Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.
Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.


Effectiveness and cost-effectiveness of daily all-over-body application of emollient during the first year of life for preventing atopic eczema in high-risk children (The BEEP trial): protocol for a randomised controlled trial.
Atopic eczema (AE) is a common skin problem that impairs quality of life and is associated with the development of other atopic diseases including asthma, food allergy and allergic rhinitis. AE treatment is a significant cost burden for health care providers. The purpose of the trial is to investigate whether daily application of emollients for the first year of life can prevent AE developing in high-risk infants (first-degree relative with asthma, AE or allergic rhinitis). This is a protocol for a pragmatic, two-arm, randomised controlled, multicentre trial. Up to 1400 term infants at high risk of developing AE will be recruited through the community, primary and secondary care in England. Participating families will be randomised in a 1:1 ratio to receive general infant skin-care advice, or general skin-care advice plus emollients with advice to apply daily to the infant for the first year of life. Families will not be blinded to treatment allocation. The primary outcome will be a blinded assessment of AE at 24 months of age using the UK Working Party Diagnostic Criteria for Atopic Eczema. Secondary outcomes are other definitions of AE, time to AE onset, severity of AE (EASI and POEM), presence of other allergic diseases including food allergy, asthma and hay fever, allergic sensitisation, quality of life, cost-effectiveness and safety of the emollients. Subgroup analyses are planned for the primary outcome according to filaggrin genotype and the number of first-degree relatives with AE and other atopic diseases. Families will be followed up by online and postal questionnaire at 3, 6, 12 and 18 months with a face-to-face visit at 24 months. Long-term follow-up until 60 months will be via annual questionnaires. This trial will demonstrate whether skin-barrier enhancement through daily emollient for the first year of life can prevent AE from developing in high-risk infants. If effective, this simple and cheap intervention has the potential to result in significant cost savings for health care providers throughout the world by preventing AE and possibly other associated allergic diseases. ISRCTN registry; ID: ISRCTN21528841 . Registered on 25 July 2014.


Derivation of retardation phase in computer-aided photoelasticity by using carrier fringe phase shifting.
Previous phase-shifting schemes in computer-aided photoelasticity required the processing of six fringe patterns to derive the phase difference due to retardation. A technique in which a carrier fringe is used is demonstrated to reduce to four the number of fringe patterns required. The use of fewer fringe patterns lowers the computation time and the number of phase-step errors. The basis of the technique is outlined in detail, and experimental results are presented as well.


Free transverse rectus abdominis myocutaneous flap reconstruction of a massive lumbosacral defect using superior gluteal artery perforator vessels.
Despite significant advances in reconstructive surgery, the repair of massive lumbosacral defects poses significant challenges. When the extent of soft tissue loss, tumor resection, and/or radiation therapy preclude the use of traditional local options, such as gluteal advancement flaps or pedicled thigh flaps, then distant flaps are required. We report a case of a 64-year-old male who presented with a large sacral Marjolin's ulcer secondary to recurrent pilonidal cysts and ulcerations. The patient underwent wide local composite resection, which resulted in a wound measuring 450 cm(2) with exposed rectum and sacrum. The massive defect was successfully covered with a free transverse rectus abdominis myocutaneous flap, providing a well-vascularized skin paddle and obviating the need for a latissimus flap with skin graft. The free-TRAM flap proved to be a very robust flap in this situation and would be one of our flaps of choice for similar defects.


Variable Changes in Body Composition, Strength and Lower-Body Power During an International Rugby Sevens Season.
This study determined whether body composition, strength, and power changes that occur during preseason can be maintained during an international rugby sevens season. Fourteen male international rugby sevens players (age 21.4 ± 2.2 years; mean ± SD) were categorized as forward (n = 7) or back (n = 7), and assessed for height, mass (M), skinfolds (S) (∑7), upper-body (UB) strength, lower-body (LB) strength, and LB power. Bench press, back squat, and a countermovement jump were used to measure strength and power at 3 time points: initial, early season, and late season. Forwards were taller (185 ± 4 cm), heavier (95 ± 6 kg), and possessed a greater lean body mass (55.5 ± 4.0 M·S) than backs (181 ± 8 cm, 88.5 ± 5.5 kg, and 51.9 ± 3.4 M·S). Over the full season, small (∼5 ± 5%; mean ± 90% confidence limits) positive changes occurred in body composition. Lower-body strength gained during the preseason, decayed in-season, whereas UB strength increased moderately (∼10 ± 3%) across the season. Power showed inconsistencies between measured variables with a moderate positive change across the season in mean velocity and relative peak power. Forwards showed a small decrease in peak power (relative and absolute). Moderate changes were observed in mean power over the season, forwards decreasing (∼6 ± 6%) and backs increasing (∼8 ± 6%). Rugby sevens forwards in this study found it difficult to maintain and improve power qualities in-season. Training loads of forwards and backs should be differentiated to maximize strength and power in-season.


The mediating role of parent-child bonding to prevent adolescent alcohol abuse among Asian American families.
The purpose of this paper is to describe unique culturally-based factors that may increase the vulnerability of Asian American adolescents to engage in alcohol use and abuse and the role of parent-child bonding as a protective factor. In particular, this paper addresses the interactions among acculturation, alcohol use, and parent-child bonding and the challenges Asian American families face in strengthening parent-child bonds. We begin by examining likely causes for alienation that occur as a result of immigration to the United States. We then present the cultural context of Asian American families that can also serve to create distance between parent and child, including the contrasting cultural orientations of individualism and collectivism, Asian traditional values, differences in Eastern and Western parenting styles, and intergenerational cultural dissonance. Next, we present a review of the research that has examined acculturation as a risk factor for alcohol use and abuse among Asian American adolescents, with special attention to the mediating role of parent-child bonding. Finally, we conclude with recommendations for future research on the risk and protective factors for adolescent substance abuse, as well as other risky health behaviors among the growing population of Asian Americans in the United States.


Temporal and Spatial Dynamics of Archaeal Communities in Two Freshwater Lakes at Different Trophic Status.
In either eutrophic Dianchi Lake or mesotrophic Erhai Lake, the abundance, diversity, and structure of archaeaplankton communities in spring were different from those in summer. In summer, archaeaplankton abundance generally decreased in Dianchi Lake but increased in Erhai Lake, while archaeaplankton diversity increased in both lakes. These two lakes had distinct archaeaplankton community structure. Archaeaplankton abundance was influenced by organic content, while trophic status determined archaeaplankton diversity and structure. Moreover, in summer, lake sediment archaeal abundance considerably decreased. Sediment archaeal abundance showed a remarkable spatial change in spring but only a slight one in summer. The evident spatial change of sediment archaeal diversity occurred in both seasons. In Dianchi Lake, sediment archaeal community structure in summer was remarkably different from that in spring. Compared to Erhai Lake, Dianchi Lake had relatively high sediment archaeal abundance but low diversity. These two lakes differed remarkably in sediment archaeal community structure. Trophic status determined sediment archaeal abundance, diversity and structure. Archaeal diversity in sediment was much higher than that in water. Water and sediment habitats differed greatly in archaeal community structure. Euryarchaeota predominated in water column, but showed much lower proportion in sediment. Bathyarchaeota was an important component of sediment archaeal community.


In vivo analysis of aquaporin 0 function in zebrafish: permeability regulation is required for lens transparency.
The zebrafish lens is well suited for studies of physiology and development due to its rapid formation in the embryo and genetic accessibility. Aquaporin 0 (AQP0), a lens-specific membrane protein, is required for lens clarity. Zebrafish have two copies of AQP0 (Aqp0a and b), whereas mammals have a single, multifunctional protein. Here we demonstrate a reliable knockdown/rescue system in zebrafish and use it to provide evidence for subfunctionalization of Aqp0a and b, as well as to show that calcium-mediated regulation of Aqp0a in zebrafish lenses is necessary for transparency. Coinjection of antisense oligonucleotides and DNA rescue constructs into zebrafish embryos, followed by evaluation of the developing fish for cataracts, was used to analyze the functions of Aqp0a and b. The water permeability and regulation characteristics of each rescue protein were tested in a Xenopus oocyte swelling assay. Both copies of AQP0 are necessary for lens clarity in the zebrafish, and neither is sufficient. Water permeability is necessary but also insufficient. Phosphorylation and regulation of Aqp0a are required for its function. In the zebrafish lens, the two closely related AQP0s have acquired distinct functions that are both necessary for lens development and clarity. Regulation of AQP0 water permeability, a well-studied phenomenon in vitro, may be physiologically relevant in the living lens.


Improved Flexible Transparent Conductive Electrodes based on Silver Nanowire Networks by a Simple Sunlight Illumination Approach.
Silver nanowire (Ag NW) networks have attracted wide attention as transparent electrodes for emerging flexible optoelectronics. However, the sheet resistance is greatly limited by large wire-to-wire contact resistances. Here, we propose a simple sunlight illumination approach to remarkably improve their electrical conductivity without any significant degradation of the light transmittance. Because the power density is extremely low (0.1 W/cm2, 1-Sun), only slight welding between Ag NWs has been observed. Despite this, a sheet resistance of <20 Ω/sq and transmittance of ~87% at wavelength of 550 nm as well as excellent mechanical flexibility have still been achieved for Ag NW networks after sunlight illumination for 1 hour or longer, which are significant upgrades over those of ITO. Slight plasmonic welding together with the associated self-limiting effect has been investigated by numerical simulations and further verified experimentally through varied solar concentrations. Due to the reduced resistance, high-performance transparent film heaters as well as efficient defrosters have been demonstrated, which are superior to the previously-reported Ag NW based film heaters. Since the sunlight is environmentally friendly and easily available, sophisticated or expensive facilities are not necessary. Our findings are particularly meaningful and show enormous potential for outdoor applications.


The Effects of Montmorency Tart Cherry Concentrate Supplementation on Recovery Following Prolonged, Intermittent Exercise.
This study investigated Montmorency tart cherry concentrate (MC) supplementation on markers of recovery following prolonged, intermittent sprint activity. Sixteen semi-professional, male soccer players, who had dietary restrictions imposed for the duration of the study, were divided into two equal groups and consumed either MC or placebo (PLA) supplementation for eight consecutive days (30 mL twice per day). On day 5, participants completed an adapted version of the Loughborough Intermittent Shuttle Test (LISTADAPT). Maximal voluntary isometric contraction (MVIC), 20 m Sprint, counter movement jump (CMJ), agility and muscle soreness (DOMS) were assessed at baseline, and 24, 48 and 72 h post-exercise. Measures of inflammation (IL-1-β, IL-6, IL-8, TNF-α, hsCRP), muscle damage (CK) and oxidative stress (LOOH) were analysed at baseline and 1, 3, 5, 24, 48 and 72 h post-exercise. Performance indices (MVIC, CMJ and agility) recovered faster and muscle soreness (DOMS) ratings were lower in the MC group (p < 0.05). Additionally, the acute inflammatory response (IL-6) was attenuated in the MC group. There were no effects for LOOH and CK. These findings suggest MC is efficacious in accelerating recovery following prolonged, repeat sprint activity, such as soccer and rugby, and lends further evidence that polyphenol-rich foods like MC are effective in accelerating recovery following various types of strenuous exercise.


CCR5 deletion protects against inflammation-associated mortality in dialysis patients.
The CC-chemokine receptor 5 (CCR5) is a receptor for various proinflammatory chemokines, and a deletion variant of the CCR5 gene (CCR5 Delta 32) leads to deficiency of the receptor. We hypothesized that CCR5 Delta 32 modulates inflammation-driven mortality in patients with ESRD. We studied the interaction between CCR5 genotype and levels of high-sensitivity C-reactive protein (hsCRP) in 603 incident dialysis patients from the multicenter, prospective NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort. CCR5 genotype and hsCRP levels were both available for 413 patients. During 5 yr of follow-up, 170 patients died; 87 from cardiovascular causes. Compared with the reference group of patients who had the wild-type CCR5 genotype and hsCRP <or= 10 mg/L (n = 225), those carrying the deletion allele with hsCRP <or= 10 mg/L (n = 55) had similar mortality, and those carrying the wild-type genotype with hsCRP > 10 mg/L (n = 108) had an increased risk for mortality (HR: 1.82; 95% CI: 1.29 to 2.58). However, those carrying the deletion allele with hsCRP > 10 mg/L (n = 25) had a mortality rate similar to the reference group; this seemingly protective effect of the CCR5 deletion was even more pronounced for cardiovascular mortality. We replicated these findings in an independent Swedish cohort of 302 ESRD patients. In conclusion, the CCR5 Delta 32 polymorphism attenuates the adverse effects of inflammation on overall and cardiovascular mortality in ESRD.


In-depth profile analysis of filled alumina and titania nanostructured templates by radiofrequency glow discharge coupled to optical emission spectrometry.
The development of highly ordered and self-assembled magnetic nanostructures such as arrays of Fe or Ni nanowires and their alloys is arousing increasing interest due to the peculiar magnetic properties of such materials at the nanoscale. These nanostructures can be fabricated using nanoporous anodic alumina membranes or self-assembled nanotubular titanium dioxide as templates. The chemical characterization of the nanostructured layers is of great importance to assist the optimization of the filling procedure or to determine their manufacturing quality. Radiofrequency glow discharge (RF-GD) coupled to optical emission spectrometry (OES) is a powerful tool for the direct analysis of either conducting or insulating materials and to carry out depth profile analysis of thin layers by multi-matrix calibration procedures. Thus, the capability of RF-GD-OES is investigated here for the in-depth quantitative analysis of self-aligned titania nanotubes and self-ordered nanoporous alumina filled with arrays of metallic and magnetic nanowires obtained using the template-assisted filling method. The samples analysed in this work consisted of arrays of Ni nanowires with different lengths (from 1.2 up to 5 microm) and multilayer nanowires of alternating layers with different thicknesses (of 1-2 microm) of Ni and Au, or Au and FeNi alloy, deposited inside the alumina and titania membranes. Results, compared with other techniques such as scanning electron microscopy and energy-dispersive X-ray spectroscopy, show that the RF-GD-OES surface analysis technique proves to be adequate and promising for this challenging application.


Comparative effectiveness of low-molecular-weight heparin versus unfractionated heparin for thromboembolism prophylaxis for medical patients.
Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are approved for venous thromboembolism (VTE) prophylaxis. Which agent is superior remains controversial. To compare the effectiveness, complications, and costs of UFH and LMWH as VTE prophylaxis for hospitalized medical patients. Retrospective cohort. Three hundred thirty-three acute care facilities in 2004-2005. Adults with 4 common medical diagnoses considered to carry moderate-to-high risk of VTE. Excluded were patients on warfarin or with hospital stays of ≤ 2 days. VTE prophylaxis was assessed from billing data. None. VTE, major bleeding or heparin-induced thrombocytopenia, mortality, and cost. Of 32,104 patients who received prophylaxis, 55% received LMWH and the remainder received UFH. The hospital where the patient obtained care was the strongest predictor of receiving LMWH. VTE was observed in 163 (0.51%) patients; complications, followed by stopping therapy, were rare (<0.2%). In analysis adjusted for the propensity for UFH and other covariates, patients treated with UFH had an odds ratio for VTE of 1.04 (95% confidence interval [CI] 0.76 to 1.43) compared to LMWH. In a grouped treatment model, the odds of VTE with UFH was 1.14 (95% CI 0.72 to 1.81). Adjusted odds of bleeding with UFH compared to LMWH were 1.64 (95% CI 0.50 to 5.33), adjusted odds of complications followed by stopping prophylaxis were 2.84 (95% CI 1.43 to 45.66), and adjusted cost ratio was 0.97 (95% CI 0.90 to 1.05). For VTE prophylaxis, the effectiveness and cost of LMWH and UFH are similar, but LMWH is associated with fewer complications.


Proapoptotic activity of cell-permeable anti-Akt single-chain antibodies.
We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage-displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzyme's substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST-anti-Akt1-MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser(473) Akt and GSK-3alpha/beta phosphorylation, suggesting that Ser(473) is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited in vitro by the anti-Akt1 fusion protein. Furthermore, GST-anti-Akt1-MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST-anti-Akt1-MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both in vitro and in vivo.


Association Between Portable Screen-Based Media Device Access or Use and Sleep Outcomes: A Systematic Review and Meta-analysis.
Sleep is vital to children's biopsychosocial development. Inadequate sleep quantity and quality is a public health concern with an array of detrimental health outcomes. Portable mobile and media devices have become a ubiquitous part of children's lives and may affect their sleep duration and quality. To conduct a systematic review and meta-analysis to examine whether there is an association between portable screen-based media device (eg, cell phones and tablet devices) access or use in the sleep environment and sleep outcomes. A search strategy consisting of gray literature and 24 Medical Subject Headings was developed in Ovid MEDLINE and adapted for other databases between January 1, 2011, and June 15, 2015. Searches of the published literature were conducted across 12 databases. No language restriction was applied. The analysis included randomized clinical trials, cohort studies, and cross-sectional study designs. Inclusion criteria were studies of school-age children between 6 and 19 years. Exclusion criteria were studies of stationary exposures, such as televisions or desktop or personal computers, or studies investigating electromagnetic radiation. Of 467 studies identified, 20 cross-sectional studies were assessed for methodological quality. Two reviewers independently extracted data. The primary outcomes were inadequate sleep quantity, poor sleep quality, and excessive daytime sleepiness, studied according to an a priori protocol. Twenty studies were included, and their quality was assessed. The studies involved 125 198 children (mean [SD] age, 14.5 [2.2] years; 50.1% male). There was a strong and consistent association between bedtime media device use and inadequate sleep quantity (odds ratio [OR], 2.17; 95% CI, 1.42-3.32) (P < .001, I2 = 90%), poor sleep quality (OR, 1.46; 95% CI, 1.14-1.88) (P = .003, I2 = 76%), and excessive daytime sleepiness (OR, 2.72; 95% CI, 1.32-5.61) (P = .007, I2 = 50%). In addition, children who had access to (but did not use) media devices at night were more likely to have inadequate sleep quantity (OR, 1.79; 95% CI, 1.39-2.31) (P < .001, I2 = 64%), poor sleep quality (OR, 1.53; 95% CI, 1.11-2.10) (P = .009, I2 = 74%), and excessive daytime sleepiness (OR, 2.27; 95% CI, 1.54-3.35) (P < .001, I2 = 24%). To date, this study is the first systematic review and meta-analysis of the association of access to and the use of media devices with sleep outcomes. Bedtime access to and use of a media device were significantly associated with the following: inadequate sleep quantity, poor sleep quality, and excessive daytime sleepiness. An integrated approach among teachers, health care professionals, and parents is required to minimize device access at bedtime, and future research is needed to evaluate the influence of the devices on sleep hygiene and outcomes.


Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease.
Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t-distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.


Molecular and cytogenetical characterization of the 10A1-2 band and adjoining region in the Drosophila melanogaster polytene X chromosome.
Some 300 kb of DNA from the 9F12-10A7 X chromosome region (seven bands) uncovered by Df(1)vL3 were cloned and 31 break points of chromosome rearrangements within the region were mapped. Positions of 12 genes found earlier in genetic saturation experiments, transcripts and P element-induced mutations were located on the physical map using either chromosome rearrangements or Southern blot hybridizations. Data on the position of the break points, genes and polytene chromosome bands allow the following conclusions to be made. (1) The size of the bands in the region varies between 4 kb (10A6 and 7) and 183-195 kb (10A1-2). The compaction ratio of DNA in bands varies from 8-36 (10A6 + 7) to 151-161 (10A1-2). Therefore, fine and thick bands appear to have different kinds of DNP packaging. (2) The bands differ in genetic content. Fine bands contain from one to three genes. In contrast, the 10A1-2 band contains three genes and at least six transcribed DNA fragments. (3) Comparison of genetic and physical maps shows that in this region 0.01 centiMorgan corresponds to 3.3 kb of DNA.


Morphological study of acoustic liposomes using transmission electron microscopy.
Sonoporation is achieved by ultrasound-mediated destruction of ultrasound contrast agents (UCA) microbubbles. For this, UCAs must be tissue specific and have good echogenicity and also function as drug carriers. Previous studies have developed acoustic liposomes (ALs), liposomes that encapsulate phosphate buffer solution and perfluoropropane (C(3)F(8)) gas and function as both UCAs and drug carriers. Few studies have examined the co-existence of gas and liquid in ALs. This study aims to elucidate AL structure using TEM. The size, zeta potential and structure of ALs were compared with those of two other UCAs, human albumin shell bubbles (ABs; Optison) and lipid bubbles (LBs). ABs and LBs encapsulate the C(3)F(8) gas. Particle size was measured by dynamic light scattering. The zeta potential was determined by the Smoluchowski equation. UCA structure was investigated by TEM. ALs were approximately 200 nm in size, smaller than LBs and ABs. ALs and LBs had almost neutral zeta potentials whereas AB values were strongly negative. The negative or double staining TEM images revealed that approximately 20% of ALs contained both liquid and gas, while approximately 80% contained liquid alone (i.e. nonacoustic). Negative staining AB images indicated electron beam scattering near the shell surface, and albumin was detected in filament form. These findings suggest that AL is capable of carrying drugs and high-molecular-weight, low-solubility gases.


Effectiveness of a grant program's efforts to promote synergy within its funded initiatives: perceptions of participants of the Southern Rural Access Program.
Foundations and public agencies commonly fund focused initiatives for individual grantees. These discrete, stand-alone initiatives can risk failure by being carried out in isolation. Fostering synergy among grantees' initiatives is one strategy proposed for promoting the success and impact of grant programs. We evaluate an explicit strategy to build synergy within the Robert Wood Johnson Foundation's Southern Rural Access Program (SRAP), which awarded grants to collaboratives within eight southeastern U.S. states to strengthen basic health care services in targeted rural counties. We interviewed 39 key participants of the SRAP, including the program director within each state and the principal subcontractors heading the program's funded initiatives that supported heath professionals' recruitment, retention and training, made loans to health care providers, and built networks among providers. Interews were recorded and transcribed. Two investigators independently coded the transcripts and a third investigator distilled the main points. Participants generally perceived that the SRAP yielded more synergies than other grant programs in which they had participated and that these synergies added to the program's impact. The synergies most often noted were achieved through relationship building among grantees and with outside agencies, sharing information and know-how, sharing resources, combining efforts to yield greater capacity, joining voices to advocate for common goals, and spotting gaps in services offered and then filling these gaps. The SRAP's strategies that participants felt fostered synergy included targeting funding to culturally and geographically similar states, supporting complementary types of initiatives, promoting opportunities to network through semi-annual meetings and regular conference calls, and the advocacy efforts of the program's leadership. Participants noted that synergies were sometimes hindered by turf issues and politics and the conflicting perspectives and cultures of participating organizations and racial groups. Inadequate funding through the SRAP, restricting program involvement to only a few needy counties, and instances of over- and under-involvement by the program's leadership were sometimes felt to inhibit synergies and/or their sustainability. Participants of the SRAP generally perceived that the SRAP's deliberate strategies yielded synergies that added to the program's impact.


Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study.
The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.


Floral Induction in a Photoperiodically Insensitive Duckweed, Lemna paucicostata LP6 : Role of Glutamate, Aspartate, and Other Amino Acids and Amides.
The effects of 20 amino acids and two amides were studied on the flowering of a photoperiodically insensitive duckweed, Lemna paucicostata LP6. Alanine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, lysine, methionine, proline, serine, and threonine induced flowering under a photoperiodic regime of 16 hours light and 8 hours darkness. Among these, glutamate and aspartate were found to be the most effective for flower induction. These acids could initiate flowering even at 5 x 10(-7) molar level, though maximal flowering (about 80%) was obtained at 10(-5) molar. Change in the photoperiodic schedule or the pH of the nutrient medium did not influence glutamate- or aspartate-induced flowering. The low concentrations at which glutamate and aspartate are effective suggests that they may have a regulatory role rather than simply acting as metabolites.


First interim analysis of the GIDEON (Global Investigation of therapeutic decisions in hepatocellular carcinoma and of its treatment with sorafeNib) non-interventional study.
Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib (GIDEON), a global, non-interventional, surveillance study, aims to evaluate the safety of sorafenib in all patients with unresectable hepatocellular carcinoma (uHCC) under real-life practice conditions, particularly Child-Pugh B patients, who were not well represented in clinical trials. Treatment decisions are determined by each physician according to local prescribing guidelines and clinical practice. Patients with uHCC who are candidates for systemic therapy, and for whom a decision has been made to treat with sorafenib, are eligible for inclusion. Demographic data and medical and disease history are recorded at entry. Sorafenib dosing and adverse events (AEs) are collected throughout the study. From January 2009 to April 2011, >3000 patients from 39 countries were enrolled. The prespecified first interim analysis was conducted when the initial approximately 500 treated patients had been followed up for ≥4 months; 479 were valid for safety evaluation. Preplanned subgroup analyses indicate differences in patient characteristics, disease aetiology and previous treatments by region. Variation in sorafenib dosing by specialty are also observed; Child-Pugh status did not appear to influence the starting dose of sorafenib. The type and incidence of AEs was consistent with findings from previous clinical studies. AE profiles were comparable between Child-Pugh subgroups. The GIDEON study is generating a large, robust database from a broad population of patients with uHCC. First interim analyses have shown global and regional differences in patient characteristics, disease aetiology and practice patterns. Subsequent planned analyses will allow further evaluation of early trends.


Parkinson's disease in Sweden-resource use and costs by severity.
To estimate resource use and costs, including direct and indirect costs, in relation to levels of severity in individuals with Parkinson's disease (PD) in a Swedish setting. Patients with idiopathic PD registered in the National Parkinson's Disease Patient Registry (PARKreg), with registrations of Hoehn and Yahr (H&Y) and "off time" in the Skåne Region, were included. Annual costs of healthcare contacts, drugs, formal and informal care, and productivity loss associated with PD were estimated using data from PARKreg linked with regional and national healthcare registers between 2013 and 2019. In total, 960 patients and 1324 observations (patient-years) were included. Total average cost per patient-year was SEK 168,982 (EUR 15,958) and ranged from SEK 62,404 (EUR 5893) for H&Y stage I to SEK 1,056,324 (EUR 99,755) in H&Y stage V. The dominating part of total costs for early stages were indirect costs accounting for 50-60% while formal care made up for 55% and 81% of total costs in H&Y IV and V, respectively. Total mean costs for formal care, informal care, and productivity loss also increased with increasing off-time. Advanced and late stages of PD are associated with significant societal costs as patients in those stages often require resource-intensive and costly formal care. Thus, there are potential savings to be made, by optimizing the pharmacological and surgical symptomatic treatment of patients with advanced disease.


HitWalker: variant prioritization for personalized functional cancer genomics.
Determining the functional relevance of identified sequence variants in cancer is a prerequisite to ultimately matching specific therapies with individual patients. This level of mechanistic understanding requires integration of genomic information with complementary functional analyses to identify oncogenic targets and relies on the development of computational frameworks to aid in the prioritization and visualization of these diverse data types. In response to this, we have developed HitWalker, which prioritizes patient variants relative to their weighted proximity to functional assay results in a protein-protein interaction network. It is highly extensible, allowing incorporation of diverse data types to refine prioritization. In addition to a ranked list of variants, we have also devised a simple shortest path-based approach of visualizing the results in an intuitive manner to provide biological interpretation. The program, documentation and example data are available as an R package from www.biodevlab.org/HitWalker.html.


How the neck affects the back: changes in regional cervical sagittal alignment correlate to HRQOL improvement in adult thoracolumbar deformity patients at 2-year follow-up.
OBJECT Regional cervical sagittal alignment (C2-7 sagittal vertical axis [SVA]) has been shown to correlate with health-related quality of life (HRQOL). The study objective was to examine the relationship between cervical and thoracolumbar alignment parameters with HRQOL among patients with operative and nonoperative adult thoracolumbar deformity. METHODS This is a multicenter prospective data collection of consecutive patients with adult thoracolumbar spinal deformity. Clinical measures of disability included the Oswestry Disability Index (ODI), Scoliosis Research Society-22 Patient Questionnaire (SRS-22), and 36-Item Short-Form Health Survey (SF-36). Cervical radiographic parameters were correlated with global sagittal parameters within the nonoperative and operative cohorts. A partial correlation analysis was performed controlling for C-7 SVA. The operative group was subanalyzed by the magnitude of global deformity (C-7 SVA ≥ 5 cm vs < 5 cm). RESULTS A total of 318 patients were included (186 operative and 132 nonoperative). The mean age was 55.4 ± 14.9 years. Operative patients had significantly worse baseline HRQOL and significantly larger C-7 SVA, pelvic tilt (PT), mismatch between pelvic incidence and lumbar lordosis (PI-LL), and C2-7 SVA. The operative patients with baseline C-7 SVA ≥ 5 cm had significantly larger C2-7 lordosis (CL), C2-7 SVA, C-7 SVA, PI-LL, and PT than patients with a normal C-7 SVA. For all patients, baseline C2-7 SVA and CL significantly correlated with baseline ODI, Physical Component Summary (PCS), SRS Activity domain, and SRS Appearance domain. Baseline C2-7 SVA also correlated with SRS Pain and SRS Total. For the operative patients with baseline C-7 SVA ≥ 5 cm, the 2-year C2-7 SVA significantly correlated with 2-year Mental Component Summary, SRS Mental, SRS Satisfaction, and decreases in ODI. Decreases in C2-7 SVA at 2 years significantly correlated with lower ODI at 2 years. Using partial correlations while controlling for C-7 SVA, the C2-7 SVA correlated significantly with baseline ODI (r = 0.211, p = 0.002), PCS (r = -0.178, p = 0.009), and SRS Activity (r = -0.145, p = 0.034) for the entire cohort. In the subset of operative patients with larger thoracolumbar deformities, the change in C2-7 SVA correlated with change in ODI (r = -0.311, p = 0.03). CONCLUSIONS Changes in cervical lordosis correlate to HRQOL improvements in thoracolumbar deformity patients at 2-year follow-up. Regional cervical sagittal parameters such as CL and C2-7 SVA are correlated with clinical measures of regional disability and health status in patients with adult thoracolumbar scoliosis. This effect may be direct or a reciprocal effect of the underlying global deformities on regional cervical alignment. However, the partial correlation analysis, controlling for the magnitude of the thoracolumbar deformity, suggests that there is a direct effect of cervical alignment on health measures. Improvements in regional cervical alignment postoperatively correlated positively with improved HRQOL.


Effects of tumor-associated macrophages on the proliferation and migration of esophageal cancer-associated lymphatic endothelial cells.
The aim of this study was to explore whether M2 macrophages can be transformed into M1 macrophages, and to investigate the effect of different types of macrophages on the proliferation, migration and ring-forming ability of esophageal cancer-related lymphatic endothelial cell (LEC). Human monocytic leukemia cell line (THP-1 cell) was induced to differentiate to M1 macrophages (M1 group) and M2 macrophages (M2 group), and co-cultured with esophageal cancer-associated LEC. The individual esophageal cancer co-cultured with LEC was used as control. Different types of macrophages were observed by Cell counting kit-8 (CCK-8). Enzyme-linked immunosorbent assay (ELISA) was used to detect the VEGF-C concentration; the expression of VEGFR-3 protein and its mRNA was detected by Western blot and qRT-PCR, respectively. The positive rate of the M1 group induced by IFN-γ and LPS was significantly higher than that of M2 macrophages (48.57%5.98% vs 25.83%1.95%). The expression of VEGF-C in the supernatant of the M2 group was higher than that in the control group, but no significant differences regarding the expression of VEGF-C between M1 and control groups were found. In addition, the expression of VEGFR-3 on both mRNA and protein in esophageal cancer-related LEC of the M2 group was significantly higher than those in the control group; however, the M1 group had a significantly lower VEGFR-3 level on both mRNA and protein than the control group. Human M2 macrophages can be transformed into M1 macrophages, and can promote the proliferation, migration and ring-forming ability of esophageal cancer-associated LEC.


Further evidence for the likely completeness of the library of solved single domain protein structures.
Recent studies questioned whether the Protein Data Bank (PDB) contains all compact, single domain protein structures. Here, we show that all quasi-spherical, QS, random protein structures devoid of secondary structure are in the PDB and are excellent templates for all native PDB proteins up to 250 residues. Because QS templates have a similar global contour as native, TASSER can refine 98% (90%) of those whose TM-score is 0.4 (0.35) to structures greater than or equal to the 0.5 TM-score threshold (0.74 (0.64) mean TM-score) for CATH/SCOP assignment. On the basis of this and the fact that, at a TM-score of 0.4, 83% (90%) of all (internal) core secondary structure elements are recovered, a 0.40 TM-score is an appropriate fold similarity assignment threshold. Despite the claims of Taylor, Trovato, and Zhou that many of their structures lack a PDB counterpart, using fr-TM-align, at a 0.45 (0.5) TM-score threshold, essentially all (most) are found in the PDB. Thus, the conclusion that the PDB is likely complete is further supported.


Safety behaviour: a reconsideration.
There is ample evidence that the use of safety behaviour can interfere with the progress of therapy, particularly if exposure is involved. As a result, it is widely asserted that safety behaviour is anti-therapeutic. However, an unqualified rejection of safety behaviour should be reconsidered because we now have theoretical justification, experimental evidence and clinical observations showing that the judicious use of safety behaviour, especially in the early stages of treatment, can be facilitative. Experiments in which escape behaviour facilitated fear reduction, and others in which the use of safety gear facilitated fear reduction, are reviewed. It also appears that safety behaviour does not necessarily prevent disconfirmatory experiences. We propose that additional investigations of the judicious use of safety behaviour will help to elucidate therapeutic uses of safety behaviour in the treatment of anxious and related types of psychopathology.


The first ReI organometallic complex with an organoimido-polyoxometalate ligand.
The spectroscopic, electrochemical and photophysical properties of the first ReI organometallic organoimido-polyoxometalate complex [n-Bu4N][Mo6O18NC6H4-CH2-N3C2H2-Re-phen(CO)3] compared with all fragments are reported. The UV-Vis spectra are analysed using experimental and theoretical tools. In contrast to the reported studies in the literature, our results show that a new more intense band is present in the spectra of the hybrid ligand obscuring the intra-polyanion charge transfer. The electrochemical results show that the strong acceptor character of the polyoxometalate fragment is quenched by the condensation of the phenyl-triazole molecule.


Preliminary development and validation of a new end-of-life patient-reported outcome measure assessing the ability of patients to finalise their affairs at the end of life.
The ability of patients to finalise their affairs at the end of life is an often neglected aspect of quality of life (QOL) measurement in palliative care effectiveness research despite compelling evidence of the high value patients place on this domain. This paper describes the preliminary development and evaluation of a new, single-item, end-of-life patient-reported outcome measure (EOLPRO) designed to capture changes in the ability of patients to finalise their affairs at the end of life. Cognitive interviews with purposively sampled Australian palliative care patients (N = 9) were analysed thematically to explore content validity. Simultaneously, secondary analysis of data from a randomised controlled trial comparing ketamine and placebo for the management of cancer pain (N = 185) evaluated: construct validity; test-retest reliability; and responsiveness. Preliminary findings suggest patients interpret the new measure consistently. The EOLPRO captures the ability to complete physical tasks and finalise practical matters although it is unclear whether emotional tasks or resolution of relationship issues are considered. Personal and financial affairs should be separated to allow for differences in ability for these two types of affairs. The significant correlation between performance status and EOLPRO scores (r = 0.41, p<0.01, n = 137) and expected relationships between EOLPRO and proximity to death and constipation demonstrated construct validity. Pre- and post-treatment EOLPRO scores moderately agreed (n = 14, κ = 0.52 [95% CI 0.19, 0.84]) supporting reliability. The measure's apparent lack of sensitivity to discriminate between treatment responders and non-responders may be confounded. Based on the preliminary findings, the EOLPRO should be separated into 'personal' and 'financial' affairs with further testing suggested, particularly to verify coverage and responsiveness. Initial evaluation suggests that the single-item EOLPRO is a useful addition to QOL outcome measurement in palliative care effectiveness research because common palliative care specific QOL questionnaires do not include or explicitly capture this domain.


Factors associated with the use of home-visit nursing services covered by the long-term care insurance in rural Japan: a cross-sectional study.
In Japan, there is a large increase in the number of elderly persons who potentially need home-visit nursing services (VNS). However, the number of persons using the VNS has increased only little in comparison to the number of individuals who use home social services, which are also covered by the Long-Term Care Insurance (LTCI) system. This cross-sectional study investigated the predictors of the VNS used under the LTCI system in Japan. We used 1,580 claim data from all the users of community-based services and 1,574 interview survey data collected in 2001 from the six municipal bodies in Japan. After we merged the two datasets, 1,276 users of community-based services under the LTCI were analyzed. Multiple logistic regression models stratified by care needs levels were used for analysis. Only 8.3% of the study subjects were VNS users. Even among study participants within the higher care-needs level, only 22.0% were VNS users. In the lower care level group, people with a higher care level (OR: 3.50, 95% CI: 1.50-8.93), those whose condition needed long term care due to respiratory or heart disease (OR: 4.31, 95% CI: 1.88-89.20), those whose period of needing care was two years or more (OR: 2.01, 95% CI: 1.14-3.48), those whose service plan was created by a medical care management agency (OR: 2.39, 95% CI: 1.31-4.33), those living with family (OR: 1.86, 95% CI: 1.00-3.42), and those who use home-help services (OR: 2.12, 95% CI: 1.17-3.83) were more likely to use the VNS. In the higher care level group, individuals with higher care level (OR: 3.63, 95% CI: 1.56-8.66), those with higher income (OR: 3.79, 95% CI: 1.01-14.25), and those who had regular hospital visits before entering the LTCI (OR: 2.36, 95% CI: 1.11-5.38) were more likely to use the VNS. Our results suggested that VNS use is limited due to management by non-medical care management agencies, due to no caregivers being around or a low income household. The findings of this study provide valuable insight for LTCI policy makers: the present provision of VNS should be reconsidered.


Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle.
The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.


Nutrition education for osteoporosis prevention in men with prostate cancer initiating androgen deprivation therapy.
Osteoporosis is a common side effect of treatment with androgen deprivation therapy (ADT) in men with prostate cancer. ADT may prolong survival; however, deterioration of bone mass density occurs soon after initiation. A systematic review of current literature revealed the importance of adequate nutrition during treatment with ADT to reduce the risk of osteoporosis. More specifically, this literature stressed achieving adequate intake of calcium and vitamin D through a combination of supplements and food. The necessity of providing nutrition education to patients with prostate cancer at initiation of ADT was identified. Healthcare professionals, including nurses, oncologists, and dietitians, can be instrumental in identifying patients with prostate cancer initiating ADT who are at risk for osteoporosis. Research on nutrition and lifestyle modification interventions to maintain bone health and reduce fracture risk for patients initiating ADT is limited. Additional research is required to develop and evaluate nutrition education interventions that will reduce the risk and prevent osteoporosis in men on ADT.


Association of radiologic findings with mortality of patients infected with 2019 novel coronavirus in Wuhan, China.
Radiologic characteristics of 2019 novel coronavirus (2019-nCoV) infected pneumonia (NCIP) which had not been fully understood are especially important for diagnosing and predicting prognosis. We retrospective studied 27 consecutive patients who were confirmed NCIP, the clinical characteristics and CT image findings were collected, and the association of radiologic findings with mortality of patients was evaluated. 27 patients included 12 men and 15 women, with median age of 60 years (IQR 47-69). 17 patients discharged in recovered condition and 10 patients died in hospital. The median age of mortality group was higher compared to survival group (68 (IQR 63-73) vs 55 (IQR 35-60), P = 0.003). The comorbidity rate in mortality group was significantly higher than in survival group (80% vs 29%, P = 0.018). The predominant CT characteristics consisted of ground glass opacity (67%), bilateral sides involved (86%), both peripheral and central distribution (74%), and lower zone involvement (96%). The median CT score of mortality group was higher compared to survival group (30 (IQR 7-13) vs 12 (IQR 11-43), P = 0.021), with more frequency of consolidation (40% vs 6%, P = 0.047) and air bronchogram (60% vs 12%, P = 0.025). An optimal cutoff value of a CT score of 24.5 had a sensitivity of 85.6% and a specificity of 84.5% for the prediction of mortality. 2019-nCoV was more likely to infect elderly people with chronic comorbidities. CT findings of NCIP were featured by predominant ground glass opacities mixed with consolidations, mainly peripheral or combined peripheral and central distributions, bilateral and lower lung zones being mostly involved. A simple CT scoring method was capable to predict mortality.


Testis-sparing surgery of unilateral testicular large-cell calcifying Sertoli cell tumor: a sporadic case.
Testicular sex cord-stromal tumors constitute a small portion of all types of testicular tumors. Only 1% of these tumors are pure Sertoli cell tumors. Sertoli cell tumors have three identified subgroups: large-cell calcifying (LCCSCT), sclerosing Sertoli cell and general type. Up to 40% of LCCSCT are associated with the genetic syndromes like Carney complex, Peutz-Jeghers syndrome. These lesions are usually benign, only 17% of the cases show malignant behaviour. Herein, we report a case of unilateral LCCSCT in a 37 year-old man without any hormonal symptoms or inherited diseases which was succesfully treated with testis-sparing surgery.


How do microRNAs regulate gene expression?
miRNAs (microRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. They generally bind to the 3'-UTR (untranslated region) of their target mRNAs and repress protein production by destabilizing the mRNA and translational silencing. The exact mechanism of miRNA-mediated translational repression is yet to be fully determined, but recent data from our laboratory have shown that the stage of translation which is inhibited by miRNAs is dependent upon the promoter used for transcribing the target mRNA. This review focuses on understanding how miRNA repression is operating in light of these findings and the questions that still remain.


Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.
The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.


Accuracy of musculoskeletal imaging for the diagnosis of polymyalgia rheumatica: systematic review.
To review the evidence for accuracy of imaging for diagnosis of polymyalgia rheumatica (PMR). Searches included MEDLINE, EMBASE and PubMed. Evaluations of diagnostic accuracy of imaging tests for PMR were eligible, excluding reports with <10 PMR cases. Two authors independently extracted study data and three authors assessed methodological quality using modified QUADAS-2 criteria. 26 studies of 2370 patients were evaluated: 10 ultrasound scanning studies; 6 MRI studies; 1 USS and MRI study; 7 18-fluorodeoxyglucose-positron emission tomography (PET) studies; 1 plain radiography and 1 technetium scintigraphy study. In four ultrasound studies, subacromial-subdeltoid bursitis had sensitivity 80% (95% CI 55% to 93%) and specificity 68% (95% CI 60% to 75%), whereas bilateral subacromial-subdeltoid bursitis had sensitivity 66% (95% CI 43% to 87%) and specificity 89% (95% CI 66% to 97%). Sensitivity for ultrasound detection of trochanteric bursitis ranged from 21% to 100%. In four ultrasound studies reporting both subacromial-subdeltoid bursitis and glenohumeral synovitis, detection of subacromial-subdeltoid bursitis was more accurate than that of glenohumeral synovitis (p=0.004). MRI and PET/CT revealed additional areas of inflammation in the spine and pelvis, including focal areas between the vertebrae and anterior to the hip joint, but the number of controls with inflammatory disease was inadequate for precise specificity estimates. Subacromial-subdeltoid bursitis appears to be the most helpful ultrasound feature for PMR diagnosis, but interpretation is limited by study heterogeneity and methodological issues, including variability in blinding and potential bias due to case-control study designs. Recent MRI and PET/CT case-control studies, with blinded readers, yielded promising data requiring validation within a diagnostic cohort study.


Interventions for replacing missing teeth: different types of dental implants.
Dental implants are available in different materials, shapes and with different surface characteristics. In particular, numerous implant designs and surface modifications have been developed for improving clinical outcome. This is an update of a Cochrane review first published in 2002, and previously updated in 2003, 2005 and 2007. Primary: to compare the clinical effects of different root-formed osseointegrated dental implant types for replacing missing teeth for the following specific comparisons: implants with different surface preparations, but having similar shape and material; implants with different shapes, but having similar surface preparation and material; implants made of different materials, but having similar surface preparation and shape; different implant types differing in surface preparation, shape, material or a combination of these.Secondary: to compare turned and roughened dental implants for occurrence of early implant failure (before prosthetic loading) and occurrence of peri-implantitis. We searched the following electronic databases: the Cochrane Oral Health Group's Trials Register (to 17 January 2014), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2013, Issue 12), MEDLINE via OVID (1946 to 17 January 2014) and EMBASE via OVID (1980 to 17 January 2014). We placed no restrictions on the language or date of publication when searching the electronic databases. We included any randomised controlled trial (RCT) comparing osseointegrated dental implants of different materials, shapes and surface properties having a follow-up in function of at least one year. Outcome measures were success of the implants, radiographic peri-implant marginal bone levels changes and incidence of peri-implantitis. At least two review authors independently conducted screening, risk of bias assessment and data extraction of eligible trials in duplicate. We expressed results using fixed-effect models (if up to three studies were present in a meta-analysis) or random-effects models (when there were more than three studies) using mean differences (MD) for continuous outcomes and risk ratios (RR) for dichotomous outcomes with 95% confidence intervals (CI). We reported the following endpoints: one, three, five and 10 years after functional loading. We identified 81 different RCTs. We included 27 of these RCTs, reporting results from 1512 participants and 3230 implants in the review. We compared 38 different implant types with a follow-up ranging from one to 10 years. All implants were made of commercially pure titanium or its alloys, and had different shapes and surface preparations. We judged two trials to be at low risk of bias, 10 to be at unclear risk of bias and 15 to be at high risk of bias. On a 'per participant' rather than 'per implant' basis, we found no significant differences between various implant types for implant failures. The only observed statistically significant difference for the primary objective regarded more peri-implant bone loss at Nobel Speedy Groovy implants when compared with NobelActive implants (MD -0.59 mm; 95% CI -0.74 to -0.44, different implant shapes). The only observed statistically significant difference for the secondary objective was that implants with turned (smoother) surfaces had a 20% reduction in risk to be affected by peri-implantitis than implants with rough surfaces three years after loading (RR 0.80; 95% CI 0.67 to 0.96). There was a tendency for implants with turned surfaces to fail early more often than implants with roughened surfaces. Based on the results of the included RCTs, we found no evidence showing that any particular type of dental implant had superior long-term success. There was limited evidence showing that implants with relatively smooth (turned) surfaces were less prone to lose bone due to chronic infection (peri-implantitis) than implants with much rougher surfaces (titanium-plasma-sprayed). These findings were based on several RCTs, often at high risk of bias, with few participants and relatively short follow-up periods.


Disintegrins from snake venoms and their applications in cancer research and therapy.
Integrins regulate diverse functions in cancer pathology and in tumor cell development and contribute to important processes such as cell shape, survival, proliferation, transcription, angiogenesis, migration, and invasion. A number of snake venom proteins have the ability to interact with integrins. Among these are the disintegrins, a family of small, non-enzymatic, and cysteine-rich proteins found in the venom of numerous snake families. The venom proteins may have a potential role in terms of novel therapeutic leads for cancer treatment. Disintegrin can target specific integrins and as such it is conceivable that they could interfere in important processes involved in carcinogenesis, tumor growth, invasion and migration. Herein we present a survey of studies involving the use of snake venom disintegrins for cancer detection and treatment. The aim of this review is to highlight the relationship of integrins with cancer and to present examples as to how certain disintegrins can detect and affect biological processes related to cancer. This in turn will illustrate the great potential of these molecules for cancer research. Furthermore, we also outline several new approaches being created to address problems commonly associated with the clinical application of peptide-based drugs such as instability, immunogenicity, and availability.


Effects of borate-based bioactive glass on neuron viability and neurite extension.
Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested to determine whether bioactive glasses have the potential for use in the treatment of peripheral nerve injury. In these experiments, degradable bioactive borate glass was fabricated into rods and microfibers. To study the compatibility with neurons, embryonic chick dorsal root ganglia (DRG) were cultured with different forms of bioactive borate glass. Cell viability was measured with no media exchange (static condition) or routine media exchange (transient condition). Neurite extension was measured within fibrin scaffolds with embedded glass microfibers or aligned rod sheets. Mixed cultures of neurons, glia, and fibroblasts growing in static conditions with glass rods and microfibers resulted in decreased cell viability. However, the percentage of neurons compared with all cell types increased by the end of the culture protocol compared with culture without glass. Furthermore, bioactive glass and fibrin composite scaffolds promoted neurite extension similar to that of control fibrin scaffolds, suggesting that glass does not have a significant detrimental effect on neuronal health. Aligned glass scaffolds guided neurite extension in an oriented manner. Together these findings suggest that bioactive glass can provide alignment to support directed axon growth.


Copper-64 radiolabeling and biological evaluation of bifunctional chelators for radiopharmaceutical development.
The development of novel bifunctional chelates for attaching copper-64 to biomolecules has been an active area of research for several years. However, many of these (64)Cu-chelates have poor in vivo stability or harsh radiolabeling conditions. In this study, two triazacyclononane analogs; C-NE3TA (4-carboxymethyl-7-[2-(carboxymethyl-amino)-3-(4-nitro-phenyl)-propyl]-[1,4,7]triazo-nan-1-yl-acetic acid) and N-NE3TA (4-carboxymethyl-7-[2-[carboxymethyl-(4-nitro-benzyl)-amino]-ethyl]-[1,4,7]triazonan-1-yl-acetic acid) were evaluated for their labeling efficiency with (64)Cu at room temperature and evaluated in vitro and in vivo. In vitro studies included complexation kinetics with Cu(II) using a spectrophotometric method and rat serum stability, while the in vivo biodistribution was evaluated using SCID mice. C-NE3TA and N-NE3TA were labeled at >95% efficiency up to ~3.4Ci/μmol. Both C-NE3TA and N-NE3TA formed complexes with Cu(II) almost immediately, with the Cu(II) complexation by C-NE3TA being faster than the formation of Cu(II)-N-NE3TA. Both (64)Cu-N-NE3TA and (64)Cu-C-NE3TA were 96.1% and 90.5% intact after 48h incubation in rat serum, respectively. This is compared to (64)Cu complexes of the control chelators, p-NH(2)-Bn-DOTA and p-NH(2)-Bn-NOTA, with 93.9% and 97.9% retention of (64)Cu in the complex, respectively. In vivo evaluation of (64)Cu-N-NE3TA and (64)Cu-C-NE3TA demonstrates good clearance from normal tissues except for the liver, where 59% and 51% of the radioactivity is retained at 24h compared to 1h for (64)Cu-N-NE3TA and (64)Cu-C-NE3TA, respectively. This compares to 78% and 3% retention for (64)Cu-p-NH(2)-Bn-DOTA and (64)Cu-p-NH(2)-Bn-NOTA. These studies demonstrate that while N-NE3TA and C-NE3TA appear to be superior chelators for (64)Cu than p-NH(2)-Bn-DOTA, they are not better than p-NH(2)-Bn-NOTA. Nevertheless, it may still be interesting to evaluate these chelators after conjugation to biomolecules.


Anti-cancer effect of metformin by suppressing signaling pathway of HER2 and HER3 in tamoxifen-resistant breast cancer cells.
Development of new therapeutic strategies is becoming increasingly important to overcome tamoxifen resistance. Recently, much interest has been focused on anti-tumor effects of metformin commonly used to treat type II diabetes. Increased protein expression and signaling of epidermal growth factor receptor (EGFR) family is a possible mechanism involved in tamoxifen resistance. Since HER2/HER3 heterodimers are able to induce strong downstream signaling and activate various biological responses such as cellular proliferation and growth, we investigated the anti-cancer effect of metformin by inhibition of signaling pathway via downregulation of HER2 and HER3 using tamoxifen-resistant MCF-7 (TR MCF-7) cells. Compared to MCF-7 cells, TR MCF-7 cells showed increased expression of EGFR, HER2, and HER3, and metformin inhibited the expression of these proteins in a dose- and time-dependent manner. Metformin inhibited activation of HER2 (Tyr1248)/HER3 (Tyr1289)/Akt (Ser473) as well as cell proliferation and colony formation by estrogenic promotion in MCF-7 and TR MCF-7 cells. Known as a HER3 ligand, heregulin (HRG)-β1-induced phosphorylation of HER2, HER3 and Akt, and protein interaction of HER2/HER3 and colony formation were inhibited by metformin in both cells. Consistent with the results in the two cell lines, we identified that metformin inhibited HER2/HER3/Akt signaling axis activated by HRG-β1 using the HER2 and HER3-overexpressing breast cancer cell line SK-BR-3. Lastly, lapatinib-induced HER3 upregulation was significantly inhibited by treatment of metformin in HER3 siRNA-transfected TR MCF-7 cells. These data suggest that metformin might overcome tamoxifen resistance through the inhibition of expression and signaling of receptor tyrosine kinase HER2 and HER3.


Cell Phone Use Habits Among the Spanish Population: Contribution of Applications to Problematic Use.
Mobile phone abuse may be associated with health problems as well as with interferences in daily life. However, beyond the mobile as a device, the contributions of specific utilities and applications to the problematic mobile phone use remains to be analyzed. To address this important question we conducted 1,126 online interviews in Spain with participants aged 16 to 65 who are representative of the general population. The aim of the study was to analyze the patterns and differences of cell phone use based on habitual use, abuse, and problematic use, considering the most frequently used utilities and applications. Additional variables used were personal perception of cell phones, intensity of use, and participant's lifestyle with regard to entertainment and the maintenance of healthy habits. Further, we aimed to analyze difference between problematic and non-problematic mobile phone users in the utilization of applications, controlling for additional variables such as age, gender, educational level, consumption of tobacco and alcohol and illegal drugs. Results show that problematic use is consistent with the self-perception of abuse and is related to internet browsing, social media, music, and mobile games; particular applications of interest include Facebook, music applications, and Twitter. Furthermore, among problematic users, a pattern of interference with other activities or inappropriate use in certain contexts is observed, with a differential pattern of entertainment and abandonment of healthy habits. Finally, two binary logistic regression analyses demonstrated that beyond the cell phones themselves, specific utilities and applications such as browsing, chatting, or downloading and listening to music contribute to the differences between problematic and non-problematic users. Specifically, the use of Facebook, Twitter, and music applications have the greatest power to discriminate between the two types of users.


Cardiovascular risk in rheumatoid arthritis versus osteoarthritis: acute phase response related decreased insulin sensitivity and high-density lipoprotein cholesterol as well as clustering of metabolic syndrome features in rheumatoid arthritis.
Rheumatoid arthritis (RA) patients experience a markedly increased frequency of cardiovascular disease. We evaluated cardiovascular risk profiles in 79 RA patients and in 39 age-matched and sex-matched osteoarthritis (OA) patients. Laboratory tests comprised ultrasensitive C-reactive protein (CRP) and fasting lipids. Insulin sensitivity (IS) was determined by the Quantitative Insulin Sensitivity Check Index (QUICKI) in all OA patients and in 39 of the RA patients. Ten RA patients were on glucocorticoids. RA patients exercised more frequently than OA patients (chi2 = 3.9, P < 0.05). Nine RA patients and one OA patient had diabetes (chi2 = 4.5, P < 0.05). The median CRP, the mean QUICKI and the mean high-density lipoprotein (HDL) cholesterol were 9 mg/l (range, 0.5-395 mg/l), 0.344 (95% confidence interval [CI], 0.332-0.355) and 1.40 mmol/l (95% CI, 1.30-1.49 mmol/l) in RA patients, respectively, as compared with 2.7 mg/l (range, 0.3-15.9 mg/l), 0.369 (95% CI, 0.356-0.383) and 1.68 mmol/l (95% CI, 1.50-1.85 mmol/l) in OA patients. Each of these differences was significant (P < 0.05). After controlling for the CRP, the QUICKI was similar in RA and OA patients (P = 0.07), while the differences in HDL cholesterol were attenuated but still significant (P = 0.03). The CRP correlated with IS, while IS was associated with high HDL cholesterol and low triglycerides in RA patients and not in OA patients. A high CRP (>/= 8 mg/l) was associated with hypertension (chi2 = 7.4, P < 0.05) in RA patients. RA glucocorticoid and nonglucocorticoid users did not differ in IS and lipids (P > 0.05). Excess cardiovascular risk in RA patients as compared with OA patients includes the presence of decreased IS and HDL cholesterol in RA patients. The latter is only partially attributable to the acute phase response. The CRP, IS, HDL cholesterol, triglycerides and hypertension are inter-related in RA patients, whereas none of these relationships were found in OA patients.


Misconceptions about breast lumps and delayed medical presentation in urban breast cancer patients.
Despite current recommendations for women to be screened for breast cancer with mammography every 1 to 2 years, less than half of all newly diagnosed breast cancers are initially detected through screening mammography. Prompt medical attention to a new breast symptom can result in earlier stage at diagnosis, yet many patients delay seeking medical care after becoming aware of a breast symptom. In a population-based study of breast cancer, we examined factors potentially associated with patient delay in seeking health care for a breast symptom among 436 symptomatic urban breast cancer patients (146 white, 197 black, and 95 Hispanic). Race/ethnicity, socioeconomic status, health care access and utilization, and misconceptions about the meaning of breast lumps were the key independent variables. Sixteen percent of patients reported delaying more than 3 months before seeking medical advice about breast symptoms. Misconceptions about breast lumps and lacking a regular provider, health insurance, and recent preventive care were all associated with prolonged patient delay (P < 0.005 for all). Misconceptions were much more common among ethnic minorities and women of lower socioeconomic status. Reducing patient delay and disparities in delay will require educating women about the importance of getting breast lumps evaluated in a timely manner and providing greater access to regular health care.


Distinct genetic architectures and environmental factors associate with host response to the γ2-herpesvirus infections.
Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr Virus (EBV) establish life-long infections and are associated with malignancies. Striking geographic variation in incidence and the fact that virus alone is insufficient to cause disease, suggests other co-factors are involved. Here we present epidemiological analysis and genome-wide association study (GWAS) in 4365 individuals from an African population cohort, to assess the influence of host genetic and non-genetic factors on virus antibody responses. EBV/KSHV co-infection (OR = 5.71(1.58-7.12)), HIV positivity (OR = 2.22(1.32-3.73)) and living in a more rural area (OR = 1.38(1.01-1.89)) are strongly associated with immunogenicity. GWAS reveals associations with KSHV antibody response in the HLA-B/C region (p = 6.64 × 10-09). For EBV, associations are identified for VCA (rs71542439, p = 1.15 × 10-12). Human leucocyte antigen (HLA) and trans-ancestry fine-mapping substantiate that distinct variants in HLA-DQA1 (p = 5.24 × 10-44) are driving associations for EBNA-1 in Africa. This study highlights complex interactions between KSHV and EBV, in addition to distinct genetic architectures resulting in important differences in pathogenesis and transmission.


Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance.
Freezing injury of plants may be caused by the deleterious reactions of active oxygen species, and free-radical scavenging systems may be important in the alleviation of freezing stress. To test the feasibility of this hypothesis, enzymes and metabolites that cooperatively scavenge O2 and H2O2 were analyzed in Scots pine (Pinus sylvestris L.) seedlings during a stepwise cold acclimation procedure. Elevated levels of enzymatic scavengers such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were found, along with increased freezing tolerance during cold acclimation, supporting the hypothesis. Induction of the scavenging systems during acclimation is discussed in relation to freezing tolerance.


Extracting Protein-Protein Interactions from MEDLINE using the Hidden Vector State model.
A major challenge in text mining for biomedicine is automatically extracting protein-protein interactions from the vast amount of biomedical literature. We have constructed an information extraction system based on the Hidden Vector State (HVS) model for protein-protein interactions. The HVS model can be trained using only lightly annotated data whilst simultaneously retaining sufficient ability to capture the hierarchical structure. When applied in extracting protein-protein interactions, we found that it performed better than other established statistical methods and achieved 61.5% in F-score with balanced recall and precision values. Moreover, the statistical nature of the pure data-driven HVS model makes it intrinsically robust and it can be easily adapted to other domains.


The solvation structure of alprazolam.
Alprazolam is a benzodiazepine that is commonly prescribed for the treatment of anxiety and other related disorders. Like other benzodiazepines, it is thought to exert its effect through interaction with GABAA receptors. However, it has also been described as a potent and selective protein interaction inhibitor of bromodomain and extra-terminal (BET) proteins. Indeed, the only crystal structure of alprazolam bound to a protein is a complex between alprazolam and the BRD4 bromodomain. The structure shows that the complex also involves many water interactions that mediate contacts between the drug and the protein, a scenario that exists in many drug-protein complexes. How such waters relate to solvation patterns of small molecules may improve our understanding of what dictates their appearance or absence in bridging positions within complexes and thus will be important in terms of future rational drug-design. Here, we use neutron diffraction in conjunction with molecular dynamics simulations to provide a detailed analysis of how water molecules interact with alprazolam in methanol/water mixtures. The agreement between the neutron diffraction and the molecular dynamics is extremely good. We discuss the results in the context of drug design.


Obesity might be a good prognosis factor for COPD patients using domiciliary noninvasive mechanical ventilation.
Cachexia is known to be a deteriorating factor for survival of patients with chronic obstructive pulmonary disease (COPD), but data related to obesity are limited. We observed that obese patients with COPD prescribed long-term noninvasive mechanical ventilation (NIMV) had better survival rate compared to nonobese patients. Therefore, we conducted a retrospective observational cohort study. Archives of Thoracic Diseases Training Hospital were sought between 2008 and 2013. All the subjects were prescribed domiciliary NIMV for chronic respiratory failure secondary to COPD. Subjects were grouped according to their body mass index (BMI). The first group consisted of subjects with BMI between 20 and 30 kg/m(2), and the second group consisted of subjects with BMI >30 kg/m(2). Data obtained at the first month's visit for the following parameters were recorded: age, sex, comorbid diseases, smoking history, pulmonary function test, 6-minute walk test (6-MWT), and arterial blood gas analysis. Hospital admissions were recorded before and after the domiciliary NIMV usage. Mortality rate was searched from the electronic database. Overall, 118 subjects were enrolled. Thirty-eight subjects had BMI between 20 and 30 kg/m(2), while 80 subjects had BMI >30 kg/m(2). The mean age was 65.8±9.4 years, and 81% were male. The median follow-up time was 26 months and mortality rates were 32% and 34% for obese and nonobese subjects (P=0.67). Improvement in 6-MWT was protective against mortality. In conclusion, survival of obese patients with COPD using domiciliary NIMV was found to be better than those of nonobese patients, and the improvement in 6-MWT in such patients was found to be related to a better survival.


Advanced Practice Registered Nurses on Therapeutic Hypothermia Response Teams.
Therapeutic hypothermia can improve neurological recovery after cardiac arrest when implemented quickly. To determine whether outcomes are improved among patients undergoing therapeutic hypothermia by adding advanced practice registered nurses to a therapeutic hypothermia response team. A pilot quality improvement project was conducted in a Midwest community teaching hospital using a retrospective chart review of all adult patients undergoing therapeutic hypothermia before and after the addition of advanced practice registered nurses to the therapeutic hypothermia response team. Outcomes evaluated included time to target core body temperature, therapeutic hypothermia protocol initiation, discharge status, and hospital length of stay. A total of 14 adult patients (preintervention n = 8, postintervention n = 6) comprised the sample. Length of stay decreased in the postintervention group (median 2.5 vs 6 days for the preintervention group; P = .05), but other outcomes did not differ. This quality improvement project provides foundational data to evaluate advanced practice registered nurses specific metrics and to compare with future data using a larger longitudinal sample.


Hemodialysis patients' preferences for the management of secondary hyperparathyroidism.
Patient engagement and patient-centered care are critical in optimally managing patients with end-stage renal disease (ESRD). Understanding patient preferences is a key element of patient-centered care and shared decision making. The objective of this study was to elicit patients' preferences for the treatment of secondary hyperparathyroidism (SHPT) associated with ESRD using a discrete-choice experiment survey. Clinical literature, nephrologist input, patient-education resources, and a patient focus group informed development of the survey instrument, which was qualitatively pretested before its administration to a broader sample of patients. The National Kidney Foundation invited individuals in the United States with ESRD who were undergoing hemodialysis to participate in the survey. Respondents chose among three hypothetical SHPT treatment alternatives (two medical alternatives and surgery) in each of a series of questions, which were defined by attributes of efficacy (effect on laboratory values and symptoms), safety, tolerability, mode of administration, and cost. The survey instrument included a best-worst scaling exercise to quantify the relative bother of the individual attributes of surgery. Random-parameters logit models were used to evaluate the conditional relative importance of the attributes. A total of 200 patients with ESRD completed the survey. The treatment attributes that were most important to the respondents were whether a treatment was a medication or surgery and out-of-pocket cost. Patients had statistically significant preferences for efficacy attributes related to symptom management and laboratory values, but placed less importance on the attributes related to mode of administration and side effects. The most bothersome attribute of surgery was the risk of surgical mortality. Patients with ESRD and SHPT who are undergoing hemodialysis understand SHPT and have clear and measurable treatment preferences. These results may help inform clinicians about patients' preferences regarding treatment options for a common complication of ESRD.


Structural elements of an NRPS cyclization domain and its intermodule docking domain.
Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.


SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.
The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.


Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content.
1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.


Hepatitis C recurrence after liver transplantation.
End stage liver disease from hepatitis C is the leading indication for liver transplantation (LT) in the United States. Recurrent hepatitis C after LT is universal and causes substantial morbidity and mortality with up to 30% patients developing cirrhosis by the fifth postoperative year. Once cirrhosis is established, the risk of hepatic decompensation is approximately 40% per year. Risk factors associated with accelerated disease recurrence are elevated high viral load prior to transplantation, older donor age, prolonged ischemic time, cytomegalovirus coinfection, intensity of immunosuppression and HIV coinfection. Although the mechanisms of accelerated HCV-induced liver damage after transplantation are poorly understood, strategies employed to limit severe recurrence include avoidance of older donors, early recognition of cytomegalovirus, minimization of immunosuppression, particularly T-cell depleting therapies and pulsed steroids for acute cellular rejection. Treatment of recurrent hepatitis C post-transplant is also problematic and fraught with controversy. As there is a paucity of evidence on when treatment should be initiated, out of necessity treatment has been empiric and often varies between centers. As prophylactic treatment immediately after transplantation is rarely effective and associated with numerous side effects, most clinicians acknowledge that treatment should be initiated once early fibrosis has developed although sustained viral rates with pegylated interferon and ribavirin are frequently less than 30%. Side effects are common and can lead to dose reduction or discontinuation of treatment. For those patients who develop develop decompensated cirrhosis from recurrent hepatitis C, retransplantation may be considered.


Reconstructing evolutionary trajectories of mutation signature activities in cancer using TrackSig.
The type and genomic context of cancer mutations depend on their causes. These causes have been characterized using signatures that represent mutation types that co-occur in the same tumours. However, it remains unclear how mutation processes change during cancer evolution due to the lack of reliable methods to reconstruct evolutionary trajectories of mutational signature activity. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data from 2658 cancers across 38 tumour types, we present TrackSig, a new method that reconstructs these trajectories using optimal, joint segmentation and deconvolution of mutation type and allele frequencies from a single tumour sample. In simulations, we find TrackSig has a 3-5% activity reconstruction error, and 12% false detection rate. It outperforms an aggressive baseline in situations with branching evolution, CNA gain, and neutral mutations. Applied to data from 2658 tumours and 38 cancer types, TrackSig permits pan-cancer insight into evolutionary changes in mutational processes.


Craniofacial morphology but not excess body fat is associated with risk of having sleep-disordered breathing--the PANIC Study (a questionnaire-based inquiry in 6-8-year-olds).
We investigated the associations of dental occlusion, other craniofacial features and body fat with paediatric sleep-disordered breathing (SDB) in a representative population sample of 491 Finnish children 6-8 years of age. Overweight and obesity were defined using age- and sex-specific body mass index cutoffs by International Obesity Task Force (IOTF) criteria. Body fat percentage was assessed by dual-energy X-ray absorptiometry. Facial proportions, dental occlusion and soft tissue structures were evaluated by an orthodontist. Sleep was assessed by a sleep questionnaire administered by the parents. SDB was defined as apnoeas, frequent or loud snoring or nocturnal mouth breathing observed by the parents. The prevalence of SDB was 9.9 % with no difference between boys and girls. The median (interquartile range) of body fat percentage was 20.6 (17.4-27.1) in girls and 15.0 (11.4-21.6) in boys. Altogether 11.4 % of boys and 15.6 % of girls were classified as having overweight or obesity according to the IOTF criteria. There was no difference in the prevalence of overweight, obesity or body fat percentage between children with SDB and those without it. Children with tonsillar hypertrophy had a 3.7 times higher risk of suffering SDB than those with normal size tonsils after adjustment for age, sex and body fat percentage. Furthermore, children with cross bite had a 3.3 times higher risk of having SDB than those without cross bite, and children with a convex facial profile had a 2.6 times higher risk of having SDB than those with a normal facial profile. Abnormal craniofacial morphology, but not excess body fat, is associated with an increased risk of having SDB in 6-8-year-old children. A simple model of necessary clinical examinations (i.e. facial profile, dental occlusion and tonsils) is recommended to recognize children with an increased risk of SDB.