A comprehensive screening system for damaged nucleotide-binding proteins.
To identify novel nucleotide pool sanitizing enzymes, we have established a comprehensive screening system for damaged nucleotide-binding proteins based on proteomics technology. In the screening system, affinity chromatography with resins carrying various damaged nucleotides is used for the purification of binding proteins, and the purified proteins are identified by mass-spectrometry. Inosine triphosphate (ITP) is a deleterious damaged nucleotide, and can be generated by nitrosative deamination of ATP or phosphorylation of inosine monophosphate (IMP). Using the above system, we performed screens for ITP-binding proteins from mouse and human cell extracts, and identified several ITP-binding enzymes. We identified both mouse inosine triphosphatase (ITPA) and human ITPA, well-known ITP hydrolyzing enzymes, as ITP-binding proteins. These results support the validity of this screening system. In addition to ITPA, we identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an ITP-binding protein. Biochemical analysis revealed that NUDT16 selectively hydrolyzes deoxyinosine diphosphate (dIDP) and IDP to deoxyinosine monophosphate (dIMP) and IMP, respectively. dITP and ITP are also hydrolyzed by NUDT16 to a lesser extent. The knockdown of NUDT16 in HeLa MR cells suppressed cell proliferation, and was accompanied by a significantly increased accumulation of strand breaks in nuclear DNA, suggesting that NUDT16 has an essential role in the maintenance of genome stability. RS21-C6, another ITP-binding protein identified in our screen, binds not only to ITP, but also to ATP. RS21-C6 hydrolyzes dCTP and 5-halo-dCTP, but does not hydrolyze ITP or ATP. It is likely that RS21-C6 may control dCTP levels or eliminate 5-halo-dCTP in the nucleotide pools. In conclusion, the results of these studies show that our screening system is applicable in studying the health effects of damaged nucleotides and cellular sanitizing systems for nucleotide pools.


B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue.
Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing. We found immunoglobulin heavy chain variable region sequences that were unique to mELT and had accumulated functionally relevant somatic mutations, together indicating localized antigen-driven affinity maturation. Our results suggest that B cells in mELT actively participate in CNS autoimmunity, which may be relevant to mELT in MS and ELT in other chronic inflammatory conditions.


Cytoskeletal influences on nuclear shape in granulocytic HL-60 cells.
During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope).


Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.
Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun.


Antimicrobial resistant Escherichia coli in Scottish wild deer: Prevalence and risk factors.
Antimicrobial resistance (AMR) is a recognised threat to global health. Obtaining data on the prevalence of AMR in environmental bacteria is key to understanding drivers and routes of transmission. Here, 325 Shiga toxin negative deer faecal samples-gathered from across the Scottish mainland-were screened for the presence of AMR Escherichia coli and investigated for potential risk factors associated with AMR occurrence. E. coli with resistance to antimicrobials of clinical health concern, including carbapenems and 3rd generation cephalosporins, were targeted. Ninety-nine percent of samples yielded E. coli, and the prevalence of resistant E. coli at the level of faecal samples was 21.8% (n = 71) for tetracycline, 6.5% (n = 21) for cefpodoxime, 0.3% for ciprofloxacin (n = 1), with no recorded resistance to meropenem. Potential risk factors for tetracycline and cefpodoxime resistance were investigated. The presence of broadleaved woodlands was significantly associated with both AMR phenotypes, which may relate to land use within or around such woodlands. Associated risk factors varied across resistance phenotype and deer species, with proximity or density of horses an indicator of significantly decreased and increased risk, respectively, or tetracycline and cefpodoxime resistance in E. coli from roe deer, but not from red deer. Distance from wastewater treatment plants was a significant risk factor for tetracycline resistance in E. coli from red deer but not from roe deer. Data indicated that AMR E. coli can occur in wild deer populations that are not directly exposed to the selective pressure exerted by antimicrobial treatment. Overall, resistance to critically important antimicrobials was found to be low in the studied population, suggesting no immediate cause for concern regarding human health. Utilising existing culling frameworks, wild deer in Scotland could function well as a sentinel species for the surveillance of AMR in the Scottish environment.


Cell-free plasma hypermethylated CASZ1, CDH13 and ING2 are promising biomarkers of esophageal cancer.
Identifying sensitive and specific biomarkers for early detection of cancer is immensely imperative for early diagnosis and treatment and better clinical outcome of cancer patients. This study aimed to construct a specific DNA methylation pattern of cancer suppressor genes and explore the feasibility of applying cell-free DNA based methylation as a biomarker for early diagnosis of esophageal squamous cell carcinoma (ESCC). We recruited early stage ESCC patients from Yangzhong County, China. The Illumina Infinium 450K Methylation BeadChip was used to construct a genome-wide DNA methylation profile. Then, differentiated genes were selected for the validation study using the Sequenom MassARRAY platform. The frequency of methylation was compared between cancer tissues, matched cell-free DNAs and normal controls. The specific methylation profiles were constructed, and the sensitivity and specificity were calculated. Seven CG sites in three genes CASZ1, CDH13 and ING2 were significantly hypermethylated in ESCC as compared with normal controls. A significant correlation was found between the methylation of DNA extracted from cancer tissues and matched plasma cell-free DNA, either for individual CG site or for cumulative methylation analysis. The sensitivity and specificity reached 100% at an appropriate cut-point using these specific methylation biomarkers. This study revealed that aberrant DNA methylation is a promising biomarker for molecular diagnosis of esophageal cancer. Hypermethylation of CASZ1, CDH13 and ING2 detected in plasma cell-free DNA can be applied as a potential noninvasive biomarker for diagnosis of esophageal cancer.


Ty3 transposes in mating populations of yeast: a novel transposition assay for Ty3.
Ty3 is a retrotransposon of Saccharomyces cerevisiae that integrates just upstream of the transcription initiation site of genes transcribed by RNA polymerase III. Ty3 transcription is pheromone-inducible in haploid cells and is mating-type regulated in diploid cells. The specificity of Ty3 integration was exploited in the design of a novel target into which transposition of Ty3 elements could be selected. The target plasmid contains divergently oriented tRNA genes with 19 base pairs separating the two tRNA gene coding sequences. An inactive ochre suppressor tRNA(Tyr) gene with a modified transcription initiation region was used as the selectable marker and a tRNA(Val) (AAC) gene was used to direct Ty3 integration into the transcription initiation region of the suppressor tRNA(Tyr) gene. Integration of Ty3 activated expression of the suppressor tRNA gene, which resulted in suppression of ochre nonsense alleles ade2-101(0) and lys2-1(0) and allowed cell growth on selective medium. Based on the activity of this target, Ty3, under control of a galactose-inducible promoter and present on a high copy-number plasmid, was estimated to transpose into the genome at a rate of 5.6 x 10(-3) per cell division. We show here that induction of Ty3 transcription from its natural promoter results in transposition. Ty3 elements in strains of the a or alpha mating-type transposed efficiently to target plasmids in cells of the opposite mating-type. Thus, natural transposition of Ty3 is regulated temporally to occur in mating populations.


Adiposity, compared with masculinity, serves as a more valid cue to immunocompetence in human mate choice.
According to the 'good genes' hypothesis, females choose males based on traits that indicate the male's genetic quality in terms of disease resistance. The 'immunocompetence handicap hypothesis' proposed that secondary sexual traits serve as indicators of male genetic quality, because they indicate that males can contend with the immunosuppressive effects of testosterone. Masculinity is commonly assumed to serve as such a secondary sexual trait. Yet, women do not consistently prefer masculine looking men, nor is masculinity consistently related to health across studies. Here, we show that adiposity, but not masculinity, significantly mediates the relationship between a direct measure of immune response (hepatitis B antibody response) and attractiveness for both body and facial measurements. In addition, we show that circulating testosterone is more closely associated with adiposity than masculinity. These findings indicate that adiposity, compared with masculinity, serves as a more important cue to immunocompetence in female mate choice.


Topography-induced symmetry transition of droplets on quasi-periodically patterned surfaces.
Quasi-periodic structures of quasicrystals yield novel effects in diverse systems. However, there is little investigation on employing quasi-periodic structures in morphology control. Here, we show the use of quasi-periodic surface structures in controlling the transition of liquid droplets. Although surface structures seem random-like, we find that on these surfaces, droplets spread to well-defined 5-fold symmetric shapes and the symmetry of droplet shapes spontaneously restores during spreading, hitherto unreported in the morphology control of droplets. To obtain physical insights into these symmetry transitions, we conduct energy analysis and perform systematic experiments by varying the properties of both liquid droplets and patterned surfaces. The results show the dominant factors in determining droplet shapes to be surface topography and the self-similarity of the surface structure. Quantified results of the droplet spreading process show distinct dynamics from the spreading experiments on periodically micropatterned surfaces. Our findings significantly advance the control capability of the droplet morphology. Such a quasi-periodic patterning strategy can offer a new method to achieve complex patterns, and may be used to model patterns in the study of rough surfaces.


Perceived morningness-eveningness predicts academic adjustment and substance use across university, but social jetlag is not to blame.
Past research has consistently found that evening-types typically report poorer academic adjustment and higher levels of substance use compared to morning-types. An important development within the morningness-eveningness and psychosocial adjustment literature has been the hypothesis that social jetlag (i.e. the asynchrony between an individual's "biological" and "social" clocks) is one factor that may explain why evening-types are at a greater risk for negative psychosocial adjustment. Yet, only a handful of studies have assessed social jetlag. Furthermore, the few studies that have assessed social jetlag have done so only with concurrent data, and thus have not been able to determine the direction of effects among morningness-eveningness, social jetlag and psychosocial adjustment. To address this important gap in the literature, the present 3-year longitudinal study employed the use of a cross-lagged auto-regressive model to specifically examine the predictive role of perceived morningness-eveningness and social jetlag on two important indices of psychosocial adjustment among university students: academic adjustment and substance use. We also assessed whether there would be an indirect effect between perceived morningness-eveningness and psychosocial adjustment through social jetlag. Participants were 942 (71.5% female; M = 19 years, SD = 0.90) undergraduates at a mid-sized university in Southern Ontario, Canada, who completed a survey at three assessments, each one year apart, beginning in first-year university. Measures were demographics (age, gender and parental education), sleep problems, perceived morningness-eveningness, social jetlag, academic adjustment and substance use. As hypothesized, results of path analyses indicated that a greater perceived eveningness preference significantly predicted higher social jetlag, poorer academic adjustment and higher substance use over time. In contrast, we found no support for social jetlag as a predictor of academic adjustment and substance use, indicating that social jetlag did not explain the link between perceived morningness-eveningness and negative psychosocial adjustment. An important finding was the significant predictive effect of higher substance use on social jetlag over time. Results of the present study highlight the importance of employing a longitudinal framework within which to specifically determine the direction of effects among the study variables in order to validate proposed theoretical models that aim to guide our understanding of how perceived morningness-eveningness, social jetlag, academic adjustment and substance use relate to each other.


Risk factors for anorexia nervosa: three integrated case-control comparisons.
Many risk factors have been implicated in the development of anorexia nervosa. Little is known about their relative contributions, nor in most cases is it clear whether they are specific to anorexia nervosa or risk factors for all eating disorders or for psychiatric disorder in general. We used a case-control design involving the comparison of 67 female subjects with a history of anorexia nervosa with 204 healthy control subjects, 102 subjects with other psychiatric disorders, and 102 subjects with bulimia nervosa. A broad range of risk factors was assessed by interview. The subjects with anorexia nervosa and the healthy controls differed in their exposure to most of the putative risk factors. There was no greater exposure to factors that increased the likelihood of dieting, once the influence of other classes of risk factors had been taken into account. Premorbid perfectionism and negative self-evaluation were especially common and more so than among the general psychiatric controls. Parental obesity and an early menarche, together with parental psychiatric disorder, distinguished those with bulimia nervosa from those with anorexia nervosa. There appears to be a broad range of risk factors for anorexia nervosa and bulimia nervosa, some of which are shared with other psychiatric disorders. Factors that increase the likelihood of dieting seem to have more important influence as risk factors for bulimia nervosa than anorexia nervosa. Perfectionism and negative self-evaluation appear to be particularly common and characteristic antecedents of both eating disorders.


Extracorporeal Membrane Oxygenation for ARDS: National Trends in the United States 2008-2012.
Recent advances in technology and protocols have made the use of extracorporeal membrane oxygenation (ECMO) a viable rescue therapy for patients with ARDS who present with refractory hypoxemia. Despite the lack of strong evidence supporting the use of ECMO in ARDS, its use seems to be increasing. We sought to determine recent trends in the use of ECMO for ARDS. We also assessed trends in mortality among patients with ARDS in whom ECMO was used. We performed a retrospective analysis using the largest all-payer in-patient healthcare database in the United States, the Healthcare Cost and Utilization project, the National In-patient Sample database from 2008 to 2012. Subjects with ARDS were identified using carefully chosen International Classification of Diseases, Ninth Revision codes. We found that in 2008, about 1 in 1,000 subjects with ARDS underwent ECMO. Over the subsequent 4-y time period, there was a 0.19% absolute increase and 70% relative increase in the use of ECMO for ARDS. The mortality rate among subjects with ARDS in whom ECMO was used declined from 78% in 2008 to 64% in 2012. We also found a trend toward a reduction in hospital stay among survivors. In the United States, between 2008 and 2012, there was an increasing trend toward the use of ECMO in patients with ARDS that coincided with a slight increase in survival among these patients.


Genetic variation in candidate genes like the HMGA2 gene in the extremely tall.
Genetic variation in several candidate genes has been associated with short stature. Recently, a high-mobility group A2 (HMGA2) gene SNP has been robustly associated with height in the general population. Only few have attempted to study these genes in extremely tall stature. We therefore studied common genetic variation in candidate genes for height in extremely tall Dutch. We included 116 constitutionally tall cases with height >2 SD and 103 controls with normally distributed height <2 SD. We genotyped 10 common polymorphisms previously associated with height variation. The HMGA2 gene SNP was significantly associated with tall stature. Using a logistic regression model, we calculated that carrying the HMGA2 (rs1042725) C allele significantly increased the odds of being tall (OR = 1.53, 95% CI 1.02-2.28; p = 0.03). In addition, controls with one or two copies of the C allele were significantly taller than controls carrying the TT genotype [TC: mean (SD) +0.61 (0.21) SDS; p = 0.004, and CC: +0.77 (0.25) SDS; p = 0.003]. Our study shows that a common polymorphism in the HMGA2 gene is not only associated with height variation in the general population but also plays an important role in one of the extremes of the height distribution.


A statistical method for the detection of alternative splicing using RNA-seq.
Deep sequencing of transcriptome (RNA-seq) provides unprecedented opportunity to interrogate plausible mRNA splicing patterns by mapping RNA-seq reads to exon junctions (thereafter junction reads). In most previous studies, exon junctions were detected by using the quantitative information of junction reads. The quantitative criterion (e.g. minimum of two junction reads), although is straightforward and widely used, usually results in high false positive and false negative rates, owning to the complexity of transcriptome. Here, we introduced a new metric, namely Minimal Match on Either Side of exon junction (MMES), to measure the quality of each junction read, and subsequently implemented an empirical statistical model to detect exon junctions. When applied to a large dataset (>200M reads) consisting of mouse brain, liver and muscle mRNA sequences, and using independent transcripts databases as positive control, our method was proved to be considerably more accurate than previous ones, especially for detecting junctions originated from low-abundance transcripts. Our results were also confirmed by real time RT-PCR assay. The MMES metric can be used either in this empirical statistical model or in other more sophisticated classifiers, such as logistic regression.


Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs.
GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Together, our results demonstrate that GRP78ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the β-γ bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.


Tamoxifen for breast cancer risk reduction: impact of alternative approaches to quality-of-life adjustment on cost-effectiveness analysis.
In cost-effectiveness analysis (CEA), the effects of health-care interventions on multiple health dimensions typically require consideration of both quantity and quality of life. To explore the impact of alternative approaches to quality-of-life adjustment using patient preferences (utilities) on the outcome of a CEA on use of tamoxifen for breast cancer risk reduction. A state transition Markov model tracked hypothetical cohorts of women who did or did not take 5 years of tamoxifen for breast cancer risk reduction. Incremental quality-adjusted effectiveness and cost-effectiveness ratios (ICERs) for models including and excluding a utility adjustment for menopausal symptoms were compared with each other and to a global utility model. Two hundred fifty-five women aged 50 and over with estimated 5-year breast cancer risk >or=1.67% participated in utility assessment interviews. Standard gamble utilities were assessed for specified tamoxifen-related health outcomes, current health, and for a global assessment of possible outcomes of tamoxifen use. Inclusion of a utility for menopausal symptoms in the outcome-specific models substantially increased the ICER; at the threshold 5-year breast cancer risk of 1.67%, tamoxifen was dominated. When a global utility for tamoxifen was used in place of outcome-specific utilities, tamoxifen was dominated under all circumstances. CEAs may be profoundly affected by the types of outcomes considered for quality-of-life adjustment and how these outcomes are grouped for utility assessment. Comparisons of ICERs across analyses must consider effects of different approaches to using utilities for quality-of-life adjustment.


Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats.
Puerarin, a major isoflavonoid derived from the Chinese medical herb Radix puerariae (kudzu root), has been reported to be useful in the treatment of various cardiovascular diseases. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of puerarin on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. Treatment of puerarin (25 and 50 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia in rats. Administration of puerarin at 50 mg/kg, showed marked reduction in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor-1alpha (HIF-1alpha), inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions as well as the mRNA expression of tumor necrosis factor-alpha (TNF-alpha) in ischemic regions. These expressions were markedly inhibited by the treatment of puerarin (50 mg/kg). In addition, puerarin (10-50 microM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by formyl-Met-Leu-Phe. On the other hand, puerarin (20-500 microM) did not significantly inhibit the thiobarbituric acid-reactive substance reaction in rat brain homogenates. An electron spin resonance (ESR) method was conducted on the scavenging activity of puerarin on the free radicals formed. Puerarin (200 and 500 microM) did not reduce the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate that puerarin is a potent neuroprotective agent on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1alpha and TNF-alpha activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, puerarin treatment may represent a novel approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.


Evaluation of the short term 12 hour toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of Venustaconcha ellipsiformis and Epioblasma triquetra and its host fish (Percina caprodes).
The present study evaluated the risk of 12-h exposures of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of the federally endangered snuffbox (Epioblasma triquetra) and its primary host fish the common logperch (Percina caprodes) as well as a surrogate to the snuffbox, the ellipse (Venustaconcha ellipsiformis). Life stages examined included free glochidia, 1-wk juveniles, and adults of the ellipse; free glochidia, glochidia on host fish, and 1-wk juveniles of the snuffbox; and adult logperch. Larval sea lampreys were also tested alongside adult ellipse and logperch for direct comparison. Survival exceeded 82% among all life stages in both mussel species at levels up to 1.8 times what would be applied during treatments, suggesting that routine sea lamprey control operations would not adversely affect mussels. However, substantial mortality of adult logperch was observed at TFM concentrations typically applied to streams, and loss of host fish could adversely affect snuffbox reproduction. In addition, TFM had no significant effect on the number of glochidia that metamorphosed on adult logperch. Although the snuffbox is not likely to be acutely affected from sea lamprey control operations, mitigation efforts to minimize impacts to the host fish should be considered.


Rapid cost-effective subtyping of meticillin-resistant Staphylococcus aureus by denaturing HPLC.
The importance of meticillin-resistant Staphylococcus aureus (MRSA) in hospital-acquired infection is widely acknowledged. The UK government has stated that MRSA bloodstream infection rates will have to be halved by 2008. Such radical improvements will require advances on several fronts. Screening for MRSA in high-risk patients on arrival at hospital allows isolation of carriers and reduces transmission to staff and other patients. Concurrent subtyping of MRSA could also inform outbreak investigations and long-term epidemiological studies. The variability within the staphylococcal protein A, or spaA, gene-repeat region can be used as a marker of short- and long-term genetic variation. A novel application is described of denaturing HPLC (DHPLC) for rapid, inexpensive characterization of spaA gene amplification products, without the need for DNA sequence determination. The method allowed rapid and precise sizing of spaA gene-repeat regions from 99 S. aureus strains and was combined with heteroduplex analysis, using reference PCR products, to indicate the spa type of the test isolate. The method allowed subtyping of strains in less than 5 h from receipt of a primary isolation plate. When applied to an outbreak that occurred during this study, the authors were able to demonstrate relatedness of the isolates more than 5 days before results were received from a reference laboratory. If combined with direct amplification from swabs, DHPLC analysis of spaA gene variation could prove extremely valuable in outbreak investigation and MRSA surveillance.


Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy.
Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-β- and S1P-induced fibroblast differentiation. S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-β increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-β-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1(+/-)) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.


Current challenges in adherence to clinical guidelines for antibiotic prophylaxis in surgery.
To study the impact of guidelines on surgical antibiotic prophylaxis in clinical practice, barriers involved in adherence to guidelines and how to overcome the same. Literature pertaining to prophylactic antibiotic usage was searched. Medscape, Medline, Cochrane, Surgical Infection Prevention (SIP) project databases were reviewed. Recent articles from relevant journals, texts, and standard guidelines were also studied. Local guidelines seem more likely to be accepted and followed than those developed nationally. Major barriers involved in adherence to guidelines include lack of awareness about the guidelines, general perception of guideline as a bureaucratic rather than educational tool. Some practitioners perceive guidelines as "cookbook medicine" that does not permit them to make their own medical decisions. Other barriers are complex, multi-step systems that create confusion, decrease accountability. Methods for guideline adherence include surveillance and data analysis, new systems to facilitate documentation and improving workflow, education regarding current evidence-based guidelines and promoting the development of local guidelines or protocol, development and implementation of reminders to facilitate adherence to the local guidelines. A multidisciplinary steering team of surgeons, infectious disease specialists, pharmacists, anesthesiologists, microbiologists and nurses should develop local guidelines suitable to their institution and methods for adherence to prevent the surgical site infections. The gap between evidence-based guidelines and practice must be addressed in order to achieve optimal practice in this domain.


Psychometric validation of the identity abuse scale among LGBTQ individuals.
Identity abuse (IA) comprises a set of abuse tactics that exploit discriminatory systems including homophobia, biphobia, and transphobia (Tesch & Berkerian, 2015). This study examined the factorial validity of the IA Scale (Woulfe & Goodman, 2018) with a large independent sample of lesbian, gay, bisexual, transgender, and queer (LGBTQ) individuals. Participants included 1,049 LGBTQ-identified participants (Mage = 27.3, 71.9% White, 52.6% cisgender women, and 18.7% as other nonheterosexual identity in their sexual orientation), recruited through listservs. Participants completed an online survey measuring past-year and adult exposure to identity, physical, and psychological abuse. Confirmatory factor analysis indicated that the measurement model had good fit to the data, and strong factor loadings were found across the seven items, confirming a unidimensional factor structure. Findings demonstrate the IA Scale's validity and reliability, supporting its use to assess the frequency of IA tactics experienced within intimate partnerships among LGBTQ individuals.


Early-generation germplasm introgression from Sorghum macrospermum into sorghum (S. bicolor).
Sorghum has been improved by public and private breeding programs utilizing germplasm mostly from within the species Sorghum bicolor. Recently, hybridization with an Australian species, S. macrospermum (AAB1B1YYZZ), has been demonstrated and the genomic relationship to S. bicolor (AAB1B1) shown to be partially compatible. For this species to be potentially useful to sorghum improvement programs, there must be documented introgression into an S. bicolor background. Fifteen BC1F1 progeny were recovered using the interspecific hybrid as a female and embryo rescue. In these progeny, chromosome numbers ranged from 35 to 70 and all were essentially male-sterile. Repeated backcrossing with S. bicolor pollen produced BC2F1 seed on 3 of the 15 BC1F1 plants. BC2F1 progeny had varying levels of male fertility; selfed seed set ranged from 0% to 95%, with only 2 individuals being completely male-sterile. Using AFLP and SSR markers, genomic introgression of S. macrospermum ranged from 0% to 18.6%. Cytogenetic analysis of 19 individuals revealed that chromosome numbers were 20, except for a single backcross that had 21 chromosomes. Molecular cytogenetic analysis confirmed the presence of recombinant introgression chromosomes as well as alien addition and alien substitution chromosomes within the BC2F1s.


Massage and touch therapy in neonates: the current evidence.
Infant massage was first introduced in China in 2nd century BC. Massaging the newborn has been a tradition in India and other Asian countries since time immemorial. Various oil-based preparations have been used depending on the regional availability. There has been a recent surge in this ancient art particularly as a therapy among parents and professionals in the Western world. Evidence exists supporting the benefits of touch and massage therapy. We reviewed the literature to look at the various techniques of providing massage, its benefits, possible mechanism of action and adverse effects. The review suggests that massage has several positive effects in terms of weight gain, better sleep-wake pattern, enhanced neuromotor development, better emotional bonding, reduced rates of nosocomial infection and thereby, reduced mortality in the hospitalized patients. Many studies have described the technique and frequency of this procedure. Massage was found to be more useful when some kind of lubricant oil was used. Harmful effects like physical injury and increased risk of infection were encountered when performed inappropriately. The review also discusses the different hypotheses put forward regarding the mechanism of action. As of now there are very few studies describing the long term impact of neonatal massage.


On the influence of baseline startle reactivity on the indexation of prepulse inhibition.
Prepulse inhibition (PPI) of the startle reflex refers to the reduction of the reflexive startle response to an intense pulse stimulus when its presentation is shortly preceded by a weak prepulse stimulus. PPI is considered as a cross-species translational model of sensorimotor gating, and deficient PPI has been reported in a number of neuropsychiatric disorders. Although a part of the literature is based on the assumption that PPI is independent of the baseline startle reaction, there is accumulating evidence (Csomor et al., 2006; Sandner & Canal, 2007; Yee, Chang, Pietropaolo, & Feldon, 2005) that argues against such an independency. The authors systematically investigated whether PPI indexed as percentage or difference score is dependent on the magnitude of baseline startle reactivity in healthy human volunteers and in C57BL/6 mice. The results revealed that both indexations of PPI were affected by the magnitude of the baseline startle. The authors highlight the pitfalls of different methods to index PPI, especially when startle reactivity differs considerably between groups under comparison, and offer practical recommendations to satisfactorily deal with such baseline differences.


Mental health research and evaluation in multicultural Australia: developing a culture of inclusion.
Cultural and linguistic diversity is a core feature of the Australian population and a valued element of national identity. The proportion of the population that will be overseas-born is projected to be 32% by 2050. While a very active process of mental health system reform has been occurring for more than two decades - at national and state and territory levels - the challenges presented by cultural and linguistic diversity have not been effectively met. A key area in which this is particularly an issue is in the collection, analysis and reporting of mental health data that reflect the reality of population diversity. The purpose of this study was to examine: what is known about the mental health of immigrant and refugee communities in Australia; whether Australian mental health research pays adequate attention to the fact of cultural and linguistic diversity in the Australian population; and whether national mental health data collections support evidence-informed mental health policy and practice and mental health reform in multicultural Australia. The study consisted of three components - a brief review of what is known about mental health in, and mental health service use by, immigrant and refugee communities; an examination of national data collections to determine the extent to which relevant cultural variables are included in the collections; and an examination of Australian research to determine the extent to which immigrant and refugee communities are included as participants in such research. The review of Australian research on mental health of immigrant and refugee communities and their patterns of mental health service use generated findings that are highly variable. The work is fragmented and usually small-scale. There are multiple studies of some immigrant and refugee communities and there are no studies of others. Although there is a broadly consistent pattern of lower rates of utilisation of specialist public mental health services by immigrants and refugees the absence of adequate population epidemiological data prevents judgments about whether the observed patterns constitute under-utilisation. There are virtually no data on quality of service outcomes. The examination of national data collections revealed multiple gaps in these data collections. The review of papers published in four key Australian journals to determine whether immigrants and refugees are included in mental health research studies revealed a high rate (9.1%) of specific exclusion from studies (usually due to low English fluency) and a much higher rate of general neglect of the issue of population diversity in study design and reporting. While there are many positive statements of policy intent in relation to immigrant and refugee communities in national mental health policies and strategies there is virtually no reporting by Commonwealth or State and Territory governments of whether policies that are relevant to immigrant and refugee communities are effectively implemented. It is not possible, on the basis of the data collected, to determine whether immigrant and refugee communities are benefiting from the mental health system reforms that are being actively carried out. The majority of Australian mental health research does not adequately include immigrant and refugee samples. On the basis of the findings of this study eight strategies have been recommended that will contribute to the development of a culture of inclusion of all Australians in the national mental health research enterprise.


A unique model for SDH-deficient GIST: an endocrine-related cancer.
We describe a unique patient-derived xenograft (PDX) and cell culture model of succinate dehydrogenase-deficient gastrointestinal stromal tumor (SDH-deficient GIST), a rare mesenchymal tumor that can occur in association with paragangliomas in hereditary and non-hereditary syndromes. This model is potentially important for what it might reveal specifically pertinent to this rare tumor type and, more broadly, to other types of SDH-deficient tumors. The primary tumor and xenografts show a very high proliferative fraction, and distinctive morphology characterized by tiny cells with marked autophagic activity. It is likely that these characteristics resulted from the combination of the germline SDHB mutation and a somatic KRAS G12D mutation. The most broadly relevant findings to date concern oxygen and oxidative stress. In paragangliomas harboring SDHx mutations, both hypoxic signaling and oxidative stress are putative drivers of tumor growth. However, there are no models for SDH-deficient paragangliomas. This related model is the first from a SDHB-mutated human tumor that can be experimentally manipulated to study mechanisms of oxygen effects and novel treatment strategies. Our data suggest that tumor growth and survival require a balance between protective effects of hypoxic signaling vs deleterious effects of oxidative stress. While reduced oxygen concentration promotes tumor cell survival, a further survival benefit is achieved with antioxidants. This suggests potential use of drugs that increase oxidative stress as novel therapies. In addition, autophagy, which has not been reported as a major finding in any type of SDH-deficient tumor, is a potential target of agents that might trigger autophagic cell death.


C-Type Natriuretic Peptide Analogue Therapy in Children with Achondroplasia.
Achondroplasia is a genetic disorder that inhibits endochondral ossification, resulting in disproportionate short stature and clinically significant medical complications. Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification. In a multinational, phase 2, dose-finding study and extension study, we evaluated the safety and side-effect profile of vosoritide in children (5 to 14 years of age) with achondroplasia. A total of 35 children were enrolled in four sequential cohorts to receive vosoritide at a once-daily subcutaneous dose of 2.5 μg per kilogram of body weight (8 patients in cohort 1), 7.5 μg per kilogram (8 patients in cohort 2), 15.0 μg per kilogram (10 patients in cohort 3), or 30.0 μg per kilogram (9 patients in cohort 4). After 6 months, the dose in cohort 1 was increased to 7.5 μg per kilogram and then to 15.0 μg per kilogram, and in cohort 2, the dose was increased to 15.0 μg per kilogram; the patients in cohorts 3 and 4 continued to receive their initial doses. At the time of data cutoff, the 24-month dose-finding study had been completed, and 30 patients had been enrolled in an ongoing long-term extension study; the median duration of follow-up across both studies was 42 months. During the treatment periods in the dose-finding and extension studies, adverse events occurred in 35 of 35 patients (100%), and serious adverse events occurred in 4 of 35 patients (11%). Therapy was discontinued in 6 patients (in 1 because of an adverse event). During the first 6 months of treatment, a dose-dependent increase in the annualized growth velocity was observed with vosoritide up to a dose of 15.0 μg per kilogram, and a sustained increase in the annualized growth velocity was observed at doses of 15.0 and 30.0 μg per kilogram for up to 42 months. In children with achondroplasia, once-daily subcutaneous administration of vosoritide was associated with a side-effect profile that appeared generally mild. Treatment resulted in a sustained increase in the annualized growth velocity for up to 42 months. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov numbers, NCT01603095, NCT02055157, and NCT02724228.).


Fiberless, Multi-Channel fNIRS-EEG System Based on Silicon Photomultipliers: Towards Sensitive and Ecological Mapping of Brain Activity and Neurovascular Coupling.
Portable neuroimaging technologies can be employed for long-term monitoring of neurophysiological and neuropathological states. Functional Near-Infrared Spectroscopy (fNIRS) and Electroencephalography (EEG) are highly suited for such a purpose. Their multimodal integration allows the evaluation of hemodynamic and electrical brain activity together with neurovascular coupling. An innovative fNIRS-EEG system is here presented. The system integrated a novel continuous-wave fNIRS component and a modified commercial EEG device. fNIRS probing relied on fiberless technology based on light emitting diodes and silicon photomultipliers (SiPMs). SiPMs are sensitive semiconductor detectors, whose large detection area maximizes photon harvesting from the scalp and overcomes limitations of fiberless technology. To optimize the signal-to-noise ratio and avoid fNIRS-EEG interference, a digital lock-in was implemented for fNIRS signal acquisition. A benchtop characterization of the fNIRS component showed its high performances with a noise equivalent power below 1 pW. Moreover, the fNIRS-EEG device was tested in vivo during tasks stimulating visual, motor and pre-frontal cortices. Finally, the capabilities to perform ecological recordings were assessed in clinical settings on one Alzheimer's Disease patient during long-lasting cognitive tests. The system can pave the way to portable technologies for accurate evaluation of multimodal brain activity, allowing their extensive employment in ecological environments and clinical practice.


Charcoal hemoperfusion in an infant with supraventricular tachycardia and seizures secondary to amitriptyline intoxication.
Tricyclic antidepressant (TCA) overdose is one of the common causes of drug poisoning and it has cardiovascular, respiratory and neurological side effects. An 18-month male infant was admitted to our pediatric emergency service due to poisoning with amitriptyline. The infant was unconscious. Tachycardia, irregular and shallow breathing, and tonic-clonic seizures were observed on physical examination. An electrocardiogram displayed a narrow complex tachycardia that was consistent with re-entrant supraventricular tachycardia (SVT). Although antiarrhythmic and anticonvulsive agents were administrated, SVT and seizures persisted. Charcoal hemoperfusion (HP) was performed for 4 hours. The infant's clinical condition has improved after the charcoal HP, seizures and SVT were not observed. It is concluded that charcoal HP can be used efficiently in patients with severe amitriptyline intoxication.


Difficulties in organizing first indoor spray programme against malaria in Angola under the President's Malaria Initiative.
Successful attempts to control malaria require understanding of its complex transmission patterns. Unfortunately malaria transmission in Africa is often assessed using routine administrative reports from local health units, which are plagued by sporadic reporting failures. In addition, the lack of microscopic analyses of blood slides in these units introduces the effects of many confounding diseases. The danger of using administrative reports was illustrated in Angola, the first country in which malaria control was attempted under the President's Malaria Initiative, a development programme of the Government of the United States of America. Each local health unit submitted monthly reports indicating the number of suspected malaria cases to their municipality. The identification of the disease was based on clinical diagnoses, without microscopic examination of blood slides. The municipal and provincial reports were then passed on to the national headquarters, with sporadic reporting lapses at all levels. After the control effort was completed, the defective municipal reports were corrected by summarizing only the data from those health units which had submitted reports for every month during the evaluation period. The corrected data, supplemented by additional observations on rainfall and mosquito habitats, indicated that there had probably been no malaria transmission before starting the control operations. Thus the expensive malaria control effort had been wasted. It is unfortunate that WHO is also trying to plan and evaluate its malaria control efforts based on these same kinds of inadequate administrative reports.


Population-based analysis of prognostic factors and survival in familial melanoma.
Familial melanoma patients are reported to present with thinner melanomas, to be younger at the time of diagnosis, and to have a greater likelihood of developing multiple primary tumors. We sought to determine whether melanomas that occur in a familial setting demonstrate different prognostic and survival statistics relative to sporadic melanoma. This population-based study used the Utah Cancer Registry and Utah Population Database to objectively evaluate prognostic and survival statistics of the familial melanoma population. From 1973 to 1999, there were 7,785 cases of invasive melanoma identified through the Utah Cancer Registry. These were linked to the Utah Population Database, resulting in 2,659 subjects with family-history information from which a familiality score could be calculated. Cases scored in the top ninth percentile were assigned as high familial risk, and the remaining 91% were considered low familial risk. Multivariate logistic-regression analysis found no association between sex, Breslow depth, Clark level, or survival and the familial status. Age at first diagnosis of invasive melanoma was slightly lower in the high-familial-risk group (57 v 60 years; P = .03). High-familial-risk subjects had more melanomas diagnosed at age 30 or younger (12% v 6%; P < .001). A significant difference in the overall number of individuals with two or more primary malignant melanomas was not detected among the groups (P = .2). These data suggest that melanomas occurring in the context of an underlying inherited susceptibility do not have a significantly different biologic behavior.


Stallion semen quality depends on major histocompatibility complex matching to teaser mare.
The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm numbers as compared to exposure to an MHC-similar mare. Sperm velocity seemed not affected by the treatment. Overall genetic similarity between stallions and mares (determined from polymorphic microsatellites on 20 different chromosomes) played no significant role here. The MHC type of the teaser mare also affected characteristics of cold-stored sperm after 24 and 48 hr. As expected from ejaculate economics, sperm viability was elevated after exposure to an MHC-dissimilar mare. However, oxidative stress and the percentage of sperm with a high DNA fragmentation were mostly increased after exposure to an MHC-dissimilar mare, depending also on whether the teaser mare was in oestrous or not. We conclude that males can quickly adjust ejaculate quality relative to a female's MHC, and that this male reaction to the social environment can also affect important characteristics of cold-stored semen.


When seeing doesn't matter: assessing the after-effects of tactile distractor processing in the blind and the sighted.
Negative priming (NP) refers to the finding that people's responses to probe targets previously presented as prime distractors are usually slower than to unrepeated stimuli. Intriguingly, the effect sizes of tactile NP were much larger than the effect sizes for visual NP. We analyzed whether the large tactile NP effect is just a side effect of the higher difficulty when processing tactile compared to visual stimuli. Thus, we analyzed tactile NP in a sample of blind participants and in a control sample of sighted participants. Although the blind participants handled the tactile stimuli with ease, we found no evidence that the size of the tactile NP effect diminished. In two control experiments with sighted participants, we varied the processing difficulty in the visual and tactile modality and found that both modality and processing difficulty had an effect on the size of NP. Taken together, our data show that the difficulty associated with processing tactile stimuli is only partially the reason for the unusual large tactile NP effect. These results suggest that non-spatial tactile distractors are processed and selected quite differently from visual distractors.


Pharmacokinetics and tissue distribution of the renin inhibitor N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-butylami ne in marmosets.
The fate of CGP 38 560 [N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-bu tylamine], a potent renin inhibitor, has been studied in marmosets. [3H]CGP 38 560 is rapidly cleared from the plasma. The elimination process is biphasic, with a t1/2 of 4.8 +/- 1.0 min (mean +/- SD) in the first phase and 26.6 +/- 8.4 min (mean +/- SD) in the second. The kinetics of elimination from plasma are similar when measured both in a radio-inhibitor binding assay and radiometrically using 3H-labeled substance. The drug is mainly eliminated in the bile, almost 73.8% of the i.v. administered dose being excreted within the first 60 min. It is detectable in bile in both unchanged (8.6%) and metabolized form. HPLC analysis of bile extracts showed at least five tritiated peaks representing constituents capable of binding human renin (A, B, C, D, and E). These fractions were isolated, purified, and analyzed by mass spectrometry. Peak E corresponded to unchanged CGP 38 560. Metabolites A, B, C, and D are more polar than the parent compound, as indicated by their retention times upon HPLC analysis. The metabolic pathways inferable from the respective molecular weights are hydroxylation, oxygenation, and, in one case, cleavage of the n-butylamino group located at the COOH-terminal. From comparisons of the pharmacokinetic parameters after iv (0.1 mg/kg) and oral (10 mg/kg) administration, it can be estimated that the bioavailability of CGP 38 560 in the marmoset is 0.3%.(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis.
A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.


Effects of dietary vitamin E on muscle vitamin E and fatty acid content in Aohan fine-wool sheep.
Increasing the polyunsaturated fatty acid (PUFA) content and decreasing the saturated fatty acid (SFA) content of mutton can help to improve its nutritional value for consumers. Several laboratories have evaluated the effects of vitamin E on the fatty acid (FA) composition of muscle in sheep. However, little information is available on wool sheep, even though wool sheep breeds are an important source of mutton, especially in northern China where sheep are extensively farmed. The present study was designed to address the effects of vitamin E on muscle FA composition in male Aohan fine-wool sheep. Forty-two male Aohan fine-wool lambs (5 mo old) with similar initial body weight were randomly divided into seven groups and fed diets supplemented with 0 (control group), 20, 100, 200, 1,000, 2,000, or 2,400 IU/sheep/d vitamin E for 12 mo. Three lambs from each group were slaughtered to measure vitamin E and FA content in the longissimus lumborum (LL) and gluteus medius (GM) muscles. Vitamin E concentrations in the LL and GM increased significantly after 12 mo of vitamin E supplementation (P < 0.05). However, this increase did not occur in a dose-dependent manner because the muscle vitamin E concentration was highest in the 200 IU/sheep/d group. Dietary vitamin E supplementation also caused a significant reduction in SFA content and an increase in monounsaturated FA (MUFA) content in the LL and GM (P < 0.05). All six doses of vitamin E significantly increased cis9 trans11-conjugated linoleic acid (c9t11-CLA) content in the LL compared with the control group (P < 0.05). Dietary supplementation with vitamin E increased muscle vitamin E content and improved the nutritional value of mutton by decreasing SFA content and increasing MUFA and c9t11-CLA contents in Aohan fine-wool sheep. These effects were greatest in sheep fed a diet containing 200 IU/sheep/d vitamin E.


Identifying Candidate Genes for Enhancing Grain Zn Concentration in Wheat.
Wheat (Triticum aestivum L.) is one of the major staple food crops worldwide. Despite efforts in improving wheat quality, micronutrient levels are still below the optimal range for human nutrition. In particular, zinc (Zn) deficiency is a widespread problem in human nutrition in countries relying mainly on a cereal diet; hence improving Zn accumulation in grains is an imperative need. This study was designed to understand the genetic architecture of Zn grain concentrations in wheat grains. We performed a genome-wide association study (GWAS) for grain Zn concentrations in 369 European wheat genotypes, using field data from 3 years. The complete wheat panel was genotyped by high-density arrays of single nucleotide polymorphic (SNP) markers (90k iSELECT Infinium and 35k Affymetrix arrays) resulting in 15,523 polymorphic markers. Additionally, a subpanel of 183 genotypes was analyzed with a novel 135k Affymetrix marker array resulting in 28,710 polymorphic SNPs for high-resolution mapping of the potential genomic regions. The mean grain Zn concentration of the genotypes ranged from 25.05-52.67 μg g-1 dry weight across years with a moderate heritability value. Notably, 40 marker-trait associations (MTAs) were detected in the complete panel of varieties on chromosomes 2A, 3A, 3B, 4A, 4D, 5A, 5B, 5D, 6D, 7A, 7B, and 7D. The number of MTAs in the subpanel was increased to 161 MTAs whereas the most significant and consistent associations were located on chromosomes 3B (723,504,241-723,611,488 bp) and 5A (462,763,758-466,582,184 bp) having major effects. These genomic regions include newly identified putative candidate genes, which are related to Zn uptake and transport or represent bZIP and mitogen-activated protein kinase genes. These findings provide the basis for understanding the genetic background of Zn concentration in wheat grains that in turn may help breeders to select high Zn-containing genotypes to improve human health and grain quality.


Bounding Bias Due to Selection.
When epidemiologic studies are conducted in a subset of the population, selection bias can threaten the validity of causal inference. This bias can occur whether or not that selected population is the target population and can occur even in the absence of exposure-outcome confounding. However, it is often difficult to quantify the extent of selection bias, and sensitivity analysis can be challenging to undertake and to understand. In this article, we demonstrate that the magnitude of the bias due to selection can be bounded by simple expressions defined by parameters characterizing the relationships between unmeasured factor(s) responsible for the bias and the measured variables. No functional form assumptions are necessary about those unmeasured factors. Using knowledge about the selection mechanism, researchers can account for the possible extent of selection bias by specifying the size of the parameters in the bounds. We also show that the bounds, which differ depending on the target population, result in summary measures that can be used to calculate the minimum magnitude of the parameters required to shift a risk ratio to the null. The summary measure can be used to determine the overall strength of selection that would be necessary to explain away a result. We then show that the bounds and summary measures can be simplified in certain contexts or with certain assumptions. Using examples with varying selection mechanisms, we also demonstrate how researchers can implement these simple sensitivity analyses. See video abstract at, http://links.lww.com/EDE/B535.


Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water.
Water-protein interactions play a central role in protein structure, dynamics, and function. These interactions, traditionally, have been studied using nuclear magnetic resonance (NMR) by measuring chemical exchange and nuclear Overhauser effect (NOE). Polarization transferred from hyperpolarized water can result in substantial transient signal enhancements of protein resonances due to these processes. Here, we use dissolution dynamic nuclear polarization and flow-NMR for measuring the pH dependence of transferred signals to the protein trypsin. A maximum enhancement of 20 is visible in the amide proton region of the spectrum at pH 6.0, and of 47 at pH 7.5. The aliphatic region is enhanced up to 2.3 times at pH 6.0 and up to 2.5 times at pH 7.5. The time dependence of these observed signals can be modeled quantitatively using rate equations incorporating chemical exchange to amide sites and, optionally, intramolecular NOE to aliphatic protons. On the basis of these two- and three-site models, average exchange (kex) and cross-relaxation rates (σ) obtained were kex = 12 s-1, σ = -0.33 s-1 for pH 7.5 and kex = 1.8 s-1, σ = -0.72 s-1 for pH 6.0 at a temperature of 304 K. These values were validated using conventional EXSY and NOESY measurements. In general, a rapid measurement of exchange and cross-relaxation rates may be of interest for the study of structural changes of the protein occurring on the same time scale. Besides protein-water interactions, interactions with cosolvent or solutes can further be investigated using the same methods.


What can global health institutions do to help strengthen health systems in low income countries?
Weaknesses in health systems contribute to a failure to improve health outcomes in developing countries, despite increased official development assistance. Changes in the demands on health systems, as well as their scope to respond, mean that the situation is likely to become more problematic in the future. Diverse global initiatives seek to strengthen health systems, but progress will require better coordination between them, use of strategies based on the best available evidence obtained especially from evaluation of large scale programs, and improved global aid architecture that supports these processes. This paper sets out the case for global leadership to support health systems investments and help ensure the synergies between vertical and horizontal programs that are essential for effective functioning of health systems. At national level, it is essential to increase capacity to manage and deliver services, situate interventions firmly within national strategies, ensure effective implementation, and co-ordinate external support with local resources. Health systems performance should be monitored, with clear lines of accountability, and reforms should build on evidence of what works in what circumstances.


Lead toxicity among traffic wardens: a high risk group exposed to atmospheric lead, is it still a cause for concern?
Traffic policemen are identified to be at a higher risk of exposure to air pollution and its contaminants such as lead. A study done prior to the introduction of unleaded petroleum in Sri Lanka revealed a mean blood lead level of 53.07 μg/dL, which was well above the Center for Disease Control defined acceptable safe levels. This study aimed to determine whether unleading of fuel has made an impact on the blood lead levels of traffic police working in an urban area with high traffic density. A cross-sectional survey of 168 traffic police personnel working within Colombo city limits of Sri Lanka, a high traffic density area, was conducted. Blood lead levels of participants were measured using nitric acid, perchloric acid ashing method and atomic absorption spectrophotometry. An interviewer administered questionnaire was used for a targeted history and examination. Mean age of the sample population was 37 years. Thirty eight percent had detectable levels of lead in blood and 24.4% of the study sample had blood lead levels above Centre for disease control defined safe limits. Sample mean was 4.82 μg/dL (95% CI 3.58-6.04), and this is a 91% overall reduction when compared to data prior to unleading. Neither symptoms nor signs of classic lead toxicity showed significant correlation with toxic lead levels. Lead poisoning though still present in the high risk traffic warden population shows a considerable reduction following unleading. The need to have a low threshold to suspect lead poisoning is highlighted by the non-specific nature of the symptoms and signs of lead poisoning and its lack of association even in those found to have elevated lead levels. Further studies are required to elucidate a cause for the prevalence of lead poisoning despite cessation of using lead as an additive in petroleum.


Association of blood lead (Pb) and plasma homocysteine: a cross sectional survey in Karachi, Pakistan.
High blood lead (Pb) and hyperhomocysteinemia have been found to be associated with cardiovascular disease (CVD). Mean blood Pb and mean plasma homocysteine levels have been reported to be high in Pakistani population. The objective of the present study was to assess the relationship of blood Pb to the risk of hyperhomocysteinemia in a low income urban population of Karachi, Pakistan. In a cross sectional survey, 872 healthy adults (355 males, 517 females; age 18-60 years) were recruited from a low income urban population of Karachi. Fasting venous blood was obtained and assessed for blood Pb and plasma/serum homocysteine, folate, pyridoxal phosphate (PLP, a coenzymic form of vitamin B6) and vitamin B12. The study population had median (IQR) blood Pb of 10.82 microg/dL (8.29-13.60). Prevalence of high blood Pb (levels>10 microg/dL) was higher in males compared to females (62.5% males vs 56% females; p value=0.05). Mean+/-SD/median (IQR) value of plasma homocysteine was significantly higher in the highest quartile of blood Pb compared to the lowest quartile 16.13+/-11.2 micromol/L vs 13.28+/-9.7micromol/L/13.15 (10.33-17.81) micromol/L vs 11.09 (8.65 14.31) micromol/L (p value<0.001). Daily consumption of fruit juice had a positive influence on both levels of plasma homocysteine and blood Pb. Compared with the lowest quartile of blood Pb, the OR for hyperhomocysteinemia was 1.69 (95% CI, 1.00 to 2.85) for the fourth quartile when the model was adjusted for age, gender, folate and vitamin B12. This study showed a relationship between blood Pb and hyperhomocysteinemia in a general population of Karachi, Pakistan. The harmful effect of Pb on cardiovascular system could be due to its association with hyperhomocysteinemia.


Iron(III) protoporphyrin IX complexes of the antimalarial Cinchona alkaloids quinine and quinidine.
The antimalarial properties of the Cinchona alkaloids quinine and quinidine have been known for decades. Surprisingly, 9-epiquinine and 9-epiquinidine are almost inactive. A lack of definitive structural information has precluded a clear understanding of the relationship between molecular structure and biological activity. In the current study, we have determined by single crystal X-ray diffraction the structures of the complexes formed between quinine and quinidine and iron(III) protoporphyrin IX (Fe(III)PPIX). Coordination of the alkaloid to the Fe(III) center is a key feature of both complexes, and further stability is provided by an intramolecular hydrogen bond formed between a propionate side chain of Fe(III)PPIX and the protonated quinuclidine nitrogen atom of either alkaloid. These interactions are believed to be responsible for inhibiting the incorporation of Fe(III)PPIX into crystalline hemozoin during its in vivo detoxification. It is also possible to rationalize the greater activity of quinidine compared to that of quinine.


Challenges in assessing spiritual distress in survivors of cancer.
Many efforts have been made to better integrate spiritual assessment into the care of patients with cancer, with varying degrees of success in different parts of the United States. Little work has been done to describe challenges that face those who seek to implement assessment in busy ambulatory settings, particularly in the northeastern section of the United States. This study sought to test the feasibility of a screening process describing spirituality, distress, and spiritual transformation in cancer survivors after chemotherapy for lung or gastrointestinal cancer.  This descriptive pilot study took place in a rural National Cancer Institute- designated comprehensive cancer center, referral center, and outpatient medical oncology clinic. A web-based questionnaire was completed by 29 survivors, and 22 declined participation.  Respondents were primarily Christian, aged 60 years or older, and an average of 18 months post-diagnosis. The mean spiritual distress score was 1.38 (SD = 2.09), and the mean psychological distress score was 3.03 (SD = 2.73). Participants reported mean spiritual well-being, positive degree of spiritual growth, and little spiritual decline. The opportunity for spiritual growth among survivors creates a need for effective assessment and intervention to promote spiritual growth and mitigate spiritual decline and spiritual distress.


Magnetic resonance measurements of renal blood flow and disease progression in autosomal dominant polycystic kidney disease.
Whether changes in renal blood flow (RBF) are associated with and possibly contribute to cystic disease progression in autosomal dominant polycystic kidney disease (ADPKD) has not been ascertained. The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) was created to develop imaging techniques and analyses to evaluate progression. A total of 131 participants with early ADPKD had measurements of RBF and total kidney (TKV) and cyst (TCV) volumes by magnetic resonance and of GFR by iothalamate clearance at baseline and 1, 2, and 3 yr. The effects of age, gender, body mass index, hypertension status, mean arterial pressure (MAP), TKV, TCV, RBF, renal vascular resistance (RVR), GFR, serum uric acid, HDL and LDL cholesterol, 24-h urine volume, sodium (UNaE) and albumin (UAE) excretions, and estimated protein intake were examined at baseline on TKV, TCV, and GFR slopes. TKV and TCV increased, RBF decreased, and GFR remained stable. TKV, TCV, RVR, serum uric acid, UAE, UNaE, age, body mass index, MAP, and estimated protein intake were positively and RBF and GFR negatively correlated with TKV and TCV slopes. TKV, RBF, UNaE, and UAE were independent predictors of TKV and TCV slopes (structural disease progression). TKV, TCV, RVR, and MAP were negatively and RBF positively correlated with GFR slopes. Regression to the mean confounded the analysis of GFR slopes. TKV and RBF were independent predictors of GFR decline (functional disease progression). In ADPKD, RBF reduction (1) parallels TKV increase, (2) precedes GFR decline, and (3) predicts structural and functional disease progression.


Effect of a Terminated Cover Crop and Aldicarb on Cotton Yield and Meloidogyne incognita Population Density.
Terminated small grain cover crops are valuable in light textured soils to reduce wind and rain erosion and for protection of young cotton seedlings. A three-year study was conducted to determine the impact of terminated small grain winter cover crops, which are hosts for Meloidogyne incognita, on cotton yield, root galling and nematode midseason population density. The small plot test consisted of the cover treatment as the main plots (winter fallow, oats, rye and wheat) and rate of aldicarb applied in-furrow at-plant (0, 0.59 and 0.84 kg a.i./ha) as subplots in a split-plot design with eight replications, arranged in a randomized complete block design. Roots of 10 cotton plants per plot were examined at approximately 35 days after planting. Root galling was affected by aldicarb rate (9.1, 3.8 and 3.4 galls/root system for 0, 0.59 and 0.84 kg aldicarb/ha), but not by cover crop. Soil samples were collected in mid-July and assayed for nematodes. The winter fallow plots had a lower density of M. incognita second-stage juveniles (J2) (transformed to Log(10) (J2 + 1)/500 cm(3) soil) than any of the cover crops (0.88, 1.58, 1.67 and 1.75 Log(10)(J2 + 1)/500 cm(3) soil for winter fallow, oats, rye and wheat, respectively). There were also fewer M. incognita eggs at midseason in the winter fallow (3,512, 7,953, 8,262 and 11,392 eggs/500 cm(3) soil for winter fallow, oats, rye and wheat, respectively). Yield (kg lint per ha) was increased by application of aldicarb (1,544, 1,710 and 1,697 for 0, 0.59 and 0.84 kg aldicarb/ha), but not by any cover crop treatments. These results were consistent over three years. The soil temperature at 15 cm depth, from when soils reached 18 degrees C to termination of the grass cover crop, averaged 9,588, 7,274 and 1,639 centigrade hours (with a minimum threshold of 10 degrees C), in 2005, 2006 and 2007, respectively. Under these conditions, potential reproduction of M. incognita on the cover crop did not result in a yield penalty.


Ethical preparedness and developments in genomic healthcare.
Considerations of the notion of preparedness have come to the fore in the recent pandemic, highlighting a need to be better prepared to deal with sudden, unexpected and unwanted events. However, the concept of preparedness is also important in relation to planned for and desired interventions resulting from healthcare innovations. We describe ethical preparedness as a necessary component for the successful delivery of novel healthcare innovations, and use recent advances in genomic healthcare as an example. We suggest that practitioners and organisations charged with delivering innovative and ambitious healthcare programmes can only succeed if they are able to exhibit the attribute of ethical preparedness.


The effect of 5-fluorouracil on rat adrenal function.
This study was undertaken to determine the effects of 5-fluorouracil on corticosteroidogenesis and adrenal size in the rat. The rats were injected intraperitoneally with 5-FU (12.5 mg kg-1 or 25 mg kg-1), normal saline or actinomycin D (0.02 mg kg-1), daily for 10 days. High-dose 5-FU induces adrenal hyperplasia, in association with mild impairment of corticosteroidogenesis (manifest by lower corticosterone and higher ACTH levels). The response to low-dose 5-FU and actinomycin D is of lesser adrenal hyperplasia, relative to high-dose 5-FU (P less than 0.05) and elevated corticosterone levels. There may be a dose-related effect on the suppression of corticosteroidogenesis in the rat by 5-FU.


Sparing aortic valve techniques.
Prosthetic valve implantation is the most widely used therapeutic option for aortic regurgitation (AR), but complications associated with prosthetic valves, with an incidence of 3-5% patient/year, and the need for anticoagulation in mechanical valves, force us to consider aortic valve repair as an alternative to the prosthetic implant, especially in young people with many potential years of life for these complications. Aortic valve repair techniques are not excessively complicated, but there is no standardization, so they are not reproducible in all medical centers. There are multiple repair procedures, but to study them we can divide, arbitrarily, into two large groups, techniques for treatment of aortic root dilatation (reimplantation and remodeling); and repair techniques without treatment of the aortic root, that act on annulus and on aortic leaflets. The two sets of techniques are often used together, but it is easier to understand them if this arbitrary division is made. Our goal is to publish our experience and knowledge of repair techniques to facilitate their learning by other professionals. In this article, we will focus on the procedures on the aortic annulus and leaflets, because they can be applied in patients without aortic root dilatation, but they are also complementary to the reimplantation and remodeling techniques. We will present the procedures describing them individually, with an explanation in text accompanied by three videos in which the application of each technique is visualized in several cases. The standardization of sparing aortic valve techniques is difficult, because each patient requires its individualized application, yet we have tried to illustrate reproducible procedures so that professionals have more tools to deal with aortic valve-preserving surgery.


A Real-World Study of Dexamethasone Implant in Treatment-Naïve Patients with Diabetic Macular Edema: Efficacy and Correlation Between Inflammatory Biomarkers and Treatment Outcome.
There has been an increasing clinical interest in specific retinal parameters as non-invasive biomarkers of retinal inflammation in diabetic macular edema (DME) that have been shown to have prognostic value, such as hyperreflective retinal fields (HRFs) and subfoveal neuroretinal detachment (SND). We conducted a prospective, non-comparative study of treatment-naïve patients with DME to evaluate the efficacy of a Pro Re Nata (PRN) regimen of intravitreal dexamethasone implant 0.7 mg (DexI, Ozurdex™). After administration, patients underwent subsequent injections according to PRN criteria in case of edema relapse, but not earlier than 4 months after the previous treatment. Patients were evaluated at baseline, within 15 days of injection, and every month thereafter. During all visits, best-corrected visual acuity (BCVA) was recorded; central retinal thickness (CRT), type of edema, presence of SND, and presence and number of HRFs were evaluated using swept-source optical coherence tomography (SS-OCT) 3D. Treatment outcome was defined as changes in BCVA, CRT, SND and HRFs at 12 (T12) and 24 (T24) months compared with baseline (T0). The study enrolled 24 eyes of 18 patients. The mean duration of follow-up was 18±6.6 months; for all eyes, T12 data were available, while follow-up reached T24 for 12 eyes. BCVA improved significantly and CRT decreased significantly during treatment; the edema was no longer detectable in 13/24 eyes at T12 and 8/12 eyes at T24. No patient presented SND at T12 and T24, and the mean number of HRFs decreased significantly during treatment. Results with CRT and HRFs correlated with BCVA at 12 and 24 months. No significant adverse events were observed. In patients with DME, the intravitreal dexamethasone implant was effective and safe in improving both functional and tomographic parameters. This result is consistent with improvement in biomarkers of inflammation.


Genetic mapping of the female mimic morph locus in the ruff.
Ruffs (Aves: Philomachus pugnax) possess a genetic polymorphism for male mating behaviour resulting in three permanent alternative male reproductive morphs: (i) territorial 'Independents', (ii) non-territorial 'Satellites', and (iii) female-mimicking 'Faeders'. Development into independent or satellite morphs has previously been shown to be due to a single-locus, two-allele autosomal Mendelian mode of inheritance at the Satellite locus. Here, we use linkage analysis to map the chromosomal location of the Faeder locus, which controls development into the Faeder morph, and draw further conclusions about candidate genes, assuming shared synteny with other birds. Segregation data on the Faeder locus were obtained from captive-bred pedigrees comprising 64 multi-generation families (N = 381). There was no evidence that the Faeder locus was linked to the Satellite locus, but it was linked with microsatellite marker Ppu020. Comparative mapping of ruff microsatellite markers against the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) genomes places the Ppu020 and Faeder loci on a region of chromosome 11 that includes the Melanocortin-1 receptor (MC1R) gene, which regulates colour polymorphisms in numerous birds and other vertebrates. Melanin-based colouration varies with life-history strategies in ruffs and other species, thus the MC1R gene is a strong candidate to play a role in alternative male morph determination. Two unlinked loci appear to control behavioural development in ruffs. The Faeder locus is linked to Ppu020, which, assuming synteny, is located on avian chromosome 11. MC1R is a candidate gene involved in alternative male morph determination in ruffs.


Prognostic factors of lung cancer in lymphoma survivors (the LuCiLyS study).
Second cancer is the leading cause of death in lymphoma survivors, with lung cancer representing the most common solid tumor. Limited information exists about the treatment and prognosis of second lung cancer following lymphoma. Herein, we evaluated the outcome and prognostic factors of Lung Cancer in Lymphoma Survivors (the LuCiLyS study) to improve the patient selection for lung cancer treatment. This is a retrospective multicentre study including consecutive patients treated for lymphoma disease that subsequently developed non-small cell lung cancer (NSCLC). Data regarding lymphoma including age, symptoms, histology, disease stage, treatment received and lymphoma status at the time of lung cancer diagnosis, and data on lung carcinoma as age, smoking history, latency from lymphoma, symptoms, histology, disease stage, treatment received, and survival were evaluated to identify the significant prognostic factors for overall survival. Our study population included 164 patients, 145 of which underwent lung cancer resection. The median overall survival was 63 (range, 58-85) months, and the 5-year survival rate 54%. At univariable analysis no-active lymphoma (HR: 2.19; P=0.0152); early lymphoma stage (HR: 1.95; P=0.01); adenocarcinoma histology (HR: 0.59; P=0.0421); early lung cancer stage (HR: 3.18; P<0.0001); incidental diagnosis of lung cancer (HR: 1.71; P<0.0001); and lung cancer resection (HR: 2.79; P<0.0001) were favorable prognostic factors. At multivariable analysis, no-active lymphoma (HR: 2.68; P=0.004); early lung cancer stage (HR: 2.37; P<0.0001); incidental diagnosis of lung cancer (HR: 2.00; P<0.0001); and lung cancer resection (HR: 2.07; P<0.0001) remained favorable prognostic factors. Patients with non-active lymphoma (n=146) versus those with active lymphoma (n=18) at lung cancer diagnosis presented better median survival (64 vs. 37 months; HR: 2.4; P=0.02), but median lung cancer specific survival showed no significant difference (27 vs. 19 months; HR: 0.3; P=0.17). The presence and/or a history of lymphoma should not be a contraindication to resection of lung cancer. Inclusion of lymphoma survivors in a lung cancer-screening program may lead to early detection of lung cancer, and improve the survival.


Adjusting Linking Strands to Form Size-Controllable DNA Origami Rings.
DNA origami is a powerful tool in nanotechnology that can be used to construct arbitrary structures for several nanoengineering applications. Generally, the more complex and sophisticated the construction, the greater is the number of origamis and connection strands that will be needed. Therefore, developing an effective and low-cost method for multiform DNA architecture is important in nanoengineering. Here, we adopted an oblique linking strategy to connect cross-shaped DNA origami with a controlled curing angle. The size of the DNA rings ranged from four blocks of approximately 200 nm to eleven blocks of c.a. 600 nm. We observed that the minimum size of the DNA ring structure was limited by the width of a single block. The largest rings were negatively affected by thermodynamic randomness, and thus, DNA rings consisting of more than eleven blocks were not observed. This strategy facilitates the generation of various DNA origami rings, whose size can be controlled by adjusting the length of the connection strands.


Enrichment of a set of microRNAs during the cotton fiber development.
Cotton (Gossypium hirsutum) is one of the most important economic crops and provides excellent fibers for textile manufacture. In addition to its industrial and agricultural importance, the fiber cell (plant trichome) also is a biological model system for exploring gene expression and regulation. Small RNAs regulate many aspects of plant growth and development. However, whether small RNAs are involved in regulation of fiber cell development is unknown. We adopted a deep sequencing approach developed by Solexa (Illumina Inc.) to investigate global expression and complexity of small RNAs during cotton fiber initiation and development. We constructed two small RNA libraries prepared from wild type (WT) and fuzz/lintless (fl Mutant in the WT background) cotton ovules, respectively. Each library was sequenced individually and generated more than 6-7 million short sequences, resulting in a total of over 13 million sequence reads. At least 22 conserved candidate miRNA families including 111 members were identified. Seven families make up the vast majority of expressed miRNAs in developing cotton ovules. In total 120 unique target genes were predicted for most of conserved miRNAs. In addition, we identified 2 cell-type-specific novel miRNA candidates in cotton ovules. Our study has demonstrated significant differences in expression abundance of miRNAs between the wild-type and mutant, and suggests that these differentially expressed miRNAs potentially regulate transcripts distinctly involved in cotton fiber development. The present study is the first to deep sequence the small RNA population of G. hirsutum ovules where cotton fibers initiate and develop. Millions of unique miRNA sequences ranging from 18 to approximately 28 nt in length were detected. Our results support the importance of miRNAs in regulating the development of different cell types and indicate that identification of a comprehensive set of miRNAs in cotton fiber cells would facilitate our understanding of the regulatory mechanisms for fiber cell initiation and elongation.


Antibodies to the M(r) 64,000 (64K) protein in islet cell antibody positive non-diabetic individuals indicate high risk for impaired beta-cell function.
A prospective study of a normal childhood population identified 44 islet cell antibody positive individuals. These subjects were typed for HLA DR and DQ alleles and investigated for the presence of antibodies to the M(r) 64,000 (64K) islet cell antigen, complement-fixing islet cell antibodies and radiobinding insulin autoantibodies to determine their potency in detecting subjects with impaired Beta-cell function. At initial testing 64K antibodies were found in six of 44 islet cell antibody positive subjects (13.6%). The same sera were also positive for complement-fixing islet cell antibodies and five of them had insulin autoantibodies. During the follow-up at 18 months, islet cell antibodies remained detectable in 50% of the subjects studied. In all six cases who were originally positive, 64K antibodies were persistently detectable, whereas complement-fixing islet cell antibodies became negative in two of six and insulin autoantibodies in one of five individuals. HLA DR4 (p less than 0.005) and absence of asparic acid (Asp) at position 57 of the HLA DQ beta chain (p less than 0.05) were significantly increased in subjects with 64K antibodies compared with control subjects. Of 40 individuals tested in the intravenous glucose tolerance test, three had a first phase insulin response below the first percentile of normal control subjects. Two children developed Type 1 (insulin-dependent) diabetes mellitus after 18 and 26 months, respectively. Each of these subjects was non-Asp homozygous and had persistent islet cell and 64K antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)


Impact of the palliative care clinical nurse specialist on patients and relatives: a stakeholder evaluation.
Within the last decade, there has been a rapid expansion in the United Kingdom of clinical nurse specialists (CNSs), yet there has been limited evidence of their effectiveness. The clinical area of palliative care has one of the largest numbers of CNSs which has particularly started to escalate in the acute hospital setting. Following the Calman-Hine Report and the more recent NHS Cancer Plan, it is anticipated that the number of CNSs in palliative and cancer care is likely to grow. In light of the current policy emphasis of Clinical Governance and Clinical Effectiveness, there is an urgent need to demonstrate the contribution that CNSs make. This paper presents the qualitative findings of an evaluation study on the impact of the CNS within a palliative care team in a large acute hospital in the North-West of England. A stakeholder evaluation that encompassed tape-recorded semi-structured interviews to explore how the stakeholders saw the impact of the CNS within palliative care was performed. Twenty-seven interviews were conducted with stakeholders across the hospital setting including senior nurses, consultants, junior doctors and nurses representing the different grades. Data were analysed for emerging themes utilizing a case and cross-case analysis. The results indicated that the CNSs within palliative care in an acute hospital are seen to be potentially beneficial for both patients and relatives. This paper will focus on exploring these perceived benefits, which included symptom control, psychological care and being an advocate for patients and relatives.


Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.
The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.