Purification of a primase activity associated with DNA polymerase alpha from HeLa cells.
Highly purified preparations of eukaryotic DNA polymerase alpha have been shown to contain primase activity (Kaguni, L.S., Rossignol, J-M., Conaway, R.C. Banks, G.R., and Lehman, I.R. (1983) J. Biol. Chem. 258, 9037-9039; Yagura, T., Kozu, T., and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127; Shioda, M., Nelson, E.M., Bayne, M.L., and Benbow, R.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7209-7213). We have investigated the de novo synthesis of DNA by a primase-DNA polymerase alpha preparation isolated from human HeLa cells using the synthetic homopolymers poly(dT) and poly(dC) as templates. In the presence of poly(dT), synthesis of poly(dA) required ATP in addition to dATP while synthesis of poly(dG) in the presence of poly(dC) required GTP in addition to dGTP. The primase activity required a much lower GTP concentration (Km = 0.1 mM) than ATP (Km = 0.8 mM) for the synthesis of DNA. Guanosine 5'-O-(3-thiotriphosphate), 5'-guanylyl-beta, gamma-imidodiphosphate, and 5'-guanylyl methylenediphosphonate substituted for GTP but the corresponding ATP analogues did not substitute for ATP. Furthermore, ATP and ATP analogues inhibited the GTP-dependent reaction while GTP and GTP analogues inhibited the ATP-dependent reaction. DNase treatment of products labeled with [alpha-32P] GTP revealed that an RNA oligomer was covalently linked to newly synthesized DNA. Alkaline hydrolysis of these products yielded GMP and pppGp, indicating that the primer was initiated with GTP. Alkaline hydrolysis of [alpha-32P]dGTP-labeled products yielded 2'- and 3'-GMP showing that DNA chains are covalently linked to the 3' ends of RNA chains. The primase activity could not be separated from DNA polymerase alpha through a 200-fold enrichment involving phosphocellulose, DNA-cellulose, hydroxylapatite, DEAE-cellulose and glycerol gradient purification steps. However, primase activity was found to be less stable than DNA polymerase alpha activity under a variety of conditions.


Encoding difficulty and memory enhancement for young and older readers.
Memory for text that required active construction (generation) on the part of the reader to comprehend some of the information was examined. (Letters were deleted from the words composing some of the idea units of the text). For both high school students and older adults, idea units that had letters deleted were remembered better than idea units with intact words on a cued-recall test. This pattern held for easier as well as for harder letter deletions. It was concluded that retention of textually presented information can be improved in older adults if the presentation format of the text induces or guides mental operations that support good encoding. Production-deficiency accounts and attentional/resource deficiency accounts of age-related text memory decrements are discussed in light of the present results.


A note on the estimation of relative risks of rare genetic susceptibility markers.
The comparison of an incident case series with an incident series of second primary cancers, using either a case-control or follow-up study design, is proposed as an efficient method for evaluating the relative risk of a rare genetic susceptibility marker and its prevalence in the population, and for evaluating gene-environment interactions. The relative efficiency of this design versus a conventional case-control study is highly dependent on the population prevalence of the marker and its relative risk. However, for relatively rare but highly penetrant genes, the relative efficiency can be very high. In an example presented regarding a planned study of the p16 gene and its role in melanoma, a conventional case-control study may require up to 70 times as many subjects to achieve equivalent precision to the study of second primaries. The use of second primary cancers in this way requires assumptions about the validity of the classification of a new tumor as a second primary, the extent to which risk of a second cancer is influenced by treatment of the first cancer, and the nature and extent of surveillance bias. However, the problems of ascertaining a valid series of population controls are avoided. The study of second cancers represents an important and underused tool in molecular and genetic epidemiology.


Efficient Gaussian process regression for large datasets.
Gaussian processes are widely used in nonparametric regression, classification and spatiotemporal modelling, facilitated in part by a rich literature on their theoretical properties. However, one of their practical limitations is expensive computation, typically on the order of n3 where n is the number of data points, in performing the necessary matrix inversions. For large datasets, storage and processing also lead to computational bottlenecks, and numerical stability of the estimates and predicted values degrades with increasing n. Various methods have been proposed to address these problems, including predictive processes in spatial data analysis and the subset-of-regressors technique in machine learning. The idea underlying these approaches is to use a subset of the data, but this raises questions concerning sensitivity to the choice of subset and limitations in estimating fine-scale structure in regions that are not well covered by the subset. Motivated by the literature on compressive sensing, we propose an alternative approach that involves linear projection of all the data points onto a lower-dimensional subspace. We demonstrate the superiority of this approach from a theoretical perspective and through simulated and real data examples.


Does Metabolic Syndrome Increase the Risk of Postoperative Complications in Patients Undergoing Colorectal Cancer Surgery?
Metabolic syndrome is associated with poorer postoperative outcomes after various abdominal operations. However, the impact of metabolic syndrome on outcomes after colorectal cancer surgery remains poorly described. The purpose of this study was to determine the association between metabolic syndrome and short-term postoperative outcomes in patients undergoing elective colorectal cancer surgery. This was a retrospective cohort study. This study used a national multicenter database. Adult patients who underwent elective colectomy for colorectal cancer from 2010 to 2016 were identified in the American College of Surgeons National Surgical Quality Improvement Program database. Thirty-day postoperative mortality and morbidity, unplanned reoperation, unplanned readmission, operative time, and length of stay were measured. A total of 91,566 patients were analyzed; 7603 (8.3%) had metabolic syndrome. On unadjusted analysis, metabolic syndrome was associated with an increased risk of 30-day overall morbidity, pulmonary complications, renal complications, septic complications, cardiac complications, wound complications, blood transfusion, longer length of stay, and unplanned readmissions. On multivariable analysis, metabolic syndrome remained significantly associated with renal complications (OR = 1.44 (95% CI, 1.29-1.60)), superficial surgical site infection (OR = 1.46 (95% CI, 1.32-1.60)), deep surgical site infection (OR = 1.40 (95% CI, 1.15-1.70)), wound dehiscence (OR = 1.47 (95% CI, 1.20-1.80)), and unplanned readmissions (HR = 1.24 (95% CI, 1.15-1.34)). The risks of overall morbidity, cardiac and septic complications, and prolonged length of stay for laparoscopic procedures were significantly associated with diabetes mellitus rather than metabolic syndrome as a composite entity. This study was limited by its retrospective design and inability to analyze outcomes beyond 30 days. Patients with metabolic syndrome undergoing elective surgery for colorectal cancer have an increased risk of 30-day postoperative renal complications, wound complications, and unplanned hospital readmissions. A multidisciplinary approach involving lifestyle modifications and pharmacologic interventions to improve the components of metabolic syndrome should be implemented preoperatively for these patients. See Video Abstract at http://links.lww.com/DCR/A909.


Utilizing Interactive Surfaces to Enhance Learning, Collaboration and Engagement: Insights from Learners' Gaze and Speech.
Interactive displays are becoming increasingly popular in informal learning environments as an educational technology for improving students' learning and enhancing their engagement. Interactive displays have the potential to reinforce and maintain collaboration and rich-interaction with the content in a natural and engaging manner. Despite the increased prevalence of interactive displays for learning, there is limited knowledge about how students collaborate in informal settings and how their collaboration around the interactive surfaces influences their learning and engagement. We present a dual eye-tracking study, involving 36 participants, a two-staged within-group experiment was conducted following single-group time series design, involving repeated measurement of participants' gaze, voice, game-logs and learning gain tests. Various correlation, regression and covariance analyses employed to investigate students' collaboration, engagement and learning gains during the activity. The results show that collaboratively, pairs who have high gaze similarity have high learning outcomes. Individually, participants spending high proportions of time in acquiring the complementary information from images and textual parts of the learning material attain high learning outcomes. Moreover, the results show that the speech could be an interesting covariate while analyzing the relation between the gaze variables and the learning gains (and task-based performance). We also show that the gaze is an effective proxy to cognitive mechanisms underlying collaboration not only in formal settings but also in informal learning scenarios.


UKMenCar4: A cross-sectional survey of asymptomatic meningococcal carriage amongst UK adolescents at a period of low invasive meningococcal disease incidence.
Carriage of Neisseria meningitidis, the meningococcus, is a prerequisite for invasive meningococcal disease (IMD), a potentially devastating infection that disproportionately afflicts infants and children. Humans are the sole known reservoir for the meningococcus, and it is carried asymptomatically in the nasopharynx of ~10% of the population. Rates of carriage are dependent on age of the host and social and behavioural factors. In the UK, meningococcal carriage has been studied through large, multi-centre carriage surveys of adolescents in 1999, 2000, and 2001, demonstrating carriage can be affected by immunisation with the capsular group C meningococcal conjugate vaccine, inducing population immunity against carriage. Fifteen years after these surveys were carried out, invasive meningococcal disease incidence had declined from a peak in 1999. The UKMenCar4 study was conducted in 2014/15 to investigate rates of carriage amongst the adolescent population during a period of low disease incidence. The protocols and methodology used to perform UKMenCar4, a large carriage survey, are described here.


Climate variability and trends at a national scale.
Climate variability and trends have significant environmental and socioeconomic impacts. Global challenges such as food security, biodiversity loss, water scarcity and human health are affected by reference evapotranspiration, temperature, solar radiation, and precipitation together, but nonlinear dynamics of these four climatic factors have not been assessed simultaneously at the national scale. This leads to unclear climatic dynamics and limited applications. To address this knowledge gap, we analyzed the daily variability and trends of four climatic factors (reference evapotranspiration, temperature, solar radiation, and precipitation) in China simultaneously using high spatial resolution data from 1960 to 2013. The results indicate that the daily variability of climate system dynamics (quantified by multiplying fractal dimensions of the four climatic factors) in north China was higher than that in south China. For example, the climate system dynamics were more chaotic and with higher nonlinear variation in north China, most notably in Heilongjiang Province, the major grain base of China, posing threats to food security in the context of growing national population. Spatial distribution of variability varies among different climatic factors. Our study highlights the need for a more holistic study of climate variability and trends in other countries with multiple climate types to address challenges of sustainable development.


β-Elemene Reverses Chemoresistance of Breast Cancer Cells by Reducing Resistance Transmission via Exosomes.
Currently, exosomes that act as mediators of intercellular communication are being researched extensively. Our previous studies confirmed that these exosomes contain microRNAs (miRNAs) that could alter chemo-susceptibility, which is partly attributed to the successful intercellular transfer of multidrug resistance (MDR)-specific miRNAs. We also confirmed that β-elemene could influence MDR-related miRNA expression and regulate the expression of the target genes PTEN and Pgp, which may lead to the reversal of the chemoresistant breast cancer (BCA) cells. We are the first to report these findings, and we propose the following logical hypothesis: β-elemene can mediate MDR-related miRNA expression in cells, thereby affecting the exosome contents, reducing chemoresistance transmission via exosomes, and reversing the drug resistance of breast cancer cells. MTT-cytotoxic, miRNA microarray, real-time quantitative PCR, Dual Luciferase Activity Assay, and Western blot analysis were performed to investigate the impact of β-elemene on the expression of chemoresistance specific miRNA and PTEN as well as Pgp in chemoresistant BCA exosomes. Drug resistance can be reversed by β-elemene related to exosomes. There were 104 differentially expressed miRNAs in the exosomes of two chemoresistant BCA cells: adriacin (Adr) - resistant MCF-7 cells (MCF-7/Adr) and docetaxel (Doc) - resistant MCF-7 cells (MCF-7/Doc) that underwent treatment. Of these, 31 miRNAs were correlated with the constant changes in the MDR. The expression of miR-34a and miR-452 can lead to changes in the characteristics of two chemoresistant BCA exosomes: MCF-7/Adr exosomes (A/exo) and MCF-7/Doc exosomes (D/exo). The PTEN expression affected by β-elemene was significantly increased, and the Pgp expression affected by β-elemene was significantly decreased in both cells and exosomes. β-elemene induced a significant increase in the apoptosis rate in both MCF-7/Doc and MCF-7/Adr cells. Drug resistance can be reversed by β-elemene, which can alter the expression of some MDR-related miRNAs, including PTEN and Pgp in MCF-7/Adr and MCF-7/Doc in cells. It can therefore affect the exosome contents and induce the reduction of resistance transmission via exosomes.


BEST: a novel computational approach for comparing gene expression patterns from early stages of Drosophila melanogaster development.
Embryonic gene expression patterns are an indispensable part of modern developmental biology. Currently, investigators must visually inspect numerous images containing embryonic expression patterns to identify spatially similar patterns for inferring potential genetic interactions. The lack of a computational approach to identify pattern similarities is an impediment to advancement in developmental biology research because of the rapidly increasing amount of available embryonic gene expression data. Therefore, we have developed computational approaches to automate the comparison of gene expression patterns contained in images of early stage Drosophila melanogaster embryos (prior to the beginning of germ-band elongation); similarities and differences in gene expression patterns in these early stages have extensive developmental effects. Here we describe a basic expression search tool (BEST) to retrieve best matching expression patterns for a given query expression pattern and a computational device for gene interaction inference using gene expression pattern images and information on the associated genotypes and probes. Analysis of a prototype collection of Drosophila gene expression pattern images is presented to demonstrate the utility of these methods in identifying biologically meaningful matches and inferring gene interactions by direct image content analysis. In particular, the use of BEST searches for gene expression patterns is akin to that of BLAST searches for finding similar sequences. These computational developmental biology methodologies are likely to make the great wealth of embryonic gene expression pattern data easily accessible and to accelerate the discovery of developmental networks.


Cuff Pressure Pain Detection Is Associated with Both Sex and Physical Activity Level in Nonathletic Healthy Subjects.
The aim of this study was to evaluate pressure pain sensitivity on leg and arm in 98 healthy persons (50 women) using cuff algometry. Furthermore, associations with sex and physical activity level were investigated. Normal physical activity level was defined as Godin Leisure-Time Exercise Questionnaire (GLTEQ) score ≤ 45 and high activity level as GLTEQ > 45. A pneumatic double-chamber cuff was placed around the arm or leg where a single chamber was inflated. The cuff inflation rate (1 kPa/s) was constant, and pain intensity was registered continuously on a 10 cm electronic visual analogue scale (VAS). The pain detection threshold (PDT) was defined as when the pressure was perceived as painful, and pain tolerance (PTT) was when the subject terminated the cuff inflation. For PTT, the corresponding VAS score was recorded (VAS-PTT). The protocol was repeated with two chambers inflated. Only single cuff results are given. For women compared with men, the PDT was lower when assessed in the arm ( P = 0.002), PTTs were lower in the arm and leg ( P < 0.001), and the VAS-PTT was higher in the arm and leg ( P < 0.033). Highly active participants compared with less active had higher PDT ( P = 0.027) in the leg. Women showed facilitated spatial summation ( P < 0.014) in the arm and leg and a steeper VAS slope (i.e., the slope of the VAS pressure curve between PDT and PPT) in the arm and leg ( P < 0.003). This study indicates that reduced pressure pain sensitivity is associated both with male sex and physical activity level.


Multisensory processing in children with autism: high-density electrical mapping of auditory-somatosensory integration.
Successful integration of signals from the various sensory systems is crucial for normal sensory-perceptual functioning, allowing for the perception of coherent objects rather than a disconnected cluster of fragmented features. Several prominent theories of autism suggest that automatic integration is impaired in this population, but there have been few empirical tests of this thesis. A standard electrophysiological metric of multisensory integration (MSI) was used to test the integrity of auditory-somatosensory integration in children with autism (N=17, aged 6-16 years), compared to age- and IQ-matched typically developing (TD) children. High-density electrophysiology was recorded while participants were presented with either auditory or somatosensory stimuli alone (unisensory conditions), or as a combined auditory-somatosensory stimulus (multisensory condition), in randomized order. Participants watched a silent movie during testing, ignoring concurrent stimulation. Significant differences between neural responses to the multisensory auditory-somatosensory stimulus and the unisensory stimuli (the sum of the responses to the auditory and somatosensory stimuli when presented alone) served as the dependent measure. The data revealed group differences in the integration of auditory and somatosensory information that appeared at around 175 ms, and were characterized by the presence of MSI for the TD but not the autism spectrum disorder (ASD) children. Overall, MSI was less extensive in the ASD group. These findings are discussed within the framework of current knowledge of MSI in typical development as well as in relation to theories of ASD.


Study of low temperature chlorine atom initiated oxidation of methyl and ethyl butyrate using synchrotron photoionization TOF-mass spectrometry.
The initial oxidation products of methyl butyrate (MB) and ethyl butyrate (EB) are studied using a time- and energy-resolved photoionization mass spectrometer. Reactions are initiated with Cl˙ radicals in an excess of oxygen at a temperature of 550 K and a pressure of 6 Torr. Ethyl crotonate is the sole isomeric product that is observed from concerted HO2-elimination from initial alkylperoxy radicals formed in the oxidation of EB. Analysis of the potential energy surface of each possible alkylperoxy radical shows that the CH3CH(OO)CH2C([double bond, length as m-dash]O)OCH2CH3 (RγO2) and CH3CH2CH(OO)C([double bond, length as m-dash]O)OCH2CH3 (RβO2) radicals are the isomers that could undergo this concerted HO2-elimination. Two lower-mass products (formaldehyde and acetaldehyde) are observed in both methyl and ethyl butyrate reactions. Secondary reactions of alkylperoxy radicals with HO2 radicals can decompose into the aforementioned products and smaller radicals. These pathways are the likely explanation for the formation of formaldehyde and acetaldehyde.


RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5.
The Golgi-associated RAB GTPases, RAB6A and RAB6A', regulate anterograde and retrograde transport pathways from and to the Golgi. In vitro, RAB6A/A' control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described in vivo Here, we generated BlgCre; Rab6aF/F mice presenting a specific deletion of Rab6a in the mammary luminal secretory lineage during gestation and lactation. Rab6a loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon Rab6a deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A' in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A' depend on cell-type specialization and tissue context.


Chytrid rhizoid morphogenesis resembles hyphal development in multicellular fungi and is adaptive to resource availability.
Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor.


Nerve growth factor infusion into the denervated adult rat hippocampal formation promotes its cholinergic reinnervation.
The well-documented but little-understood failure of lengthy axonal regeneration after injury of the adult mammalian CNS may be caused by an insufficient availability of local growth-promoting factors. If so, identifying and supplying the missing factors may result in better central axonal regeneration. This hypothesis was tested in an adult rat CNS model in which peripheral nerve grafts were placed into a lesion cavity between the septum and hippocampal formation. Continuous infusion of nerve growth factor (NGF) into the dorsal hippocampal tissue dramatically enhanced and accelerated the regrowth and penetration of cholinergic axons into the hippocampal formation. Thus, NGF can overcome the apparent resistance of the hippocampal CNS tissue to cholinergic reinnervation.


Pruritus in adult burn survivors: postburn prevalence and risk factors associated with increased intensity.
Pruritus (itching) is a common and distressing complaint after injury. The purpose of this study was to investigate self-reported postburn pruritus in a large, multisite cohort study of adult burn survivors. Descriptive statistics, general linear regression, and mixed model repeated measures analyses were employed to test statistical significance. Two cohorts of adult burn survivors were studied. Group 1 participants (n = 637) were injured from 2006 to 2010 and were followed up prospectively for 2 years from the time of injury. Prevalence and severity of pruritus were compared across multiple subgroups. Prevalence of pruritus at discharge, 6, 12, and 24 months following injury were 93, 86, 83, and 73%, respectively. Regression results established that %TBSA-burn and %TBSA-grafted were correlated to itch intensity values. Group 2 participants (n = 336) were injured 4 to 10 years before an assessment using the validated 5-D Itch Scale. Many patients (44.4%) reported itching in the area of the burn, graft, or donor site. Within this group, 76% reported itching for <6 hours/day, and 52 and 29% considered itch intensity to be mild or moderate, respectively. This study confirms that the prevalence of burn pruritus is high, initially affecting >90% and persisting for >40% of long-term burn survivors. New predictors for postburn itch were identified to include younger age, dry skin, and raised/thick scars. Characterization of the impact of postburn pruritus on leisure, vocation, and sleep are quantified for those long-term survivors suffering from postburn pruritus.


Clinical and neuroradiological differences of paediatric acute disseminating encephalomyelitis with and without antibodies to the myelin oligodendrocyte glycoprotein.
Myelin oligodendrocyte glycoprotein (MOG) antibodies have been recently described in children with acute disseminating encephalomyelitis (ADEM), but the clinical and neuroradiological characterisation of this subgroup is lacking. To compare the clinical and neuroradiological features of paediatric ADEM with and without MOG antibodies. Clinical course, cerebrospinal fluid (CSF)-, MRI studies, outcome and MOG status of 33 paediatric ADEM prospectively studied were reviewed. MOG antibodies (median 1:2560; range 1:160-1:20 480) were detected in 19 children with ADEM. The majority of children showed a decline of serum MOG-IgG titres over time. Children with MOG antibodies did not differ in their age at presentation, sex ratio, the presence of oligoclonal bands, clinical symptoms or initial severity, apart from a higher CSF cell count (p=0.038), compared with children without MOG antibodies. In addition, further relapsing demyelinating episodes associated with MOG antibodies were observed only in children with MOG antibodies. All 19 children with MOG antibodies had a uniform MRI pattern, characterised by large, hazy and bilateral lesions and the absence of atypical MRI features (eg, mainly small lesions, well-defined lesions), which was significantly different compared to that of children without MOG antibodies (p=0.003; and p=0.032, respectively). In addition, children with MOG antibodies had involvement of more anatomical areas (p=0.035) including the myelon characterised by a longitudinally extensive transverse myelitis (p=0.003), more often a complete resolution of lesions (p=0.036) and a better outcome (p=0.038). Patients with ADEM with MOG antibodies in our cohort had a uniform MRI characterised by large, bilateral and widespread lesions with an increased frequency of longitudinal extensive transverse myelitis and a favourable clinical outcome in contrast to children lacking MOG antibodies.


Two developmental modules establish 3D beak-shape variation in Darwin's finches.
Bird beaks display tremendous variation in shape and size, which is closely associated with the exploitation of multiple ecological niches and likely played a key role in the diversification of thousands of avian species. Previous studies have demonstrated some of the molecular mechanisms that regulate morphogenesis of the prenasal cartilage, which forms the initial beak skeleton. However, much of the beak diversity in birds depends on variation in the premaxillary bone. It forms later in development and becomes the most prominent functional and structural component of the adult upper beak/jaw, yet its regulation is unknown. Here, we studied a group of Darwin's finch species with different beak shapes. We found that TGFβIIr, β-catenin, and Dickkopf-3, the top candidate genes from a cDNA microarray screen, are differentially expressed in the developing premaxillary bone of embryos of species with different beak shapes. Furthermore, our functional experiments demonstrate that these molecules form a regulatory network governing the morphology of the premaxillary bone, which differs from the network controlling the prenasal cartilage, but has the same species-specific domains of expression. These results offer potential mechanisms that may explain how the tightly coupled depth and width dimensions can evolve independently. The two-module program of development involving independent regulating molecules offers unique insights into how different developmental pathways may be modified and combined to induce multidimensional shifts in beak morphology. Similar modularity in development may characterize complex traits in other organisms to a greater extent than is currently appreciated.


An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq.
Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.


Mobile phone use and risk of glioma in 5 North European countries.
Public concern has been expressed about the possible adverse health effects of mobile telephones, mainly related to intracranial tumors. We conducted a population-based case-control study to investigate the relationship between mobile phone use and risk of glioma among 1,522 glioma patients and 3,301 controls. We found no evidence of increased risk of glioma related to regular mobile phone use (odds ratio, OR = 0.78, 95% confidence interval, CI: 0.68, 0.91). No significant association was found across categories with duration of use, years since first use, cumulative number of calls or cumulative hours of use. When the linear trend was examined, the OR for cumulative hours of mobile phone use was 1.006 (1.002, 1.010) per 100 hr, but no such relationship was found for the years of use or the number of calls. We found no increased risks when analogue and digital phones were analyzed separately. For more than 10 years of mobile phone use reported on the side of the head where the tumor was located, an increased OR of borderline statistical significance (OR = 1.39, 95% CI 1.01, 1.92, p trend 0.04) was found, whereas similar use on the opposite side of the head resulted in an OR of 0.98 (95% CI 0.71, 1.37). Although our results overall do not indicate an increased risk of glioma in relation to mobile phone use, the possible risk in the most heavily exposed part of the brain with long-term use needs to be explored further before firm conclusions can be drawn.


Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease: A Randomized, Single-Blind, Controlled Trial.
Current penile traction therapy devices have significant limitations, including the need to use them for 3 to 8 hours per day. Given these issues, the novel RestoreX® penile traction therapy device was developed in cooperation with Mayo Clinic. We performed a randomized, controlled, single-blind, intent to treat trial (ClinicalTrials.gov NCT03389854) in men with Peyronie's disease who were assigned to the penile traction therapy device for 30 to 90 minutes per day or to no therapy for 3 months. Study inclusion criteria were curvature 30 degrees or greater with no study exclusions due to complex curvature, hourglass deformity, prior Peyronie's disease therapy or surgery, or calcification. The primary outcome was safety and secondary outcomes were penile length and curvature, and questionnaire responses. A total of 110 men were randomized 3:1 to the penile traction therapy group or the control group. The cohorts were well matched with a mean age of 58.4 years, a 49.7-month Peyronie's disease history and 59.3 degree curvature. Overall penile traction therapy was well tolerated with only transient and mild adverse events reported. At 3 months men undergoing penile traction therapy demonstrated significant improvements over controls in penile length (1.5 vs 0 cm, p <0.001), curvature (-11.7 vs 1.3 degrees, p <0.01) and erectile function (4.3 vs -0.7, p = 0.01) according to the International Index of Erectile Function-Erectile Function among men with erectile dysfunction. Overall 77% of the men on penile traction therapy experienced improved curvature (mean -17.2 degrees in 28.2% of responders) while 94% achieved increased length (1.6 cm in 10.9%). Counter bending and the white line indicator improved efficacy, validating key device innovations. Of men who had previously used other penile traction therapy devices 100% preferred the RestoreX device. Penile traction therapy with the RestoreX device for 30 to 90 minutes per day was safe, and resulted in significant and clinically meaningful improvements in penile curvature and length in men with Peyronie's disease, and in erectile function in men with erectile dysfunction and Peyronie's disease with no significant adverse events. To our knowledge these data represent the only reported improvements for any device used less than 3 to 8 hours per day.


Development and validation of a universal blood donor genotyping platform: a multinational prospective study.
Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.


Identification of new mechanisms of cellular response to chemotherapy by tracking changes in post-translational modifications by ubiquitin and ubiquitin-like proteins.
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive malignancy characterized by an excessive resistance to all known anticancer therapies, a still largely elusive phenomenon. To identify original mechanisms, we have explored the role of post-translational modifications (PTMs) mediated by members of the ubiquitin family. Although alterations of these pathways have been reported in different cancers, no methodical search for these kinds of anomalies has been performed so far. Therefore, we studied the ubiquitin-, Nedd8-, and SUMO1-specific proteomes of a pancreatic cancer cell line (MiaPaCa-2) and identified changes induced by gemcitabine, the standard PDAC's chemotherapeutic drug. These PTMs profiles contained both known major substrates of all three modifiers as well as original ones. Gemcitabine treatment altered the PTM profile of proteins involved in various biological functions, some known cancer associated genes, many potentially cancer-associated genes, and several cancer-signaling networks, including canonical and noncanonical WNT and PI3K/Akt/MTOR pathways. Some of these altered PTMs formed groups of functionally and physically associated proteins. Importantly, we could validate the gemcitabine-induced PTMs variations of relevant candidates and we could demonstrate the biological significance of such altered PTMs by studying in detail the sumoylation of SNIP1, one of these new targets.


Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood-Brain Barrier.
Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation. Stem Cells 2018;36:1816-12.


The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human.
We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor-specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.


Factors Contributing to 50-ft Walking Speed and Observed Ethnic Differences in Older Community-Dwelling Mexican Americans and European Americans.
Mexican Americans comprise the most rapidly growing segment of the older US population and are reported to have poorer functional health than European Americans, but few studies have examined factors contributing to ethnic differences in walking speed between Mexican Americans and European Americans. The purpose of this study was to examine factors that contribute to walking speed and observed ethnic differences in walking speed in older Mexican Americans and European Americans using the disablement process model (DPM) as a guide. This was an observational, cross-sectional study. Participants were 703 Mexican American and European American older adults (aged 65 years and older) who completed the baseline examination of the San Antonio Longitudinal Study of Aging (SALSA). Hierarchical regression models were performed to identify the contribution of contextual, lifestyle/anthropometric, disease, and impairment variables to walking speed and to ethnic differences in walking speed. The ethic difference in unadjusted mean walking speed (Mexican Americans=1.17 m/s, European Americans=1.29 m/s) was fully explained by adjustment for contextual (ie, age, sex, education, income) and lifestyle/anthropometric (ie, body mass index, height, physical activity) variables; adjusted mean walking speed in both ethnic groups was 1.23 m/s. Contextual variables explained 20.3% of the variance in walking speed, and lifestyle/anthropometric variables explained an additional 8.4%. Diseases (ie, diabetes, stroke, chronic obstructive pulmonary disease) explained an additional 1.9% of the variance in walking speed; impairments (ie, FEV1, upper leg pain, and lower extremity strength and range of motion) contributed an additional 5.5%. Thus, both nonmodifiable (ie, contextual, height) and modifiable (ie, impairments, body mass index, physical activity) factors contributed to walking speed in older Mexican Americans and European Americans. The study was conducted in a single geographic area and included only Mexican American Hispanic individuals. Walking speed in older Mexican Americans and European Americans is influenced by modifiable and nonmodifiable factors, underscoring the importance of the DPM framework, which incorporates both factors into the physical therapist patient/client management process.


Comparative evaluation of 3 microbond strength tests using 4 adhesive systems: Mechanical, finite element, and failure analysis.
Bond strength (BS) values from in vitro studies are useful when dentists are selecting an adhesive system, but there is no ideal measuring method. The purpose of this in vitro study was to investigate the influence of the evaluation method in the BS between dentin and composite resin. Molars with exposed superficial dentin (N=240) were divided into 3 groups according to the test: microtensile (μTBS), microshear (μSBS), and micropush-out (μPBS). Each one was subdivided into 4 groups according to the adhesive system: total etch, 3- and 2-step; and self-etch, 2- and 1-step). For the μPBS test, a conical cavity was prepared and restored with composite resin. An occlusal slice (1.5 mm in thickness) was obtained from each tooth. For the μSBS test, a composite resin cylinder (1 mm in diameter) was built on the dentin surface of each tooth. For the μTBS test, a 2-increment composite resin cylinder was built on the dentin surface, and beams with a sectional area of 0.5 mm2 were obtained. Each subgroup was divided into 2 (n=10) as the specimens were tested after 7 days and 1 year of water storage. The specimens were submitted to load, and the failure recorded in units of megapascals. Original BS values from the μTBS and μSBS tests were normalized for the area from μPBS specimens. Original and normalized results were submitted to a 3-way ANOVA (α=.05). The correlation among mechanical results, stress distribution, and failure pattern was investigated. Significant differences (P<.05) were found among the adhesive systems and methods within both the original and normalized data but not between the storage times (P>.05). Within the 7 days of storage, the original BS values from μTBS were significantly higher (P<.001) than those from μPBS and μSBS. After 1 year, μSBS presented significantly lower results (P<.001). However, after the normalization for area, the BS values of the μTBS and μPBS tests were similar, and both were higher (P<.001) than that of μSBS in both storage times. In the μSBS and μTBS specimens, cohesive and adhesive failures were observed, whereas μPBS presented 100% of adhesive failures. The failure modes were compatible with the stress distribution. The storage time did not affect the results, but differences were found among the adhesives and methods. For comparisons of bond strength from tests with different bonding areas, the normalization for area seemed essential. The microshear bond test should not be used for bond strength evaluation, and the microtensile test needs improvement to enable reliable results regarding stress concentration and failure mode. The micropush-out test may be considered more reliable than the microtensile in the bond strength investigation, as demonstrated by the uniform stress concentration and adhesive failure pattern.


Timing parameter optimization for comparison experiments of TSIM.
The optimization problem of an absolute radiometer's timing parameters is investigated for comparison experiments of Total Solar Irradiance Monitor (TSIM). Comparison experiments were performed to establish the TSIM's contact with other space radiometers for measuring total solar irradiance (TSI). Since the comparison experiments had to be performed on a tight schedule under the impact of weather conditions, the measurement parameters for the comparison experiments were selected carefully using optimized solutions of timing parameters. The optimized solutions were identified by a genetic algorithm (GA) based on a thermal model of an absolute radiometer. The thermal model includes terms of heat radiation, air conduction, etc. Fitness value function and constraints of GA are constructed using the thermal model. The experimental results indicate that the selected measurement parameters are sufficient to implement accurate calibration of TSI, providing more opportunities for solar observation.


Human immunodeficiency virus type 1-infected individuals make autoantibodies that bind to CD43 on normal thymic lymphocytes.
Sera from human immunodeficiency virus type 1 (HIV-1)-infected and -noninfected individuals were screened for antibodies that could bind to native T cell differentiation antigens. Antibodies that could immunoprecipitate CD43 (sialophorin, leukosialin) from a T cell lymphoma line were detected in sera from 27% of patients, and antibodies that could bind specifically to transfected cells expressing CD43 were detected in 47% of patients. The anti-CD43 antibodies were related to HIV-1 infection in that no patients with other chronic viral infections or systemic lupus erythematosus contained such antibodies in their sera. The anti-CD43 autoantibodies bound to a partially sialylated form of CD43 expressed by normal human thymocytes, but not by normal, circulating T lymphocytes. However, the determinant(s) recognized by the anti-CD43 autoantibodies was present on a large proportion of circulating T lymphocytes, but masked from antibody recognition by sialic acid residues. These results demonstrate that HIV-1 infection is specifically associated with the production of autoantibodies that bind to a native T cell surface antigen.


Sodium dichloroisocyanurate tablets for routine treatment of household drinking water in periurban Ghana: a randomized controlled trial.
We conducted a randomized, placebo-controlled, triple-blinded trial to determine the health impact of daily use of sodium dichloroisocyanurate (NaDCC) tablets for household drinking water treatment in periurban Ghana. We randomized 240 households (3,240 individuals) to receive either NaDCC or placebo tablets. All households received a 20-liter safe water storage vvessel. Over 12 weeks, 446 diarrhea episodes (2.2%) occurred in intervention and 404 (2.0%) in control households (P = 0.38). Residual free chlorine levels indicated appropriate tablet use. Escherichia coli was found in stored water at baseline in 96% of intervention and 88% of control households and at final evaluation in 8% of intervention and 54% of control households (P = 0.002). NaDCC use did not prevent diarrhea but improved water quality. Diarrhea rates were low and water quality improved in both groups. Safe water storage vessels may have been protective. A follow-up health impact study of NaDCC tablets is warranted.


First-photon imaging.
Imagers that use their own illumination can capture three-dimensional (3D) structure and reflectivity information. With photon-counting detectors, images can be acquired at extremely low photon fluxes. To suppress the Poisson noise inherent in low-flux operation, such imagers typically require hundreds of detected photons per pixel for accurate range and reflectivity determination. We introduce a low-flux imaging technique, called first-photon imaging, which is a computational imager that exploits spatial correlations found in real-world scenes and the physics of low-flux measurements. Our technique recovers 3D structure and reflectivity from the first detected photon at each pixel. We demonstrate simultaneous acquisition of sub-pulse duration range and 4-bit reflectivity information in the presence of high background noise. First-photon imaging may be of considerable value to both microscopy and remote sensing.


Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.
Infectivity studies with highly infectious RNA inocula generated by ribozyme cleavage were used to compare the biological properties of three apparently nonviable mutants of potato spindle tuber viroid (PSTVd). One of these mutants (PSTVd-P) contains three nucleotide substitutions in the left terminal loop, and mechanical inoculation of tomato seedlings with RNA transcripts at levels equivalent to 10(3)-10(5) times the ID50 for PSTVd-Intermediate failed to result in systemic infection. Viable progeny containing a spontaneous C-->G change at position 4 could, however, be recovered from transgenic Nicotiana benthamiana plants that constitutively expressed PSTVd-P RNA. The initial mutations in PSTVd-P led to an overall weakening of its native structure in vitro, and the precisely-full-length molecule released by ribozyme cleavage in vivo was also unstable. Even RT-PCR analysis failed to reveal detectable amounts of circularized PSTVd-P among the RNAs isolated from uninfected plants. Predicted stabilizing effects of a spontaneous mutation at position 4 suggest that the appearance of viable progeny was dependent on a combination of events: errors by host RNA polymerase II during transcription of the mutant transgene coupled with a strong selective pressure against alterations in the native structure of PSTVd.


The relationship between anxiety and the social judgements of approachability and trustworthiness.
The aim of the current study was to examine the relationship between individual differences in anxiety and the social judgements of trustworthiness and approachability. We assessed levels of state and trait anxiety in eighty-two participants who rated the trustworthiness and approachability of a series of unexpressive faces. Higher levels of trait anxiety (controlling for age, sex and state anxiety) were associated with the judgement of faces as less trustworthy. In contrast, there was no significant association between trait anxiety and judgements of approachability. These findings indicate that trait anxiety is a significant predictor of trustworthiness evaluations and illustrate the importance of considering the role of individual differences in the evaluation of trustworthiness. We propose that trait anxiety may be an important variable to control for in future studies assessing the cognitive and neural mechanisms underlying trustworthiness. This is likely to be particularly important for studies involving clinical populations who often experience atypical levels of anxiety.


Psychometric Properties and Demographic Correlates of the Smartphone Addiction Scale-Short Version Among Chinese Children and Adolescents in Hong Kong.
Nearly all children and teens in Hong Kong own a smartphone. There is currently no validated instrument that measures whether they use their phone too much. This study tested the psychometric properties of a translated Chinese version of the Smartphone Addiction Scale-Short Version (SAS-SV) and examined the demographic correlates of smartphone addiction among Hong Kong children and adolescents. A total of 1,901 primary school children and secondary school pupils were recruited from 15 Hong Kong schools. Furthermore, 1,797 primary caregivers were asked to complete a self-administered questionnaire on their socioeconomic status and educational attainment. The study used exploratory factor analysis (EFA) to identify the factor structure of SAS-SV for half the participants (n = 951), while confirmatory factor analysis (CFA) was used to assess the goodness-of-fit of EFA models for the remaining half (n = 951). Spearman correlations were used to assess the convergent validity of the SAS-SV, taking account of time spent by subjects on phones per day, the Smart Device Addiction Screening Tool (SDAST), the Pittsburgh Sleep Quality Index (PSQI), the Multidimensional Scale of Perceived Social Support (MSPSS), and the Center for Epidemiological Studies Depression Scale for Children (CES-DC). EFA generated a three-factor model (with factors labeled "dependency," the incidence of a "problem," and "time spent"). CFA confirmed this model yielded an acceptable goodness-of-fit (Comparative Fit Index = 0.96, Tucker Lewis Index = 0.95, and root-mean-square error of approximation = 0.06). SAS-SV was positively correlated with SDAST (ρ = 0.59), PSQI (ρ = 0.29), and CES-D (ρ = 0.35), and negatively correlated with MSPSS (ρ = -0.10). A linear regression model showed that female adolescents, those with highly educated caregivers and those who spent more time using smartphones on their holidays, had on average higher SAS-SV scores, meaning they showed greater vulnerability to becoming addicted. The study found that SAS-SV is a valid scale for estimating excessive smartphone use among Hong Kong children and adolescents.


The quality of nursing home care: do the opinions of family members change after implementation of emotion-oriented care?
The present study focuses on opinions on the quality of nursing home care of family members of nursing home residents with dementia. Furthermore, we examined whether family members' appreciation of the care increased as a result of the implementation of emotion-oriented care. Randomized clinical trial. An 18-item questionnaire was developed. The following subjects were addressed: communication activities between staff and family members; satisfaction regarding contacts with staff; the extent to which family members can participate in care; the contact that family members experience with the person with dementia, and opinions about the way in which nursing staff treat residents. Most family members already had a positive opinion on the nursing home care prior to the implementation of emotion-oriented care. The most positive assessment concerned the way in which nursing staff treated residents. The lowest scores concerned communication activities between ward staff and family members. Comparison of the first and end measurements showed that in general opinions on the quality of care did not change. A large number of incomplete questionnaires made it impossible to conduct factor analysis on the classification of the questions in various sections and therefore allowed us only to make statements at the item level. For the most part family members had a positive opinion on the nursing home care. In general, implementation of emotion-oriented care did not lead to a more positive assessment. Despite the generally accepted notion that involving family members in care is important, family members were regularly treated as outsiders. This demonstrates that there is room for improvement in the communication by nursing home staff with family members (e.g. more frequent contacts and information about the illness).


Recovery from spinal cord injury using naturally occurring antiinflammatory compound curcumin: laboratory investigation.
Spinal cord injury (SCI) is a debilitating disease. Primary SCI results from direct injury to the spinal cord, whereas secondary injury is a side effect from subsequent edema and ischemia followed by activation of proinflammatory cytokines. These cytokines activate the prosurvival molecule nuclear factor-κB and generate obstacles in spinal cord reinnervation due to gliosis. Curcumin longa is an active compound found in turmeric, which acts as an antiinflammatory agent primarily by inhibiting nuclear factor-κB. Here, the authors study the effect of curcumin on SCI recovery. Fourteen female Sprague-Dawley rats underwent T9-10 laminectomy and spinal cord contusion using a weight-drop apparatus. Within 30 minutes after contusion and weekly thereafter, curcumin (60 mg/kg/ml body weight in dimethyl sulfoxide) or dimethyl sulfoxide (1 ml/kg body weight) was administered via percutaneous epidural injection at the injury site. Spinal cord injury recovery was assessed weekly by scoring hindlimb motor function. Animals were killed 6 weeks postcontusion for histopathological analysis of spinal cords and soleus muscle weight evaluation. Curcumin-treated rats had improved motor function compared with controls starting from Week 1. Body weight gain significantly improved, correlating with improved Basso-Beattie-Bresnahan scores. Soleus muscle weight was greater in curcumin-treated rats than controls. Histopathological analysis validated these results with increased neural element mass with less gliosis at the contusion site in curcumin-treated rats than controls. Epidural administration of curcumin resulted in improved recovery from SCI. This occurred with no adverse effects noted in experimental animals. Therefore, curcumin treatment may translate into a novel therapy for humans with SCI.


Ecoinformatics can reveal yield gaps associated with crop-pest interactions: a proof-of-concept.
Farmers and private consultants execute a vast, decentralized data collection effort with each cropping cycle, as they gather pest density data to make real-time pest management decisions. Here we present a proof of concept for an ecoinformatics approach to pest management research, which attempts to harness these data to answer questions about pest-crop interactions. The impact of herbivory by Lygus hesperus on cotton is explored as a case study. Consultant-derived data satisfied a 'positive control' test for data quality by clearly resolving the expected negative relationship between L. hesperus density and retention of flower buds. The enhanced statistical power afforded by the large ecoinformatics dataset revealed an early-season window of crop sensitivity, during which L. hesperus densities as low as 1-2 per sample were associated with yield loss. In contrast, during the mid-season insecticide use by farmers was often unnecessary, as cotton compensated fully for moderate L. hesperus densities. Because the dataset emerged from the commercial production setting, it also revealed the limited degree to which farmers were willing to delay crop harvest to provide opportunities for compensatory fruiting. Observational approaches to pest management research have strengths and weaknesses that complement those of traditional, experimental approaches; combining these methods can contribute to enhanced agricultural productivity.


Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).
HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.


Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.
Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules that selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits that potently inhibit DSB repair, and we have validated their functional activity in a comprehensive panel of orthogonal secondary assays. A selection of these inhibitors was found to radiosensitize cancer cell lines in vitro, which suggests that they may be useful as novel chemo- and radio sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker mibefradil dihydrochloride, that demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a phase I clinical trial testing mibefradil as a glioma radiosensitizer.


Association between age-related reductions in testosterone and risk of prostate cancer-An analysis of patients' data with prostatic diseases.
The relationship between serum total testosterone and prostate cancer (PCa) risk is controversial. The hypothesis that faster age-related reduction in testosterone is linked with increased PCa risk remains untested. We conducted our study at a tertiary-level hospital in southeast of the USA, and derived data from the Medical Registry Database of individuals that were diagnosed of any prostate-related disease from 2001 to 2015. Cases were those diagnosed of PCa and had one or more measurements of testosterone prior to PCa diagnosis. Controls were those without PCa and had one or more testosterone measurements. Multivariable logistic regression models for PCa risk of absolute levels (one-time measure and 5-year average) and annual change in testosterone were respectively constructed. Among a total of 1,559 patients, 217 were PCa cases, and neither one-time measure nor 5-year average of testosterone was found to be significantly associated with PCa risk. Among the 379 patients with two or more testosterone measurements, 27 were PCa cases. For every 10 ng/dL increment in annual reduction of testosterone, the risk of PCa would increase by 14% [adjusted odds ratio, 1.14; 95% confidence interval (CI), 1.03-1.25]. Compared to patients with a relatively stable testosterone, patients with an annual testosterone reduction of more than 30 ng/dL had 5.03 [95% CI: 1.53, 16.55] fold increase in PCa risk. This implies a faster age-related reduction in, but not absolute level of serum total testosterone as a risk factor for PCa. Further longitudinal studies are needed to confirm this finding.


Association of IL-10-promoter genetic variants with the rate of CD4 T-cell loss, IL-10 plasma levels, and breadth of cytotoxic T-cell lymphocyte response during chronic HIV-1 infection.
Interleukin-10 (IL-10) is a potent immunoregulatory cytokine. IL-10-promoter polymorphisms have been shown to affect human immunodeficiency virus type 1 (HIV-1) clinical outcomes but the underlying mechanisms are poorly understood. We investigated the relationship between IL-10-promoter variants, plasma cytokine levels, immune responses and markers of disease outcome in antiretroviral-naïve HIV-1 chronically infected individuals from South Africa. Two IL-10-promoter single nucleotide polymorphisms (SNPs) were genotyped in 451 participants. Baseline plasma levels of select cytokines were measured for 112 individuals. Viral load, CD4(+) T-cell counts and HIV-1-specific interferon-gamma CD8(+) T-cell immune responses were measured at baseline. CD4(+) T-cell counts were measured longitudinally and rates of CD4(+) T-cell decline computed for 300 study subjects. The minor IL-10-1082G and -592A variants occurred at frequencies of 0.31 and 0.34, respectively. The -592AA genotype associated significantly with attenuated loss of CD4(+) T cells (P = .0496). Individuals possessing -1082GG had significantly higher IL-10 levels compared to -1082AA/AG (P = .0006). The -592AA genotype was associated with greater breadth of virus-specific CD8(+) T-cell responses compared to CC and CA (P = .002 and .004 respectively). IL-10-promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels and the breadth of CD8(+) T-cell immune responses.


High-risk patients with hematuria are not evaluated according to guideline recommendations.
To determine whether high-risk patients with hematuria receive evaluation according to guideline recommendations. We recently performed a screening study for bladder cancer using a urine-based tumor marker in 1502 subjects at high risk based on aged > or = 50 years, > or = 10-year smoking history, and/or a 15-year or more environmental exposure. We evaluated use of urinalysis (UA) within 3 years preceding the screening study. Chart review was performed to determine if this subset with microhematuria received any additional evaluation. Of 1502 study participants, routine urinalysis was performed in 73.2% and 164 (14.9%) subjects had documented hematuria (>3 red blood cells / high-power field) before inclusion. Of these, 42.1% had no further evaluation. Additional testing included repeat urinalysis (36%), urine culture (15.2%), cytology (10.4%), imaging (22.6% overall: 15.9% computed tomography, 4.3% intravenous pyelography; 2.4% magnetic resonance imaging), and cystoscopy (12.8%). Three subjects with microscopic hematuria (2%) were subsequently found to have bladder cancer during the screening study but were not referred for evaluation based on their hematuria. The source of hematuria was unknown in 65%, infection in 22%, benign prostatic enlargement in 10%, and renal stone disease in 4%, but these results are based on incomplete evaluation since only 12.8% underwent cystoscopy. Subjects at high risk for bladder cancer based on > or = 10 years of smoking or environmental exposure with microscopic hematuria are rarely evaluated thoroughly and only 12.8% were referred for urologic evaluation. Further studies are needed to evaluate both the utilization and effectiveness of guidelines for hematuria.


Tablet Formulation of a Synthesized Celecoxib Potassium Salt and Development of a Validated Method for Its Analysis.
Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) and cyclooxygenase-2 (COX-2) inhibitor. It is used for the treatment of rheumatoid arthritis, osteoarthritis, juvenile arthritis, and acute pain. Celecoxib has low systemic bioavailability due to its low water solubility. This study aimed to improve water solubility and dissolution profile by synthesizing a suitable celecoxib potassium salt (celecoxib-K salt). Four celecoxib salts were synthesized, and the solubility of these four salts was determined. Celecoxib- K monohydrate salt was chosen for tablet formulation. A simple and feasible reversed-phase high-performance liquid chromatography (HPLC) method was developed for the analysis of the formulated tablet and then validated according to international guidelines. The dissolution profile, shelf life, and accelerated stability studies on the formulated tablet were conducted. Celecoxib-K monohydrate salt exhibited increased water solubility by more than 140-folds (0.464 mg/ml) compared with celecoxib. The in vitro dissolution profile of the formulated celecoxib-K salt tablet was totally dissolved after 10 min. The developed analytical HPLC method was a reliable and valid method with good linearity, accuracy, and precision. Moreover, it was sensitive, and the limit of detection and quantification (LOD and LOQ) were 0.001 and 0.1 mg/L, respectively. The formulated celecoxib-K monohydrate salt tablet was stable under room temperature and accelerated condition for 60 days. The potassium salt of celecoxib was highly increased, and the formulated tablet of celecoxib-K salt exhibited a good dissolution profile in the water. In addition, the developed HPLC method was valid and reliable for the analysis and quantification of the formulated tablet. The formulated tablet was stable both at room temperature and under stress conditions.


Dating genomic variants and shared ancestry in population-scale sequencing data.
The origin and fate of new mutations within species is the fundamental process underlying evolution. However, while much attention has been focused on characterizing the presence, frequency, and phenotypic impact of genetic variation, the evolutionary histories of most variants are largely unexplored. We have developed a nonparametric approach for estimating the date of origin of genetic variants in large-scale sequencing data sets. The accuracy and robustness of the approach is demonstrated through simulation. Using data from two publicly available human genomic diversity resources, we estimated the age of more than 45 million single-nucleotide polymorphisms (SNPs) in the human genome and release the Atlas of Variant Age as a public online database. We characterize the relationship between variant age and frequency in different geographical regions and demonstrate the value of age information in interpreting variants of functional and selective importance. Finally, we use allele age estimates to power a rapid approach for inferring the ancestry shared between individual genomes and to quantify genealogical relationships at different points in the past, as well as to describe and explore the evolutionary history of modern human populations.


Occupational health risks for Mexican women: the case of the maquiladora along the Mexican-United States border.
International reports of morbidity among female workers in Mexico's border zone have raised concern about the occupational health of female workers in maquiladora plants (foreign-owned border industries with special tariff benefits). Commentators have suggested that U.S. industries may be exploiting workers by transferring work to nations with less stringent health and safety regulation through the maquiladora program. Using data from a larger evaluation of the effectiveness of Project Concern and a specially developed questionnaire, this study investigated the extent to which female workers reported higher morbidity rates than women with other employment and women not employed outside the home in seven colonias (communities) in Tijuana, Baja California, Mexico. Results showed essentially no difference in many short-term self-reported symptoms of illness among maquiladora workers and two other groups. Women who worked exclusively in the home reported the greatest number of symptoms. These results suggest that additional primary care services may be needed for women who have primarily domestic responsibilities. Additional research is needed to assess the risks for long-latency morbidity.


Searching for Atrial Fibrillation Poststroke: A White Paper of the AF-SCREEN International Collaboration.
Cardiac thromboembolism attributed to atrial fibrillation (AF) is responsible for up to one-third of ischemic strokes. Stroke may be the first manifestation of previously undetected AF. Given the efficacy of oral anticoagulants in preventing AF-related ischemic strokes, strategies of searching for AF after a stroke using ECG monitoring followed by oral anticoagulation (OAC) treatment have been proposed to prevent recurrent cardioembolic strokes. This white paper by experts from the AF-SCREEN International Collaboration summarizes existing evidence and knowledge gaps on searching for AF after a stroke by using ECG monitoring. New AF can be detected by routine plus intensive ECG monitoring in approximately one-quarter of patients with ischemic stroke. It may be causal, a bystander, or neurogenically induced by the stroke. AF after a stroke is a risk factor for thromboembolism and a strong marker for atrial myopathy. After acute ischemic stroke, patients should undergo 72 hours of electrocardiographic monitoring to detect AF. The diagnosis requires an ECG of sufficient quality for confirmation by a health professional with ECG rhythm expertise. AF detection rate is a function of monitoring duration and quality of analysis, AF episode definition, interval from stroke to monitoring commencement, and patient characteristics including old age, certain ECG alterations, and stroke type. Markers of atrial myopathy (eg, imaging, atrial ectopy, natriuretic peptides) may increase AF yield from monitoring and could be used to guide patient selection for more intensive/prolonged poststroke ECG monitoring. Atrial myopathy without detected AF is not currently sufficient to initiate OAC. The concept of embolic stroke of unknown source is not proven to identify patients who have had a stroke benefitting from empiric OAC treatment. However, some embolic stroke of unknown source subgroups (eg, advanced age, atrial enlargement) might benefit more from non-vitamin K-dependent OAC therapy than aspirin. Fulfilling embolic stroke of unknown source criteria is an indication neither for empiric non-vitamin K-dependent OAC treatment nor for withholding prolonged ECG monitoring for AF. Clinically diagnosed AF after a stroke or a transient ischemic attack is associated with significantly increased risk of recurrent stroke or systemic embolism, in particular, with additional stroke risk factors, and requires OAC rather than antiplatelet therapy. The minimum subclinical AF duration required on ECG monitoring poststroke/transient ischemic attack to recommend OAC therapy is debated.


Bisphosphonate therapy for osteogenesis imperfecta.
Osteogenesis imperfecta is caused by a genetic defect resulting in an abnormal type I collagen bone matrix which typically results in multiple fractures with little or no trauma. Bisphosphonates are used in an attempt to increase bone mineral density and reduce these fractures in people with osteogenesis imperfecta. To assess the effectiveness and safety of bisphosphonates in increasing bone mineral density, reducing fractures and improving clinical function in people with osteogenesis imperfecta. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Inborn Errors of Metabolism Trials Register which comprises references identified from comprehensive electronic database searches, handsearches of journals and conference proceedings. We additionally searched PubMed and major conference proceedings.Date of the most recent search: 07 April 2014. Randomised and quasi-randomised controlled trials comparing bisphosphonates to placebo, no treatment, or comparator interventions in all types of osteogenesis imperfecta. Two authors independently extracted data and assessed the risk of bias of the included trials. Fourteen trials (819 participants) were included. Overall, the trials were mainly at a low risk of bias, although selective reporting was an issue in several of the trials. Data for oral bisphosphonates versus placebo could not be aggregated; a statistically significant difference favouring oral bisphosphonates in fracture risk reduction and number of fractures was noted in two trials. No differences were reported in the remaining three trials which commented on fracture incidence. Five trials reported data for spine bone mineral density; all found statistically significant increased lumbar spine density z scores for at least one time point studied. For intravenous bisphosphonates versus placebo, aggregated data from two trials showed no statistically significant difference for the number of participants with at least one fracture, risk ratio 0.56 (95% confidence interval 0.30 to 1.06). In the remaining trial no statistically significant difference was noted in fracture incidence. For spine bone mineral density, no statistically significant difference was noted in the aggregated data from two trials, mean difference 9.96 (95% confidence interval -2.51 to 22.43). In the remaining trial a statistically significant difference in mean per cent change in spine bone mineral density z score favoured intravenous bisphosphonates at six and 12 months. Data describing growth, bone pain, and functional outcomes after oral or intravenous bisphosphonate therapy, or both, as compared to placebo were incomplete among all studies, but do not show consistent improvements in these outcomes. Two studies compared different doses of bisphosphonates. No differences were found between doses when bone mineral density, fractures, and height or length z score were assessed. One study compared oral versus intravenous bisphosphonates and found no differences in primary outcomes. Two studies compared the intravenous bisphosphonates zoledronic acid and pamidronate. There were no significant differences in primary outcome. However, the studies were at odds as to the relative benefit of zoledronic acid over pamidronate for lumbosacral bone mineral density at 12 months. Bisphophonates are commonly prescribed to individuals with osteogenesis imperfecta. Current evidence, albeit limited, demonstrates oral or intravenous bisphosphonates increase bone mineral density in children and adults with this condition. These were not shown to be different in their ability to increase bone mineral density. It is unclear whether oral or intravenous bisphosphonate treatment consistently decreases fractures, though multiple studies report this independently and no studies report an increased fracture rate with treatment. The studies included here do not show bisphosphonates conclusively improve clinical status (reduce pain; improve growth and functional mobility) in people with osteogenesis imperfecta. Given their current widespread and expected continued use, the optimal method, duration of therapy and long-term safety of bisphosphonate therapy require further investigation. In addition, attention should be given to long-term fracture reduction and improvement in quality of life indicators.


The main cost drivers in dementia: a systematic review.
Because of the increasing prevalence of dementia worldwide, combined with limited healthcare expenditures, a better understanding of the main cost drivers of dementia in different care settings is needed. A systematic review of cost-of-illness (COI) studies in dementia was conducted from 2003 to 2012, searching the following databases: PubMed (Medline), Cochrane Library, ScienceDirect (Embase) and National Health Service Economic Evaluations Database. Costs (per patient) by care setting were analyzed for total, direct, indirect and informal costs and related to the following: (1) cost perspective and (2) disease severity. In total, 27 studies from 14 different healthcare systems were evaluated. In the included studies, total annual costs for dementia of up to $70,911 per patient (mixed setting) were estimated (average estimate of total costs = $30,554). The shares of cost categories in the total costs for dementia indicate significant differences for different care settings. Overall main cost drivers of dementia are informal costs due to home based long term care and nursing home expenditures rather than direct medical costs (inpatient and outpatient services, medication). The results of this review highlight the significant economic burden of dementia for patients, families and healthcare systems and thus are important for future health policy planning. The significant variation of cost estimates for different care settings underlines the need to understand and address the financial burden of dementia from both perspectives. For health policy planning in dementia, future COI studies should follow a quality standard protocol with clearly defined cost components and separate estimates by care setting and disease severity.


Efficient parameters of vagus nerve stimulation to enhance extinction learning in an extinction-resistant rat model of PTSD.
Vagus nerve stimulation (VNS) has shown promise as an adjuvant treatment for posttraumatic stress disorder (PTSD), as it enhances fear extinction and reduces anxiety symptoms in multiple rat models of this condition. Yet, identification of the optimal stimulation paradigm is needed to facilitate clinical translation of this potential therapy. Using an extinction-resistant rat model of PTSD, we tested whether varying VNS intensity and duration could maximize extinction learning while minimizing the total amount of stimulation. We confirmed that sham rats failed to extinguish after a week of extinction training. Delivery of the standard LONG VNS trains (30 s) at 0.4 mA enhanced extinction and reduced anxiety but did not prevent fear return. Increasing the intensity of LONG VNS trains to 0.8 mA prevented fear return and attenuated anxiety symptoms. Interestingly, delivering 1, 4 or 16 SHORT VNS bursts (0.5 s) at 0.8 mA during each cue presentation in extinction training also enhanced extinction. LONG VNS trains or multiple SHORT VNS bursts at 0.8 mA attenuated fear renewal and reinstatement, promoted extinction generalization and reduced generalized anxiety. Delivering 16 SHORT VNS bursts also facilitated extinction in fewer trials. This study provides the first evidence that brief bursts of VNS can enhance extinction training, reduce relapse and support symptom remission using much less VNS than previous protocols. These findings suggest that VNS parameters can be adjusted in order to minimize total charge delivery and maximize therapeutic effectiveness.


Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.
We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.


Testosterone Treatment and Sexual Function in Older Men With Low Testosterone Levels.
The Testosterone Trials are a coordinated set of seven trials to determine the efficacy of T in symptomatic men ≥65 years old with unequivocally low T levels. Initial results of the Sexual Function Trial showed that T improved sexual activity, sexual desire, and erectile function. To assess the responsiveness of specific sexual activities to T treatment; to relate hormone changes to changes in sexual function; and to determine predictive baseline characteristics and T threshold for sexual outcomes. A placebo-controlled trial. Twelve academic medical centers in the United States. A total of 470 men ≥65 years of age with low libido, average T <275 ng/dL, and a partner willing to have sexual intercourse at least twice a month. Men were assigned to take T gel or placebo for 1 year. Sexual function was assessed by three questionnaires every 3 months: the Psychosexual Daily Questionnaire, the Derogatis Interview for Sexual Function, and the International Index of Erectile Function. Compared with placebo, T administration significantly improved 10 of 12 measures of sexual activity. Incremental increases in total and free T and estradiol levels were associated with improvements in sexual activity and desire, but not erectile function. No threshold T level was observed for any outcome, and none of the 27 baseline characteristics predicted responsiveness to T. In older men with low libido and low T levels, improvements in sexual desire and activity in response to T treatment were related to the magnitude of increases in T and estradiol levels, but there was no clear evidence of a threshold effect.


Group Yoga Effects on Cancer Patient and Caregiver Symptom Distress: Assessment of Self-reported Symptoms at a Comprehensive Cancer Center.
Complementary and integrative health approaches such as yoga provide support for psychosocial health. We explored the effects of group-based yoga classes offered through an integrative medicine center at a comprehensive cancer center. Patients and caregivers had access to two yoga group classes: a lower intensity (YLow) or higher intensity (YHigh) class. Participants completed the Edmonton Symptom Assessment System (ESAS; scale 0-10, 10 most severe) immediately before and after the class. ESAS subscales analyzed included global (GDS; score 0-90), physical (PHS; 0-60), and psychological distress (PSS; 0-20). Data were analyzed examining pre-yoga and post-yoga symptom scores using paired t-tests and between types of classes using ANOVAs. From July 18, 2016, to August 8, 2017, 282 unique participants (205 patients, 77 caregivers; 85% female; ages 20-79 years) attended one or more yoga groups (mean 2.3). For all participants, we observed clinically significant reduction/improvement in GDS, PHS, and PSS scores and in symptoms (ESAS decrease ≥1; means) of anxiety, fatigue, well-being, depression, appetite, drowsiness, and sleep. Clinically significant improvement for both patients and caregivers was observed for anxiety, depression, fatigue, well-being, and all ESAS subscales. Comparing yoga groups, YLow contributed to greater improvement in sleep versus YHigh (-1.33 vs -0.50, P = .054). Improvement in fatigue for YLow was the greatest mean change (YLow -2.12). A single yoga group class resulted in clinically meaningful improvement of multiple self-reported symptoms. Further research is needed to better understand how yoga class content, intensity, and duration can affect outcomes.


Accessory proteins function as matchmakers in the assembly of the T4 DNA polymerase holoenzyme.
During bacteriophage T4 DNA replication, the 44/62 and 45 accessory proteins combine with the DNA polymerase to form a processive holoenzyme complex. Formation of this complex is dependent upon ATP hydrolysis by the 44/62 protein. It is uncertain, however, whether the 44/62 protein remains with the 45 protein as part of this protein 'sliding clamp' during DNA synthesis, or whether it is required only for complex assembly. To address this tissue, we have stoichiometrically assembled a processive T4 DNA polymerase holoenzyme complex, capable of strand-displacement synthesis, on a forked primer/template. By titrating the 44/62 protein to substoichiometric concentrations, we have shown that it can act catalytically to load on to the primer/template the 45 protein, which, in turn, combines with the DNA polymerase to form a processive complex. Two distinct complex species are formed: most of the complexes are highly stable, with a half life of 7 minutes, whereas the remainder have a half-life of 0.4 minutes. Precipitation of the protein-DNA complexes, followed by western blot analysis, verified that the complexes contain the DNA polymerase and 45 proteins, but not the 44/62 protein. Using physiological protein concentrations, we have shown that the composition of the T4 protein sliding clamp consists solely of the 45 protein. The role of the 44/62 protein is that of a molecular matchmaker, in that it serves to load the 45 protein onto the DNA but does not remain an essential component of the processive complex.


Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry.
Lumpy skin disease (LSD) is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. Transmission of the virus has been associated with haematophagous insects such as Stomoxys calcitrans as well as Aedes and Culex species of mosquitoes. Recent studies have reported the transmission of the virus by Amblyomma hebraeum, Rhipicephalus appendiculatus, and Rhipicephalus decoloratus ticks and the presence of LSDV in saliva of A. hebraeum and R. appendiculatus ticks. The aim of this study was to determine which tick organs become infected by LSDV following intrastadial infection and transstadial persistence of the virus in A. hebraeum and R. appendiculatus ticks. Nymphal and adult ticks were orally infected by feeding them on LSDV-infected cattle. Partially fed adult ticks were processed for testing while nymphs were fed to repletion and allowed to moult to adults before being processed for testing. The infection in tick organs was determined by testing for the presence of the viral antigen using monoclonal antibodies with immunohistochemical staining. The viral antigen was detected in salivary glands, haemocytes, synganglia, ovaries, testes, fat bodies, and midgut. Since the virus was shown to be able to cross the midgut wall and infect various tick organs, this may indicate potential for biological development and transmission of LSDV in ticks. This study strengthens the previously reported evidence of the occurrence of LSDV in tick saliva.


Long-term risk of BP values above normal for cardiovascular mortality: a 24-year observation of Japanese aged 30 to 92 years.
In Western populations, blood pressure (BP) measured at baseline has been reported to predict long-term (over 20 years) risk of mortality from cardiovascular diseases (CVDs). However, corresponding evidence is scarce in Asia where stroke is dominant. We investigated the association between baseline BP and 24-year mortality risk due to CVD, in a representative Japanese general population. We followed up a nationwide sample of 8592 Japanese, aged 30 years or above without a history of CVD and antihypertensive medication at baseline, for 24 years. Hazard ratios for CVD mortality in BP categories defined according to JCN7 criteria were estimated using Cox model adjusted for potential confounding factors with normal BP treated as the reference category. We observed 689 CVD deaths. Hazard ratios for CVD mortality were progressively and significantly increased from the category of prehypertension. Population-attributable fraction (PAF) demonstrated that 43 and 48% of CVD and stroke deaths were explained by non-normal BP at baseline. Hazard ratios and PAF were remarkably higher in younger participants (aged 30-59 years) than those in the elderly (aged 60 years or above). Particularly, in younger men, 81% of CVD deaths were explained by non-normal BP. In sensitivity analysis, participants with antihypertensive medication showed the highest hazard ratio for CVD morality compared with the other categories. BP levels above normal at baseline retained significant relative and absolute risks of CVD and stroke mortality during 24 years. Long-lasting burden of non-normal BP particularly in younger individuals suggests the importance of primary prevention of high BP from younger generation.


Aging Increases Compensatory Saccade Amplitude in the Video Head Impulse Test.
Rotational vestibular function declines with age resulting in saccades as a compensatory mechanism to improve impaired gaze stability. Small reductions in rotational vestibulo-ocular reflex (VOR) gain that would be considered clinically normal have been associated with compensatory saccades. We evaluated whether compensatory saccade characteristics varied as a function of age, independent of semicircular canal function as quantified by VOR gain. Horizontal VOR gain was measured in 243 participants age 27-93 from the Baltimore Longitudinal Study of Aging using video head impulse testing. Latency and amplitude of the first saccade (either covert - occurring during head impulse, or overt - occurring following head impulse) were measured for head impulses with compensatory saccades (n = 2230 head impulses). The relationship between age and saccade latency, as well as the relationship between age and saccade amplitude, were evaluated using regression analyses adjusting for VOR gain, gender, and race. Older adults (mean age 75.9) made significantly larger compensatory saccades relative to younger adults (mean age 45.0). In analyses adjusted for VOR gain, there was a significant association between age and amplitude of the first compensatory covert saccade (β = 0.015, p = 0.008). In analyses adjusted for VOR gain, there was a significant association between age and amplitude of the first compensatory overt saccade (β = 0.02, p < 0.001). Compensatory saccade latencies did not vary significantly by age. We observed that aging increases the compensatory catch-up saccade amplitude in healthy adults after controlling for VOR gain. Size of compensatory saccades may be useful in addition to VOR gain for characterizing vestibular function in aging adults.