA expression. Heifers from LOWDENS had more (P<0.01) steps/week compared with HIDENS. No treatment effects were detected (P=0.82) for heifer BW gain. Plasma cortisol concentrations were greater (P⩽0.05) in LOWDENS compared with HIDENS heifers on days 84, 140, 161 and 182 (treatment×day interaction; P<0.01). Hair cortisol concentrations were greater (P<0.01) in HIDENS compared with LOWDENS heifers beginning on day 98 (treatment×day interaction; P<0.01). Heifers from LOWDENS had greater (P=0.04) mean mRNA expression of HSP72, and tended (P=0.09) to have greater mean mRNA expression of HSP70 compared with HIDENS. Heifers from HIDENS experienced delayed puberty attainment and had less (P<0.01) proportion of pubertal heifers on day 182 compared with LOWDENS (treatment×day interaction; P<0.01). In summary, HIDENS altered heifer stress-related and physiological responses, and delayed puberty attainment compared with LOWDENS.


Biosynthesis and possible functions of inositol pyrophosphates in plants.
Inositol phosphates (InsPs) are intricately tied to lipid signaling, as at least one portion of the inositol phosphate signaling pool is derived from hydrolysis of the lipid precursor, phosphatidyl inositol (4,5) bisphosphate. The focus of this review is on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e., PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals (referred to as phytic acid). Even though plants produce huge amounts of precursor InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. Recent work has delineated that Arabidopsis has two genes capable of PP-InsP5 synthesis, and PPx-InsPs have been detected across the plant kingdom. This review will detail the known roles of PPx-InsPs in yeast and animal systems, and provide a description of recent data on the synthesis and accumulation of these novel molecules in plants, and potential roles in signaling.


The effect of power in sexual relationships on sexual and reproductive health: an examination of the evidence.
This article reviews what has been learned to date about the role of gender-based power in sexual relationships in determining sexual and reproductive health outcomes. A framework for assessing the relationship between power relations and reproductive health is outlined and measurement issues are critically discussed. A summary is included of the main types of intervention approaches that have been implemented, as are a discussion of the programmatic, methodological, and ethical implications of the findings and recommendations for further experimentation and research. Although many challenges remain, results to date suggest that when the role of gender-based power is made an integral feature of sexual and reproductive health programs, there is a considerable payoff for both women and men.


Barriers to the Accessibility and Continuity of Health-Care Services in People with Multiple Sclerosis: A Literature Review.
Individuals with multiple sclerosis (MS) face a range of barriers to accessing and using health-care services. The aim of this review was to identify specific barriers to accessing and using health-care services based on a continuum of the health-care delivery system. Literature searches were conducted in the PubMed, PsycINFO, CINAHL, and Web of Science databases. The following terms were searched as subject headings, key words, or abstracts: health care, access, barriers, physical disability, and multiple sclerosis. The literature search produced 361 potentially relevant citations. After screening titles, abstracts, and citations, eight citations were selected for full-text review. Health-care barriers were divided into three continuous phases of receiving health care. In the before-visit phase, the most commonly identified barrier was transportation. In the during-visit phase, communication quality was the major concern. In the after-visit phase, discontinued referral was the major barrier encountered. There are multiple interrelated barriers to accessing and using health-care services along the health-care delivery continuum for people with MS and its associated physical disabilities, ranging from complex and long-recognized barriers that will likely require extended advocacy to create policy changes to issues that can and should be addressed through relatively minor changes in health-care delivery practices, improved care coordination, and increased provider awareness, education, and responsiveness to patients' needs.


Phytoniosome: a Novel Drug Delivery for Myrtle Extract.
Traditionally, Myrtus communis (myrtle) has been used for treatment of several kinds of disorders. However, there are some factors, namely, low solubility and permeability, which restrict use of myrtle extract (ME) in medical applications. Regarding these limitations, the aim of the present study was to develop a new niosomal formulation to enhance ME stability and permeability. Briefly, several niosomal formulations were prepared by non-ionic surfactants and cholesterol with different molar ratios. Afterward, size, entrapment efficiency (EE%), release and stability of niosomal myrtle extract (nME) were investigated. The effect of ME and nME on viability of 3T3 cells was evaluated using MTT assay. Antibacterial activity of ME and nME was also assessed against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Micrococcus luteus, and Bacillus subtilis. Sizes of niosomes were 5.3 ± 0.3 to 15.9 ± 2.2 µm with 4.1 ± 0.3 to 26.9 ± 1.7 mV zeta potential. The EE% of niosomes was varied from 45.4% to 93.4%. An in-vitro release study on F5 formulation (Span60: Tween60: cholesterol (3:3:4 molar ratio)) revealed that about 36.9%, 38.5% and 26.7% of phytoconstituents were released within 12 h from acetate cellulose membrane, 0.45 µm, regenerated cellulose membrane, 0.45 µm, and cellophane dialysis sack, 12000 Da, respectively. F5 formulation significantly showed lower toxicity on cells. It had higher antibacterial activity that has been shown by lower MICs and higher zone of inhibition compared to ME. Overall, F5 formulation in the presence of 4% ME produced stable multi lamellar vesicles with optimal in-vitro release and EE%. This formulation also exhibited better antibacterial activity than ME.


[Vitamin A, E and C serum concentration in patients with Borrelia burgdorferi antibodies--non-symptomatic carriers].
To estimate vitamin A, E and C serum concentrations among forestry workers showing antibodies against Borrelia burgdorferi presence. Vitamins A, E and C concentrations were evaluated in 117 sera of forestry workers. 78 persons aged 18-63 (x=43.07) showed antibodies against Borrelia burgdorferi presence. In this group 13 persons showed presence of IgM, 42 persons with IgG and 23 with IgM and IgG. Control group consisted of 39 persons aged 18-56 years (x=40,97), with no detectable anti-Borrelia burgdorferi antibodies in serum. Serologic diagnosis was performed with use of ELISA kit - Borrelia recombinant IgM, IgG (Biomedica, Austria). Vitamins A and E serum concentrations were detected by RP-HPLC method with spectrophotometric detection (De Leenheet and co.). Vitamin C was detected by RP-HPLS method with spectrophotometric method (Ivanovic and co). Obtains results were statistically analysed. Significantly lower of vitamin A and E serum concentration of persons with anti-borrelia antibodies presence. The lowest concentration was observed in group showing presence of IgM and IgG. No significant difference in vitamin C serum concentration in examined groups was observed. These results may suggest that low serum concentrations of vitamin A and E may have influence on Borrelia burgdorferi infection development.


Telomere directed fragmentation of mammalian chromosomes.
Cloned human telomeric DNA can integrate into mammalian chromosomes and seed the formation of new telomeres. This process occurs efficiently in three established human cell lines and in a mouse embryonic stem cell line. The newly seeded telomeres appear to be healed by telomerase. The seeding of new telomeres by cloned telomeric DNA is either undetectable or very inefficient in non-tumourigenic mouse or human somatic cell lines. The cytogenetic consequences of the seeding of new telomeres include large chromosome truncations but most of the telomere seeding events occur close to the pre-existing ends of natural chromosomes.


Ten-year experience with surgical repair of mycotic aortic aneurysms.
Mycotic aneurysm remains a lethal pathologic entity, especially when rupture occurs. It may result from primary aortitis, be induced by septic emboli, or be secondary to an adjacent infection, such as pancreatitis or a psoas muscle abscess. Surgical intervention is the only way to treat such disease. Even when successful repair is achieved by insertion of an interposition in situ graft or by performance of an extra-anatomic bypass, the prognosis is poor. The aim of this study was to present our experience of managing mycotic aortic aneurysms during the past 10 years. From January 1994 to June 2004, a total of 734 patients with aortic aneurysms underwent surgical repair at our institution. Among these cases, 17 (2.3%) were shown to be mycotic aneurysms of the ascending aorta (n = 1), aortic arch (2), thoracic and thoracoabdominal aorta (3), or abdominal aorta (11); 14 patients (mean age, 58.8 years) were male. Preoperative imaging studies were performed in all patients. Mycotic aortic aneurysms were suspected in 12 of the 17 patients (70.6%) preoperatively, and 4 of these 12 patients were found to have ruptures on imaging. At the time of surgery, 9 of the 17 aneurysms (52.9%) were ruptured. Fifteen patients had an interposition graft inserted after meticulous debridement, 1 underwent an aorto-aortic bypass, and 1 underwent an extra-anatomic (axillo-femoral) bypass. An omentum patch was applied to wrap the graft in 8 of 11 mycotic aortic aneurysms of the abdominal aorta. The most common pathogens were Salmonella spp. (n = 7) and Staphylococcus spp. (4). All patients received antibiotic therapy, according to the culture report, for about 4-6 weeks postoperatively. In-hospital mortality was 11.8% (n = 2). Another patient died from massive upper gastrointestinal bleeding 6 months after operation because of complications involving an aorto-duodenal fistula, and another died from stomach cancer 6 years after surgery. Long-term follow-up (mean, 37 months; range, 3-111 months) revealed that, at the time of writing, the remaining 13 patients were alive and well, without any recurrence of aneurysm. Mycotic aneurysm of the aorta is a life-threatening disease, especially when rupture occurs. The high mortality rate is due not only to the high rupture rate, but also to sepsis. When mycotic aortic aneurysm is diagnosed, early surgical intervention is mandatory.


In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status.
Treatment of advanced metastatic colorectal cancer (mCRC) patients is associated with a poor prognosis and significant morbidity. Moreover, targeted therapies such as anti-epidermal growth factor receptor (EGFR) have no effect in metastatic patients with tumors harboring a mutation in the RAS gene. The failure of conventional treatment to improve outcomes in mCRC patients has prompted the development of adoptive immunotherapy approaches including natural killer (NK)-based therapies. In this study, after confirmation that patients' NK cells were not impaired in their cytotoxic activity, evaluated against long-term tumor cell lines, we evaluated their interactions with autologous mCRC cells. Molecular and phenotypical evaluation of mCRC cells, expanded in vitro from liver metastasis, showed that they expressed high levels of polio virus receptor and Nectin-2, whereas UL16-binding proteins were less expressed in all tumor samples evaluated. Two different patterns of MICA/B and HLA class I expression on the membrane of mCRC were documented; approximately half of mCRC patients expressed high levels of these molecules on the membrane surface, whereas, in the remaining, very low levels were documented. Resting NK cells were unable to display sizeable levels of cytotoxic activity against mCRC cells, whereas their cytotoxic activity was enhanced after overnight or 5-day incubation with IL-2 or IL-15. The susceptibility of NK-mediated mCRC lysis was further significantly enhanced after coating with cetuximab, irrespective of their RAS mutation and HLA class I expression. These data open perspectives for combined NK-based immunotherapy with anti-epidermal growth factor receptor antibodies in a cohort of mCRC patients with a poor prognosis refractory to conventional therapies.


Distribution of human papillomavirus types 16 and 18 variants in squamous cell carcinomas and adenocarcinomas of the cervix.
The distributions of human papillomavirus (HPV) types detected in cervical adenocarcinomas and squamous cell tumors differ. However, whether the distributions of intratypic HPV variants seen in these two histological forms of cervical disease differ is unknown. Our objective was to compare the distribution of HPV intratypic variants observed in squamous cell carcinomas (SCC) and cervical tumors of glandular origin (e.g., adenocarcinomas; AC) for two HPV types commonly observed in cervical tumors, HPV16 and HPV18. Participants in a multicenter case-control study of AC and SCC conducted in the eastern United States were studied. A total of 85 HPV16 and/or HPV18 positive individuals (31 diagnosed with AC, 43 diagnosed with SCC, and 11 population controls) were included. For HPV16-positive individuals, both the noncoding long control region and the E6 open reading frame were sequenced, and classified into phylogenetic-based lineage groups (European, Asian-American, African1, and African2). For HPV18-positive individuals, the long control region region only was sequenced and classified into known intratypic lineages (European, Asian-Amerindian, and African). The distribution of these different intratypic lineages among AC cases, SCC cases, and population controls was compared using standard methods. Non-European HPV16 and/or HPV18 intratypic variants were observed in 42% of ACs compared with 16% of SCCs and 18% of population controls (P = 0.04). Intratypic variants from the Asian-American lineage of HPV16 accounted for the differences seen between histological groups. The differences observed between AC and SCC cases were strongest for HPV16, and persisted in analysis restricted to Caucasian women, suggesting that the effect cannot be explained by differences in the ethnic make-up of AC versus SCC cases. Cervical AC and SCC differ not only with respect to the distribution of HPV types detected but also with respect to intratypic variants observed. Non-European HPV16 and/or HPV18 variants are commonly seen in AC. A possible hormonal mechanism is suggested to explain the observed findings.


Proteo-Transcriptomic Characterization of Sirex nitobei (Hymenoptera: Siricidae) Venom.
The wood-boring woodwasp Sirex nitobei is a native pest in Asia, infecting and weakening the host trees in numerous ecological and commercial coniferous forest plantations. In China, hosts of S. nitobei are diverse, so the pest has spread to several provinces of China, resulting in considerable economic and ecological damage. During female oviposition, S. nitobei venom along with arthrospores of the symbiotic fungus Amylostereum areolatum or A. chaetica is injected into host trees, and the combination of these two biological factors causes the death of xylem host trees. The presence of venom alone causes only the yellowing and wilting of needles. In this study, we constructed the venom gland transcriptome of S. nitobei for the first time and a total of 15,036 unigenes were acquired. From the unigenes, 11,560 ORFs were identified and 537 encoding protein sequences with signal peptides at the N-terminus. Then, we used the venomics approach to characterize the venom composition of female S. nitobei and predicted 1095 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We focused on seven proteins that were both highly expressed in the venom gland transcriptome and predicted in the crude venom proteome. These seven proteins are laccase-2, laccase-3, a protein belonging to the Kazal family, chitooligosaccharidolytic β-N-acetylglucosaminidase, beta-galactosidase, icarapin-like protein, and waprin-Thr1-like protein. Using quantitative real-time PCR (qRT-PCR), we also proved that the genes related to these seven proteins are specifically expressed in the venom glands. Finally, we revealed the functional role of S. nitobei venom in the physiological response of host trees. It can not only promote the colonization of symbiotic fungus but contribute to the development of eggs and larvae. This study provides a deeper understanding of the molecular mechanism of the woodwasp-pine interaction.


Emergence of switch-like behavior in a large family of simple biochemical networks.
Bistability plays a central role in the gene regulatory networks (GRNs) controlling many essential biological functions, including cellular differentiation and cell cycle control. However, establishing the network topologies that can exhibit bistability remains a challenge, in part due to the exceedingly large variety of GRNs that exist for even a small number of components. We begin to address this problem by employing chemical reaction network theory in a comprehensive in silico survey to determine the capacity for bistability of more than 40,000 simple networks that can be formed by two transcription factor-coding genes and their associated proteins (assuming only the most elementary biochemical processes). We find that there exist reaction rate constants leading to bistability in ∼90% of these GRN models, including several circuits that do not contain any of the TF cooperativity commonly associated with bistable systems, and the majority of which could only be identified as bistable through an original subnetwork-based analysis. A topological sorting of the two-gene family of networks based on the presence or absence of biochemical reactions reveals eleven minimal bistable networks (i.e., bistable networks that do not contain within them a smaller bistable subnetwork). The large number of previously unknown bistable network topologies suggests that the capacity for switch-like behavior in GRNs arises with relative ease and is not easily lost through network evolution. To highlight the relevance of the systematic application of CRNT to bistable network identification in real biological systems, we integrated publicly available protein-protein interaction, protein-DNA interaction, and gene expression data from Saccharomyces cerevisiae, and identified several GRNs predicted to behave in a bistable fashion.


The Database of Cross-Linguistic Colexifications, reproducible analysis of cross-linguistic polysemies.
Advances in computer-assisted linguistic research have been greatly influential in reshaping linguistic research. With the increasing availability of interconnected datasets created and curated by researchers, more and more interwoven questions can now be investigated. Such advances, however, are bringing high requirements in terms of rigorousness for preparing and curating datasets. Here we present CLICS, a Database of Cross-Linguistic Colexifications (CLICS). CLICS tackles interconnected interdisciplinary research questions about the colexification of words across semantic categories in the world's languages, and show-cases best practices for preparing data for cross-linguistic research. This is done by addressing shortcomings of an earlier version of the database, CLICS2, and by supplying an updated version with CLICS3, which massively increases the size and scope of the project. We provide tools and guidelines for this purpose and discuss insights resulting from organizing student tasks for database updates.


Psychological and physiological characteristics of a proposed object-referral/self-referral continuum of self-awareness.
This research extends and confirms recent brainwave findings that distinguished an individual's sense-of-self along an Object-referral/Self-referral Continuum of self-awareness. Subjects were interviewed and were given tests measuring inner/outer orientation, moral reasoning, anxiety, and personality. Scores on the psychological tests were factor analyzed. The first unrotated PCA component of the test scores yielded a "Consciousness Factor," analogous to the intelligence "g" factor, which accounted for over half of the variance among groups. Analysis of unstructured interviews of these subjects revealed fundamentally different descriptions of self-awareness. Individuals who described themselves in terms of concrete cognitive and behavioral processes (predominantly Object-referral mode) exhibited lower Consciousness Factor scores, lower frontal EEG coherence, lower alpha and higher gamma power during tasks, and less efficient cortical preparatory responses (contingent negative variation). In contrast, individuals who described themselves in terms of an abstract, independent sense-of-self underlying thought, feeling and action (predominantly Self-referral mode) exhibited higher Consciousness Factor scores, higher frontal coherence, higher alpha and lower gamma power during tasks, and more efficient cortical responses. These data suggest that definable states of brain activity and subjective experiences exist, in addition to waking, sleeping and dreaming, that may be operationally defined by psychological and physiological measures along a continuum of Object-referral/Self-referral Continuum of self-awareness.


Osteoimmunology at the nexus of arthritis, osteoporosis, cancer, and infection.
Over the past decade and a half, the biomedical community has uncovered a previously unappreciated reciprocal relationship between cells of the immune and skeletal systems. Work in this field, which has been termed "osteoimmunology," has resulted in the development of clinical therapeutics for seemingly disparate diseases linked by the common themes of inflammation and bone remodeling. Here, the important concepts and discoveries in osteoimmunology are discussed in the context of the diseases bridging these two organ systems, including arthritis, osteoporosis, cancer, and infection, and the targeted treatments used by clinicians to combat them.


Hepatoblastoma and APC gene mutation in familial adenomatous polyposis.
Hepatoblastoma is a rare, rapidly progressive, usually fatal childhood malignancy, which if confined to the liver can be cured by radical surgical resection. An association between hepatoblastoma and familial adenomatous polyposis (FAP), which is due to germline mutation of the APC (adenomatous polyposis coli) gene, has been confirmed, but correlation with site of APC mutation has not been studied. To analyse the APC mutational spectrum in FAP families with hepatoblastoma as a possible basis to select kindreds for surveillance. Eight patients with hepatoblastoma in seven FAP kindreds were compared with 97 families with identified APC gene mutation in a large Registry. APC gene mutation was evaluated by RNase protection assay or in vitro synthesis protein assay. The chi 2 test and correlation were used for data analysis. APC gene mutation was identified in all seven FAP kindreds in which an at risk member developed hepatoblastoma. A male predominance was noted (six of eight), similar to literature cases (18 of 25, p < 0.01. Mutations were restricted to codons 141 to 1230, but no significant difference in site of mutation between pedigrees with and without hepatoblastoma was identified. Hepatoblastoma occurs primarily in boys in FAP kindreds and is associated with germline APC mutation in the 5' end of the gene. However, the site of APC mutation cannot be used to predict occurrence of this extracolonic cancer in FAP pedigrees.


Modeling crawling cell movement on soft engineered substrates.
Self-propelled motion, emerging spontaneously or in response to external cues, is a hallmark of living organisms. Systems of self-propelled synthetic particles are also relevant for multiple applications, from targeted drug delivery to the design of self-healing materials. Self-propulsion relies on the force transfer to the surrounding. While self-propelled swimming in the bulk of liquids is fairly well characterized, many open questions remain in our understanding of self-propelled motion along substrates, such as in the case of crawling cells or related biomimetic objects. How is the force transfer organized and how does it interplay with the deformability of the moving object and the substrate? How do the spatially dependent traction distribution and adhesion dynamics give rise to complex cell behavior? How can we engineer a specific cell response on synthetic compliant substrates? Here we generalize our recently developed model for a crawling cell by incorporating locally resolved traction forces and substrate deformations. The model captures the generic structure of the traction force distribution and faithfully reproduces experimental observations, like the response of a cell on a gradient in substrate elasticity (durotaxis). It also exhibits complex modes of cell movement such as "bipedal" motion. Our work may guide experiments on cell traction force microscopy and substrate-based cell sorting and can be helpful for the design of biomimetic "crawlers" and active and reconfigurable self-healing materials.


TIP-1 translocation onto the cell plasma membrane is a molecular biomarker of tumor response to ionizing radiation.
Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors. In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment. This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.


Early Application of Next-Generation Sequencing Identifies Pancreatic Mass as Metastasis From an EGFR-Mutated Lung Adenocarcinoma.
Pancreatic metastasis of primary lung adenocarcinoma is a rare occurrence, accounting for <0.3% of all pancreatic malignancies. Given that the prognosis and treatment options for primary pancreatic cancer differ greatly from pancreatic metastases from a primary site, an accurate diagnosis is critical. This report presents a unique case of a 65-year-old man who was admitted with significant unintentional weight loss, fatigue, abdominal pain, and jaundice, and found to have a pancreatic mass initially thought to be primary pancreatic adenocarcinoma and subsequently diagnosed as an EGFR-mutated lung adenocarcinoma with metastases to the pancreas via early application of next-generation sequencing (NGS). The use of NGS early in the patient's clinical course not only changed the treatment strategy but also drastically altered the prognosis. Although metastatic pancreatic adenocarcinoma has a poor prognosis and survival rate, treatment of EGFR-mutated non-small cell lung cancer with EGFR tyrosine kinase inhibitors is associated with high response rates. Importantly, our case demonstrates that timely application of NGS very early in the disease course is paramount to the diagnosis, management, and prognosis of solid malignancies.


Effects of tumor suppressor gene (p53) on brain tumor angiogenesis and expression of angiogenic modulators.
p53 retarded tumor growth by several known mechanisms, including suppression of cell proliferation and inhibition of tumor angiogenesis. Vascular endothelial growth factors (VEGF) and angiopoietins (Ang-1, Ang-2) are major angiogeneic modulators. The current study examined the effect of p53 on the expression of these factors in conjunction with tumor growth and vascular formation. Growth characteristics of rat glioma cells (RT-2) infected with retrovirus (MSCV) encoding a full-length human wild-type p53 gene were examined by clonogenic assay. Expression of the transgene in vitro was verified by RT-PCR and immunoprecipitation. Tumor morphology, vascular architecture and the expression of VEGF, Ang-1, Ang-2 and Tie-2 were examined by immunohistochemistry and semi-quantitative RT-PCR. p53-infected cells showed retardation in growth and colony formation. In vivo, expression of the transgene resulted in prolonged survival and reduction of tumor volume (62%) and reduced the expression of VEGF (57.8%) and Tie-2 (15.4%) but not Ang-1 and Ang-2. The tumor exhibited increased necrosis (38%), hemorrhage and abnormal vascular architecture. p53 causes tumor regression by suppressing tumor proliferation and indirectly induces involution of tumor vessels by fostering unopposed activity of Ang-2 in an environment of diminishing VEGF.


Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin.
Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Delta22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Delta22 with Trx, under oxidizing conditions, with an IC50 of approximately 10 microM.


Association of caffeine intake and CYP1A2 genotype with ovarian cancer.
Coffee and caffeine consumption has been associated with ovarian cancer risk in several epidemiological studies. CYP1A2 is a key enzyme in the metabolism of coffee and in the activation of heterocyclic aromatic compounds that may be carcinogenic. Data from a preliminary investigation conducted in Hawaii of 164 epithelial ovarian cancer cases and 194 controls were used to examine the hypothesis that coffee and caffeine intake increases the risk of ovarian cancer and that these relations are modified by the CYP1A2 high-inducibility A/A genotype. A personal interview and blood specimen were collected in the subjects' homes. A significant positive trend (p = 0.02) in the odds ratios (ORs) was found with increasing intake of caffeine but not with tea or soda. Regular coffee drinkers were at significantly increased risk (OR = 1.8, 95% confidence interval, CI = 1.1-2.8) of ovarian cancer compared with women who did not drink regular coffee. Women with any CYP1A2 C allele were at similar risk of ovarian cancer (OR = 1.1, 95% CI = 0.7-1.7) compared with women with the A/A genotype. The associations of caffeine and coffee intake with risk were stronger among women with the A/A genotype than among women with any C allele. Somewhat stronger relations of coffee and caffeine intake to risk were found among women with cruciferous vegetable consumption above the median and among cases with mucinous histology. These preliminary data suggest a modest positive association of caffeine and coffee consumption with the OR for ovarian cancer that may be modified by CYP1A2 genotype and exposures, such as cruciferous vegetable consumption, that influence CYP1A2 expression.


The evolutionary history of protein fold families and proteomes confirms that the archaeal ancestor is more ancient than the ancestors of other superkingdoms.
The entire evolutionary history of life can be studied using myriad sequences generated by genomic research. This includes the appearance of the first cells and of superkingdoms Archaea, Bacteria, and Eukarya. However, the use of molecular sequence information for deep phylogenetic analyses is limited by mutational saturation, differential evolutionary rates, lack of sequence site independence, and other biological and technical constraints. In contrast, protein structures are evolutionary modules that are highly conserved and diverse enough to enable deep historical exploration. Here we build phylogenies that describe the evolution of proteins and proteomes. These phylogenetic trees are derived from a genomic census of protein domains defined at the fold family (FF) level of structural classification. Phylogenomic trees of FF structures were reconstructed from genomic abundance levels of 2,397 FFs in 420 proteomes of free-living organisms. These trees defined timelines of domain appearance, with time spanning from the origin of proteins to the present. Timelines are divided into five different evolutionary phases according to patterns of sharing of FFs among superkingdoms: (1) a primordial protein world, (2) reductive evolution and the rise of Archaea, (3) the rise of Bacteria from the common ancestor of Bacteria and Eukarya and early development of the three superkingdoms, (4) the rise of Eukarya and widespread organismal diversification, and (5) eukaryal diversification. The relative ancestry of the FFs shows that reductive evolution by domain loss is dominant in the first three phases and is responsible for both the diversification of life from a universal cellular ancestor and the appearance of superkingdoms. On the other hand, domain gains are predominant in the last two phases and are responsible for organismal diversification, especially in Bacteria and Eukarya. The evolution of functions that are associated with corresponding FFs along the timeline reveals that primordial metabolic domains evolved earlier than informational domains involved in translation and transcription, supporting the metabolism-first hypothesis rather than the RNA world scenario. In addition, phylogenomic trees of proteomes reconstructed from FFs appearing in each of the five phases of the protein world show that trees reconstructed from ancient domain structures were consistently rooted in archaeal lineages, supporting the proposal that the archaeal ancestor is more ancient than the ancestors of other superkingdoms.


Improvements in blood pressure among undiagnosed hypertensive participants in a community-based lifestyle intervention, Mississippi, 2010.
Effective strategies are needed to reach and treat people who lack awareness of or have uncontrolled hypertension. We used data from a community-based participatory research initiative, Hub City Steps, to quantify the prevalence of undiagnosed hypertension and determine the relationship between hypertension status at baseline and postintervention improvements in blood pressure and health-related quality of life. Hub City Steps was a 6-month preintervention-postintervention lifestyle intervention targeting hypertension risk factors. Outcome measures were collected at baseline, 3 months, and 6 months. Generalized linear mixed models were used to test for effects by time and hypertension status. Of the enrolled sample (N = 269), most were overweight or obese (91%), African American (94%), and women (85%). When considering hypertension status, 42% had self-reported diagnosis of hypertension (self-reported subgroup; 84% with antihypertensive medication use); 36% had no self-reported medical history of hypertension, but when blood pressure was measured they had a clinical diagnosis of prehypertension or hypertension (undiagnosed subgroup); and 22% had no self-reported or clinical hypertension diagnosis (no hypertension subgroup). From baseline to 6 months, systolic blood pressure significantly improved for participants with self-reported hypertension [8.2 (SD, 18.2) mm Hg] and undiagnosed hypertension [12.3 (SD, 16.3) mm Hg], with undiagnosed participants experiencing the greatest improvements (P < .001). Effects remained significant after controlling for covariates. Health-related quality of life significantly improved for all 3 hypertension subgroups, with no apparent subgroup differences. This study reveals advantages of a culturally appropriate community-based participatory research initiative to reach those with undetected hypertension and effectively improve blood pressure status and health-related quality of life.


The Efficacy of a Walking Intervention Using Social Media to Increase Physical Activity: A Randomized Trial.
Facebook may be a useful tool to provide a social support group to encourage increases in physical activity. This study examines the efficacy of a Facebook social support group to increase steps/day in young women. Female college freshmen (N = 63) were randomized to one of two 8-week interventions: a Facebook Social Support Group (n = 32) or a Standard Walking Intervention (n = 31). Participants in both groups received weekly step goals and tracked steps/day with a pedometer. Women in the Facebook Social Support Group were also enrolled in a Facebook group and asked to post information about their steps/day and provide feedback to one another. Women in both intervention arms significantly increased steps/day pre- to postintervention (F(8,425) = 94.43, P < .001). However, women in the Facebook Social Support Group increased steps/day significantly more (F(1,138) = 11.34, P < .001) than women in the Standard Walking Intervention, going from 5295 to 12,472 steps/day. These results demonstrate the potential effectiveness of using Facebook to offer a social support group to increase physical activity in young women. Women in the Facebook Social Support Group increased walking by approximately 1.5 miles/day more than women in the Standard Walking Intervention which, if maintained, could have a profound impact on their future health.


Sulforaphane Mediates Glutathione Depletion via Polymeric Nanoparticles to Restore Cisplatin Chemosensitivity.
Platinum (Pt)-based chemotherapy is a broadly used therapeutic regimen against various cancers. However, the insufficient cellular uptake, deactivation by thiol-containing species and nonspecific distribution of cisplatin (CDDP) result in its low chemosensitivity as well as systemic side effects, which can largely constrain the employment of CDDP in clinical treatment. To circumvent these problems, in this study, polymeric nanoparticles were utilized to codeliver a water-soluble CDDP derivative, poly(γ,l-glutamic acid)-CDDP conjugate, and a naturally occurring compound derived from broccoli, sulforaphane, which can achieve efficient glutathione (GSH) depletion, to improve the accumulation of CDDP in cancer cells. Results show that compared with combinational treatment of CDDP and SFN, the nanoparticles were more effectively internalized and could significantly reduce GSH content in breast cancer cells, leading to a notable increase in DNA-bound Pt and DNA damage-induced apoptosis. Moreover, in an orthotopic breast cancer model, the nanoparticles achieved a significantly higher tumor accumulation and exhibited a more powerful antitumor activity. Finally, this nanoenhanced chemotherapy was further confirmed in a liver cancer model with high-expression of GSH. Taken together, this sulforaphane-based nanostrategy holds great promise to enhance the sensitivity and therapeutic efficacy of Pt-based chemotherapy.


Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1β production in response to β-glucans and the fungal pathogen, Candida albicans.
Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-Glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating β-glucan-induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting β-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1β production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.


Cellular composition of bronchial brushings obtained from healthy dogs and dogs with chronic cough and cytologic composition of bronchoalveolar lavage fluid obtained from dogs with chronic cough.
To determine whether bronchial brushings from dogs with chronic cough have increased numbers of goblet cells and WBCs, compared with numbers for healthy dogs, or have differing WBC populations, compared with populations in bronchoalveolar lavage (BAL) fluid obtained from dogs with chronic cough. 9 healthy dogs and 10 dogs with chronic cough. Specimens were collected by use of bronchoscopy. Cellular composition was determined for brushings, and results from dogs with chronic cough were compared with those from healthy dogs. Cellular composition of brushings was compared with composition of BAL obtained from dogs with chronic cough. Brushings from healthy dogs contained a median of 2.9 x 10(6) epithelial cells, comprising 100% epithelial cells (96% ciliated, 3% goblet, and 1% other) and no WBCs. Brushings from dogs with chronic cough had 4.5 x 10(6) epithelial cells, comprising 93% epithelial cells (86% ciliated, 2% goblet, and 12% other). Dogs with chronic cough had significantly greater percentages of WBCs (7%) and neutrophils (6%), compared with values for healthy dogs. Five dogs with chronic cough had no neutrophilic inflammation evident in BAL, but 4 of these had evidence of neutrophilic inflammation in brushings. Neutrophils, but not goblet cells, were increased in brushings from dogs with chronic cough. Analysis of bronchial brushings provides information about airway inflammation that differs from that found by examination of BAL in some dogs with chronic cough and is a more sensitive indicator of airway inflammation than cytologic examination of BAL in these dogs.


Improved techniques for double-balloon-enteroscopy-assisted endoscopic retrograde cholangiopancreatography.
To investigate the clinical outcome of double balloon enteroscopy (DBE)-assisted endoscopic retrograde cholangiopancreatography (DB-ERCP) in patients with altered gastrointestinal anatomy. Between September 2006 and April 2011, 47 procedures of DB-ERCP were performed in 28 patients with a Roux-en-Y total gastrectomy (n = 11), Billroth II gastrectomy (n = 15), or Roux-en-Y anastomosis with hepaticojejunostomy (n = 2). DB-ERCP was performed using a short-type DBE combined with several technical innovations such as using an endoscope attachment, marking by submucosal tattooing, selectively applying contrast medium, and CO₂ insufflations. The papilla of Vater or hepaticojejunostomy site was reached in its entirety with a 96% success rate (45/47 procedures). There were no significant differences in the success rate of reaching the blind end with a DBE among Roux-en-Y total gastrectomy (96%), Billroth II reconstruction (94%), or pancreatoduodenectomy (100%), respectively (P = 0.91). The total successful rate of cannulation and contrast enhancement of the target bile duct in patients whom the blind end was reached with a DBE was 40/45 procedures (89%). Again, there were no significant differences in the success rate of cannulation and contrast enhancement of the target bile duct with a DBE among Roux-en-Y total gastrectomy (88 %), Billroth II reconstruction (89%), or pancreatoduodenectomy (100%), respectively (P = 0.67). Treatment was achieved in all 40 procedures (100%) in patients whom the contrast enhancement of the bile duct was successful. Common endoscopic treatments were endoscopic biliary drainage (24 procedures) and extraction of stones (14 procedures). Biliary drainage was done by placement of plastic stents. Stones extraction was done by lithotomy with the mechanical lithotripter followed by extraction with a basket or by the balloon pull-through method. Endoscopic sphincterotomy was performed in 14 procedures with a needle precutting knife using a guidewire. The mean total duration of the procedure was 93.6 ± 6.8 min and the mean time required to reach the papilla was 30.5 ± 3.7 min. The mean time required to reach the papilla tended to be shorter in Billroth II reconstruction (20.9 ± 5.8 min) than that in Roux-en-Y total gastrectomy (37.1 ± 4.9 min) but there was no significant difference (P = 0.09). A major complication occurred in one patient (3.5%); perforation of the long limb in a patient with Billroth II anastomosis. Short-type DBE combined with several technical innovations enabled us to perform ERCP in most patients with altered gastrointestinal anatomy.


Rate variation and estimation of divergence times using strict and relaxed clocks.
Understanding causes of biological diversity may be greatly enhanced by knowledge of divergence times. Strict and relaxed clock models are used in Bayesian estimation of divergence times. We examined whether: i) strict clock models are generally more appropriate in shallow phylogenies where rate variation is expected to be low, ii) the likelihood ratio test of the clock (LRT) reliably informs which model is appropriate for dating divergence times. Strict and relaxed models were used to analyse sequences simulated under different levels of rate variation. Published shallow phylogenies (Black bass, Primate-sucking lice, Podarcis lizards, Gallotiinae lizards, and Caprinae mammals) were also analysed to determine natural levels of rate variation relative to the performance of the different models. Strict clock analyses performed well on data simulated under the independent rates model when the standard deviation of log rate on branches, σ, was low (≤ 0.1), but were inappropriate when σ>0.1 (95% of rates fall within 0.0082-0.0121 subs/site/Ma when σ = 0.1, for a mean rate of 0.01). The independent rates relaxed clock model performed well at all levels of rate variation, although posterior intervals on times were significantly wider than for the strict clock. The strict clock is therefore superior when rate variation is low. The performance of a correlated rates relaxed clock model was similar to the strict clock. Increased numbers of independent loci led to slightly narrower posteriors under the relaxed clock while older root ages provided proportionately narrower posteriors. The LRT had low power for σ = 0.01-0.1, but high power for σ = 0.5-2.0. Posterior means of σ2 were useful for assessing rate variation in published datasets. Estimates of natural levels of rate variation ranged from 0.05-3.38 for different partitions. Differences in divergence times between relaxed and strict clock analyses were greater in two datasets with higher σ2 for one or more partitions, supporting the simulation results. The strict clock can be superior for trees with shallow roots because of low levels of rate variation between branches. The LRT allows robust assessment of suitability of the clock model as does examination of posteriors on σ2.


Chlamydia species as a cause of community-acquired pneumonia in Canada.
Chlamydia pneumoniae has been implicated as a cause of community-acquired pneumonia (CAP) in several studies. However, there has been no comprehensive study of the role of Chlamydia species (C. pneumoniae, C. psittaci (avian and feline strains) and C. pecorum) as a cause of CAP. The aim of the present study was to determine the role of C. pneumoniae, C. psittaci and C. pecorum as causes of CAP. A prospective cohort observational study of CAP was conducted at 15 teaching centres in eight Canadian provinces between January 1996-October 1997. Acute (n=539) and convalescent (n=272) serum samples were obtained for determination of antibody titres to C. pneumoniae, C. psittaci, C. pecorum, C. trachomatis, Mycoplasma pneumoniae, Legionella pneumophila serogroups I-VI, Streptococcus pneumoniae and various respiratory viruses. Twelve of 539 (2.2%) patients had acute C. pneumoniae pneumonia and an additional 32 (5.9%) had possible acute infection. C. pneumoniae was the sole pathogen in 16 of 42 (38.1%) of these patients. The most common copathogens were S. pneumoniae, respiratory syncytial virus and influenza virus type A. C. pneumoniae pneumonia patients were older and more likely to show congestive heart failure compared to bacteraemic S. pneumoniae patients. The latter had a lower mean diastolic blood pressure, a higher white blood cell count and a lower arterial carbon dioxide tension. Two patients had antibody titres suggestive of recent infection with the feline strain of C. psittaci. Although numerically Chlamydia pneumoniae is an important cause of community-acquired pneumonia, no distinctive clinical features associated with this pathogen were detected in the present study. Feline Chlamydia psittaci may cause a few cases of community-acquired pneumonia. Avian Chlamydia psittaci should be considered only if there is a compatible epidemiological history.


Quality Of Health Care In India: Challenges, Priorities, And The Road Ahead.
India's health care sector provides a wide range of quality of care, from globally acclaimed hospitals to facilities that deliver care of unacceptably low quality. Efforts to improve the quality of care are particularly challenged by the lack of reliable data on quality and by technical difficulties in measuring quality. Ongoing efforts in the public and private sectors aim to improve the quality of data, develop better measures and understanding of the quality of care, and develop innovative solutions to long-standing challenges. We summarize priorities and the challenges faced by efforts to improve the quality of care. We also highlight lessons learned from recent efforts to measure and improve that quality, based on the articles on quality of care in India that are published in this issue of Health Affairs The rapidly changing profile of diseases in India and rising chronic disease burden make it urgent for state and central governments to collaborate with researchers and agencies that implement programs to improve health care to further the quality agenda.


Monoclonal antibodies define antigenic variation in the ID variety of Venezuelan equine encephalitis virus.
Three monoclonal antibodies were generated that are specific for the E2 glycoprotein of Venezuelan equine encephalitis (VEE) virus and have useful reactivities in an enzyme-linked immunosorbent assay (ELISA). Antibody 1A1B-9 distinguished between the IC (epizootic) and ID (enzootic) varieties of VEE virus by ELISA. Clone 7A1A-1 antibody distinguished the Panamanian prototype virus (3880) from Colombian ID isolates by a 500-fold difference in titer by endpoint ELISA, and it detected antigenic variation in ID isolates from southern Colombia and Ecuador. Antibody 7A3A-4 defined a cryptic antigenic site on the latter two isolates. These monoclonal antibodies complement others in identifying VEE isolates by a simple ELISA.


A pilot study of Wii Fit exergames to improve balance in older adults.
To determine the safety and feasibility of using Nintendo Wii Fit exergames to improve balance in older adults. Seven older adults aged 84 (5) years with impaired balance (Berg Balance Scale [BBS] score < 52 points) were recruited from 4 continuing care retirement communities to participate in a single group pre- and postevaluation of Wii Fit exergames. Participants received individualized instructions (at least 5 home visits) on playing 4 exergames (basic step, soccer heading, ski slalom, and table tilt) and were asked to play these games in their homes at least 30 minutes 3 times per week for 3 months and received weekly telephone follow-up. They also completed a paper log of their exergame play and rated their enjoyment immediately after each session. Participants completed the BBS, 4-Meter Timed Walk test, and the Physical Activity Enjoyment Scale at baseline and 3 months. Semistructured interviews were conducted at the 3-month evaluation. Participants safely and independently played a mean of 50 sessions, median session duration of 31 minutes. Two of the games were modified to ensure participants' safety. Participants rated high enjoyment immediately after exergame play and expressed experiencing improved balance with daily activities and desire to play exergames with their grandchildren. Berg Balance Scores increased from 49 (2.1) to 53 (1.8) points (P = .017). Walking speed increased from 1.04 (0.2) to 1.33 (0.84) m/s (P = .018). Use of Wii Fit for limited supervised balance training in the home was safe and feasible for a selected sample of older adults. Further research is needed to determine clinical efficacy in a larger, diverse sample and ascertain whether Wii Fit exergames can be integrated into physical therapy practice to promote health in older adults.


Differential disease restriction of Moloney and Friend murine leukemia viruses by the mouse Rmcf gene is governed by the viral long terminal repeat.
Neonatal CxD2 (Rmcfr) and Balb/c (Rmcfs) mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibited approximately equivalent time course and pathology for disease. CxD2 mice showed only slightly reduced presence of Moloney mink cell focus-forming virus (M-MCF) provirus as seen by Southern blot analysis compared to Balb/c mice. This lack of restriction for disease and spread of MCF was in sharp contrast to that seen for CxD2 mice inoculated with Friend murine leukemia virus (F-MuLV), where incidence of disease and propagation of MCFs were severely restricted, as previously reported. Inoculation of CxD2 mice with FM-MuLV, a recombinant F-MuLV virus containing M-MuLV LTR sequences (U3 and R), resulted in T cell disease of time course equal to that seen in Balb/c mice; there also was little restriction for propagation of MCFs. This indicated that presence of the M-MuLV long terminal repeat (LTR) was sufficient for propagation of MCFs in CxD2 mice. Differing restriction for F-MuLV vs. M-MuLV in CxD2 mice was explained on the basis of different "MCF propagator cells" for the two viruses. It was suggested that cells propagating F-MCF (e.g., erythroid progenitors) are blocked by endogenous MCF-like gp70env protein, whereas cells propagating M-MCF (e.g., lymphoid) do not express this protein on their surface. F-MuLV disease in CxD2 mice was greatly accelerated when neonates were inoculated with a F-MuLV/F-MCF pseudotypic mixture. However, F-MCF provirus was not detectable or only barely detectable in F-MuLV/F-MCF-induced tumors, suggesting that F-MCF acted indirectly in induction of these tumors.


Molecularly imprinted polymers as selective adsorbents for ambient plasma mass spectrometry.
The application of molecularly imprinted polymers (MIPs) as molecular scavengers for ambient plasma ionization mass spectrometry has been reported for the first time. MIPs were synthesized using methacrylic acid as functional monomer; nicotine, propyphenazone, or methylparaben as templates; ethylene glycol dimethacrylate as a cross-linker; and 2,2'-azobisisobutyronitrile as polymerization initiator. To perform ambient plasma ionization experiments, a setup consisting of the heated crucible, a flowing atmospheric-pressure afterglow (FAPA) plasma ion source, and a quadrupole ion trap mass spectrometer has been used. The heated crucible with programmable temperature allows for desorption of the analytes from MIPs structure which results in their direct introduction into the ion stream. Limits of detection, linearity of the proposed analytical procedure, and selectivities have been determined for three analytes: nicotine, propyphenazone, and methylparaben. The analytes used were chosen from various classes of organic compounds to show the feasibility of the analytical procedure. The limits of detections (LODs) were 10 nM, 10, and 0.5 μM for nicotine, propyphenazone, and methylparaben, respectively. In comparison with the measurements performed for the non-imprinted polymers, the values of LODs were improved for at least one order of magnitude due to preconcentration of the sample and reduction of background noise, contributing to signal suppression. The described procedure has shown linearity in a broad range of concentrations. The overall time of single analysis is short and requires ca. 5 min. The developed technique was applied for the determination of nicotine, propyphenazone, and methylparaben in spiked real-life samples, with recovery of 94.6-98.4%. The proposed method is rapid, sensitive, and accurate which provides a new option for the detection of small organic compounds in various samples. Graphical abstract The experimental setup used for analysis.


Shaping T cell - B cell collaboration in the response to human immunodeficiency virus type 1 envelope glycoprotein gp120 by peptide priming.
Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.