Untargeted metabolic profiling identifies altered serum metabolites of type 2 diabetes mellitus in a prospective, nested case control study.
Application of metabolite profiling could expand the etiological knowledge of type 2 diabetes mellitus (T2D). However, few prospective studies apply broad untargeted metabolite profiling to reveal the comprehensive metabolic alterations preceding the onset of T2D. We applied untargeted metabolite profiling in serum samples obtained from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort comprising 300 individuals who developed T2D after a median follow-up time of 6 years and 300 matched controls. For that purpose, we used ultraperformance LC-MS with a protocol specifically designed for large-scale metabolomics studies with regard to robustness and repeatability. After multivariate classification to select metabolites with the strongest contribution to disease classification, we applied multivariable-adjusted conditional logistic regression to assess the association of these metabolites with T2D. Among several alterations in lipid metabolism, there was an inverse association with T2D for metabolites chemically annotated as lysophosphatidylcholine(dm16:0) and phosphatidylcholine(O-20:0/O-20:0). Hexose sugars were positively associated with T2D, whereas higher concentrations of a sugar alcohol and a deoxyhexose sugar reduced the odds of diabetes by approximately 60% and 70%, respectively. Furthermore, there was suggestive evidence for a positive association of the circulating purine nucleotide isopentenyladenosine-5'-monophosphate with incident T2D. This study constitutes one of the largest metabolite profiling approaches of T2D biomarkers in a prospective study population. The findings might help generate new hypotheses about diabetes etiology and develop further targeted studies of a smaller number of potentially important metabolites.


Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis.
AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some alpha-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.


Comparative Effects of Topical 0.2% Sirolimus for Angiofibromas in Adults and Pediatric Patients with Tuberous Sclerosis Complex.
Recent reports have suggested that the topical formulation of sirolimus is effective in treating facial angiofibromas in tuberous sclerosis complex (TSC). Here, we determined the safety and efficacy of 0.2% topical sirolimus for the treatment of facial angiofibroma and compared its effects based on age. This was a retrospective study which involved 36 TSC patients with facial angiofibromas who were treated with 0.2% sirolimus ointment. Its effect was evaluated using the Facial Angiofibroma Severity Index (FASI). In order to observe its comparative effect based on patient age, a subgroup analysis was performed, between the adult group (> 18 years old) and the pediatric group (≤18 years old). The total FASI as well as its subcategories (erythema, size, and extent) showed statistically significant improvements after the topical treatment with 0.2% sirolimus ointment (FASI before treatment: 7.2 ± 1.1, FASI after treatment: 4.4± 1.4, p < 0.001). Among the subcategories of FASI, the erythema was most significantly reduced with the fastest response to the treatment. In a subgroup analysis, the pediatric group showed significantly greater improvements in FASI (improvement of FASI in the pediatric group = 49.7 ± 12.2%, adult group = 27.9 ± 15.6%, p < 0.001). The serial improvement analysis also showed that the pediatric group achieved a consistently greater improvement in FASI at all visits. Its 1-year application in 3 patients demonstrated a continuous maintenance effect. No significant adverse effects were observed. 0.2% sirolimus ointment is safe and effective for facial angiofibromas. Considering its higher efficacy in younger patients, an early initiation of the treatment is recommended.


Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.
We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.


Motivation to obtain a food reward of pregnant ewes in negative energy balance: behavioural, metabolic and endocrine considerations.
Low food availability often coincides with pregnancy in grazing animals. This study investigated how chronic reductions in food intake affected feeding motivation, and metabolic and endocrine parameters in pregnant sheep, which might be indicative of compromised welfare. Ewes with an initial Body Condition Score of 2.7±0.3 (BCS; 0 indicates emaciation and 5 obesity) were fed to attain low (LBC 2.0±0.0,), medium (MBC 2.9±0.1) or high BCS (HBC 3.7±0.1) in the first trimester of pregnancy. A feeding motivation test in which sheep were required to walk a set distance for a palatable food reward was conducted in the second trimester. LBC and MBC ewes consumed more rewards (P=0.001) and displayed a higher expenditure (P=0.02) than HBC ewes, LBC ewes also tended to consume more rewards than MBC ewes (P=0.09). Plasma leptin and glucose concentrations were inversely correlated to expenditure (both P<0.05) and appear to be associated with hunger in sheep. LBC ewes were in negative energy balance, with lower muscle dimensions, plasma glucose, leptin, insulin, cortisol, and insulin-like growth factor-1 concentrations and higher free fatty acids concentrations compared to HBC ewes; metabolic and endocrine parameters of the MBC ewes were intermediate. The high feeding motivation and negative energy balance of low BCS ewes suggested an increased risk of compromised welfare. Imposing even a small cost on a food reward reduced motivation substantially in high BCS ewes (despite high intake when food was freely available). Assessment of a willingness to work for rewards, combined with measures of key metabolic and endocrine parameters, may provide sensitive barometers of welfare in energetically-taxed animals.


A First Look at PCMH Implementation for Minority Veterans: Room for Improvement.
Implementation of Patient Aligned Care Teams (PACT), a patient-centered medical home model, has been inconsistent among the >900 primary care facilities in the Veterans Health Administration. Estimate if the degree of PACT implementation at a facility varied with the percentage of minority veteran patients at the facility. Cross-sectional, facility-level analysis of PACT implementation measures in 2012. Veterans Health Administration hospital-based and community-based primary care facilities. We used a previously validated PACT Implementation Progress Index (Pi) and its 8 domains: access, continuity of care, care coordination, comprehensiveness, self-management support, and patient-centered care and communication, shared decision-making domains, and team functioning. Facilities were categorized as low (<5.2%, n=208), medium (5.2%-25.8%, n=413), and high (>25.8%, n=206) percent minority based on the percent of their own veteran population. Most minority veterans received care in high minority (69%) and medium minority facilities (29%). In adjusted analyses, medium and high minority facilities scored 0.773 (P=0.009) and 0.930 (P=0.008) points lower on the Pi score relative to low minority facilities. Relative to low minority facilities, both medium and high minority facilities were less likely of having high Pi scores (≥2) and more likely of having low Pi scores (≤-2). Both medium and high minority facilities had the same 3 domain scores lower than low minority facilities (care coordination, comprehensiveness, and self-management). Overall PACT implementation varied with respect to the racial/ethnic composition of a facility, with medium and high minority facilities having a lower implementation scores.


Survey of current perspectives on laryngopharyngeal reflux among Canadian otolaryngologists.
Laryngopharyngeal reflux (LPR) -- gastroesophageal reflux above the upper esophageal sphincter -- is a common problem encountered by otolaryngologists. Despite consensus guidelines, the presentation, diagnosis, and treatment remain controversial. We surveyed Canadian otolaryngologists to assess current perspectives. Web-based questionnaires were e-mailed to 135 otolaryngologists. Respondents were categorized by subspecialty as head and neck (H&N) or non-H&N (rhinology, otology, laryngology, facial plastics, general and pediatric otolaryngology). Data were analyzed to determine differences in proportions between groups. The response rate was 48 of 135 otolaryngologists. Symptoms considered to be strongly or moderately associated with LPR included globus sensation, excessive throat clearing, sore or burning throat, hoarseness, chronic cough, and dysphonia. The laryngoscopic signs considered strongly associated with LPR were edema, intra-arytenoid changes, and granulomata. The majority of otolaryngologists in both the H&N (12 of 15) and non-H&N groups (27 of 32) use flexible laryngoscopy for investigation and diagnosis of LPR. Proton pump inhibitors in addition to lifestyle modifications are recommended by both groups as first- and second-line therapy for an initial course of 6 to 12 weeks, with long-term therapy extended for 4 to 12 months. Canadian otolaryngologists do correlate specific signs and symptoms with LPR patients. This is consistent across subspecialties within the field. Flexible fibre-optic laryngoscopy is the preferred diagnostic tool. Although evidence based on randomized controlled trials has yet to demonstrate a reproducible, statistically significant improvement in LPR from medical therapy, first-line pharmacologic treatment (in addition to lifestyle changes) is generally provided as a proton pump inhibitor, with the duration of therapy being somewhat variable and less than that recommended by the current literature.


Detection of Human Endogenous Retrovirus K (HERV-K) Transcripts in Human Prostate Cancer Cell Lines.
Human endogenous retroviruses (HERVs) are transcribed in many cancers including prostate cancer. Human endogenous retrovirus K (HERV-K) of the HML2 subtype is the most recently integrated and most intact retrovirus in the human genome, with many of the viral genomes encoding full- or partial-length viral proteins. To assess transcripts of HERV-K in prostate cancer cell lines and identify the specific HERV-K elements in the human genome that are transcribed, reverse transcriptase-PCR (RT-PCR) and cDNA sequencing were undertaken. Strand-specific RT-PCR, plasmid subcloning, and cDNA sequencing detected the presence of HERV-K(HML2) coding strand transcripts within four prostate cell lines (LNCaP, DU145, PC3, and VCaP). RT-PCR across splice junctions revealed splicing variants for env gene mRNA in three cell lines, two involving previously undescribed alternative splice sites. To determine the HERV-K loci from which the transcripts arose, RepeatMasker was used to compile a list of over 200 HERV-K internal genome segment fragments and over 1,000 HERV-K solo long terminal repeat (LTR) fragments in the human genome. Surprisingly, the sequences identified from internal positions of the viral genome were mostly smaller segments, while the LTRs were relatively intact. Possible reasons for this are discussed. The transcripts in the cell lines tested, arose from several HERV-K loci, with some proviruses being detected in multiple cell lines and others in only one of the four used. In some instances, transcripts from viral antisense strands was also detected. In addition, transcripts from both strands of solo LTRs were detected. These data show that transcripts from HERV-K loci commonly occur in prostate cancer cell lines and that transcription of either strand can occur. They also emphasize the importance of single nucleotide level analysis to identify the specific, individual HERV-K loci that are transcribed, and indicate that HERV-K expression in prostate cancer warrants further study.


WD repeat domain of Dictyostelium myosin heavy chain kinase C functions in both substrate targeting and cellular localization.
Myosin II disassembly in Dictyostelium discoideum is regulated by three structurally related myosin heavy chain kinases (myosin II heavy chain kinase A [MHCK-A], -B, and -C). We show that the WD repeat domain of MHCK-C is unique in that it mediates both substrate targeting and subcellular localization, revealing a target for regulation that is distinct from those of the other MHCKs.


Promoting the social value of research in Kenya: examining the practical aspects of collaborative partnerships using an ethical framework.
The ethics of research continue to attract considerable debate, particularly when that research is sponsored by partners from the North and carried out in the South. Ethical research should contribute to social value in the country where research is being carried out, but there is significant debate around how this might be achieved and who is responsible. The literature suggests that researchers might employ two inter-related strategies to maximise social value: collaborative partnerships with policy makers and communities from the outset of research, and dissemination of research results to participants, policy makers and implementers once the research is over. These areas have received relatively little empirical attention. In this study, we carried out 40 in-depth interviews to explore the role of collaborative partnerships in health research priority setting, and the way in which research findings are disseminated to aid policy making and implementation in Kenya. Interviewees included policy makers, researchers, policy implementers and representatives of organisations funding health reforms in Kenya. Two policy issues were drawn upon as tracers wherever possible: (1) the introduction of Artemesinin-based Combination Therapies (ACTs), an anti-malarial treatment policy; and (2) Haemophilus influenzae (Hib) vaccine for the prevention of pneumococcal diseases among children. The findings point to significant gaps in the 'research to policy to practice' pathway, particularly for national research institutions with a focus on clinical/biomedical research. These gaps reflect poorly effective partnerships among stakeholders and limit the potential social value of much research. While more investment is needed to establish strong structures for promoting and directing collaboration and partnership, how to target this investment is not entirely clear, especially in the context of the considerable power of the global health agenda and the research financing tied to it.


Plasmonic interferometers for label-free multiplexed sensing.
We report a plasmonic interferometric biosensor based on a simple slit-groove metallic nanostructure that monitors the phase changes of surface plasmon polaritons resulting from biomolecular adsorptions. The proposed sensing scheme integrates the strengths of miniaturized plasmonic architectures with sensitive optical interferometry techniques. Sensing peak linewidths as narrow as 7 nm and refractive index resolutions of 1 × 10(-5) RIU were experimentally measured from a miniaturized sensing area of 10 × 30 µm(2) using a collinear transmission setup and a low-cost compact spectrometer. A high-density array of such interferometric sensors was also fabricated to demonstrate its potential for real-time multiplexed sensing using a CCD camera for intensity interrogation. A self-referencing method was introduced to increase the sensitivity and reduce sensor noise for multiplexing measurements. The enhanced sensing performance, small sensor footprint, and simple instrumentation and optical alignment suggest promise to integrate this platform into low-cost label-free biosensing devices with high multiplexing capabilities.


Structure and interdomain dynamics of apoptosis-associated speck-like protein containing a CARD (ASC).
The human protein ASC is a key mediator in apoptosis and inflammation. Through its two death domains (pyrin and CARD) ASC interacts with cell death executioners, acts as an essential adapter for inflammasome integrity, and oligomerizes into functional supramolecular assemblies. However, these functions are not understood at the structural-dynamic level. This study reports the solution structure and interdomain dynamics of full-length ASC. The pyrin and CARD domains are structurally independent six-helix bundle motifs connected by a 23-residue linker. The CARD structure reveals two distinctive characteristics; helix 1 is not fragmented as in all other known CARDs, and its electrostatic surface shows a uniform distribution of positive and negative charges, whereas these are commonly separated into two areas in other death domains. The linker adopts residual structure resulting in a back-to-back orientation of the domains, which avoids steric interference of each domain with the binding site of the other. NMR relaxation experiments show that the linker is flexible despite the residual structure. This flexibility could help expand the relative volume occupied by each domain, thus increasing the capture radius for effectors. Based on the ASC structure, a tentative model is proposed to illustrate how ASC oligomerizes via CARD and pyrin homophilic interactions. Moreover, ASC oligomers have been analyzed by atomic force microscopy, showing a predominant species of disk-like particles of approximately 12-nm diameter and approximately 1-nm height. Taken together, these results provide structural insight into the behavior of ASC as an adapter molecule.


High prevalence of multiple paternity within fruits in natural populations of Silene latifolia, as revealed by microsatellite DNA analysis.
Data on multiple paternity within broods has been gathered in several animal species, and comparable data in plants would be of great importance to understand the evolution of reproductive traits in a common framework. In this study, we first isolated and characterized six microsatellite loci from the dioecious plant Silene latifolia (Caryophyllaceae). The polymorphism of the loci was assessed in 60 individual females from four different populations. Two of the investigated loci showed a pattern of inheritance consistent with X-linkage. These microsatellite loci were highly polymorphic and therefore useful tools for parentage analysis. We then used four of the markers to determine paternity within naturally pollinated fruits in four European populations. This study revealed widespread multiple paternity in all populations investigated. The minimum number of fathers per fruit varied from one to nine, with population means ranging from 3.4 to 4.9. The number of fathers per fruit was not significantly correlated with offspring sex ratios. High prevalence of multiple paternity within fruits strongly suggest that pollen competition is likely to occur in this species. This may substantially impact male reproductive success and possibly contribute to increase female and offspring fitness, either through postpollination selection or increased genetic diversity. Wide variation in outcrossing rates may be an overlooked aspect of plant mating systems.


Functional dissection of intersubunit interactions in the EspR virulence regulator of Mycobacterium tuberculosis.
The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.


Mortality among military participants at the 1957 PLUMBBOB nuclear weapons test series and from leukemia among participants at the SMOKY test.
Health effects following low doses of ionizing radiation are uncertain. Military veterans at the Nevada test site (NTS) during the SMOKY atmospheric nuclear weapons test in 1957 were reported to be at increased risk for leukemia in 1979, but this increase was not evaluated with respect to radiation dose. The SMOKY test was one of 30 tests in 1957 within the PLUMBBOB test series. These early studies led to public laws where atomic veterans could qualify for compensation for presumptive radiogenic diseases. A retrospective cohort study was conducted of 12219 veterans at the PLUMBBOB test series, including 3020 at the SMOKY nuclear test. Mortality follow-up was through 2010 and observed causes of death were compared with expected causes based on general population rates. Radiation dose to red bone marrow was based on individual dose reconstructions, and Cox proportional hazards models were used to evaluate dose response for all leukemias other than chronic lymphocytic leukemia (non-CLL leukemia). Vital status was determined for 95.3% of the 12 219 veterans. The dose to red bone marrow was low (mean 3.2 mGy, maximum 500 mGy). Military participants at the PLUMBBOB nuclear test series remained relatively healthy after 53 years and died at a lower rate than the general population. In contrast, and in comparison with national rates, the SMOKY participants showed significant increases in all causes of death, respiratory cancer, leukemia, nephritis and nephrosis, and accidents, possibly related in part to lifestyle factors common to enlisted men who made up 81% of the SMOKY cohort. Compared with national rates, a statistically significant excess of non-CLL leukemia was observed among SMOKY participants (Standardized Mortality Ratio = 1.89, 95% 1.24-2.75, n = 27) but not among PLUMBBOB participants after excluding SMOKY (SMR = 0.87, 95% 0.64-1.51, n = 47). Leukemia risk, initially reported to be significantly increased among SMOKY participants, remained elevated, but this risk diminished over time. Despite an intense dose reconstruction, the risk for leukemia was not found to increase with increasing levels of radiation dose to the red bone marrow. Based on a linear model, the estimated excess relative risk per mGy is -0.05 (95% CI -0.14, 0.04). An explanation for the observed excess of leukemia remains unresolved but conceivably could be related to chance due to small numbers, subtle biases in the study design and/or high tobacco use among enlisted men. Larger studies should elucidate further the possible relationship between fallout radiation, leukemia and cancer among atomic veterans.


Efficacy and safety of very-low-calorie ketogenic diet: a double blind randomized crossover study.
To verify safety respect to weight loss, cardiometabolic diseases of short-term Very low-calorie ketogenic diets (VLCKDs, <800 kcal day-1). Randomized cross-over trial with placebo. The study had no. 2 dietary treatment (DT), conducted in two arms: (1) VLCKD1 in which 50% of protein intake is replaced with synthetic amino acids; (2) VLCKD2 with placebo. The VLCKDs (<800 kcal day-1) were different in term of protein content and quality each arm lasted three weeks (wks). Between the two arms a 3-wks washout period was performed to avoid additive effects on DT to follow. At the baseline, at start and end of each arm, all the subjects were evaluated for their health and nutritional status, by anthropometric analysis, body composition (Dual X-ray Absorptiometry (DXA), Bioimpedentiometry, biochemical evaluation, and Peroxisome Proliferator-Activated Receptor γ (PPAR) γ expression by transcriptomic analysis. After VLCKD1 were reduced: Body Mass Index (BMI) (Δ%=-11.1%, p=0.00), Total Body Water (TBW) (p<0.05); Android Fat Percentage (AFP) (Δ%=-1.8%, p=0.02); Android Fat Mass (AFM) (Δ%=-12.7%, p=0.00); Gynoid Fat Mass (GFM) (Δ%=-6.3%, p=0.01); Intermuscular Adipose Tissue (IMAT) (Δ%= -11.1%, p=0.00); Homeostasis Model Assessment of Insulin Re-sistance (HOMA-IR) (Δ%=-62.1%, p=0.01). After VLCKD1 a significant increase of uricemia, cre-atinine and aspartate aminotransferase (AST) (respectively Δ%=35%, p=0.01; Δ%=5.9%, p=0.02; Δ%=25.5%, p=0.03). After VLCKD2 were reduced: BMI (Δ%=-11.2%, p=0.00); AFM (Δ%=-14.3%, p=0.00); GFM (Δ%=-6.3%, p=0.00); Appendicular Skeletal Muscle Mass Index (ASMMI) (Δ%=-17.5%, p=0.00); HOMA-IR (Δ%=-59,4%, p=0.02). After VLCKD2, uricemia (Δ%=63.1%, p=0.03), and Vitamin D levels (Δ%=25.7%, p=0.02) were increased. No significant changes of car-diovascular disease (CVD) indexes were observed after DTs. No significant changes of PPARγ lev-el in any DTs. 21-days VLCKDs not impair nutritional state; not cause negative changes in global measurements of nutritional state including sarcopenia, bone mineral content, hepatic, renal and lipid profile.


Health risks and benefits from calcium and vitamin D supplementation: Women's Health Initiative clinical trial and cohort study.
The Women's Health Initiative (WHI) double-blind, placebo-controlled clinical trial randomly assigned 36,282 postmenopausal women in the U.S. to 1,000 mg elemental calcium carbonate plus 400 IU of vitamin D(3) daily or placebo, with average intervention period of 7.0 years. The trial was designed to test whether calcium plus vitamin D supplementation in a population in which the use of these supplements was widespread would reduce hip fracture, and secondarily, total fracture and colorectal cancer. This study further examines the health benefits and risks of calcium and vitamin D supplementation using WHI data, with emphasis on fractures, cardiovascular disease, cancer, and total mortality. WHI calcium and vitamin D randomized clinical trial (CT) data through the end of the intervention period were further analyzed with emphasis on treatment effects in relation to duration of supplementation, and these data were contrasted and combined with corresponding data from the WHI prospective observational study (OS). Among women not taking personal calcium or vitamin D supplements at baseline, the hazard ratio [HR] for hip fracture occurrence in the CT following 5 or more years of calcium and vitamin D supplementation versus placebo was 0.62 (95 % confidence interval (CI), 0.38-1.00). In combined analyses of CT and OS data, the corresponding HR was 0.65 (95 % CI, 0.44-0.98). Supplementation effects were not apparent on the risks of myocardial infarction, coronary heart disease, total heart disease, stroke, overall cardiovascular disease, colorectal cancer, or total mortality, while evidence for a reduction in breast cancer risk and total invasive cancer risk among calcium plus vitamin D users was only suggestive. Though based primarily on a subset analysis, long-term use of calcium and vitamin D appears to confer a reduction that may be substantial in the risk of hip fracture among postmenopausal women. Other health benefits and risks of supplementation at doses considered, including an elevation in urinary tract stone formation, appear to be modest and approximately balanced.


Cell stress in cortical organoids impairs molecular subtype specification.
Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3-5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.


Pentraxin 3 in acute respiratory distress syndrome: an early marker of severity.
Pentraxin 3 is a fluid phase receptor involved in innate immunity. It belongs to the Pentraxins family, as C-reactive protein does. Pentraxin 3 is produced by a variety of tissue cells, whereas only the liver produces C-reactive protein. Pentraxin 3 plays a unique role in the regulation of inflammation. Acute lung injury and acute respiratory distress syndrome are characterized by an important inflammatory reaction. We investigated the role of pentraxin 3 as a marker of severity and outcome predictor of acute lung injury and acute respiratory distress syndrome. We measured circulating pentraxin 3 and C-reactive protein levels within 24 hrs from intubation (day 1), after 24 hrs from the first sample, then every 3 days for the first month and then once a week, until discharge from the intensive care unit. Pentraxin 3 was also measured in bronchoalveolar lavages, performed when clinically indicated. One university medical center general intensive care unit. The study included 21 patients affected by acute lung injury and acute respiratory distress syndrome (1994 Consensus Conference criteria). None. Pentraxin 3 plasma levels were high with a peak on the first day (median 71.05 ng/mL, interquartile range 52.37-117.38 ng/mL, normal values <2 ng/mL), declining thereafter. C-reactive protein peaked later and remained at relatively high values. Out of several day 1 parameters, pentraxin 3 was the only significant difference between survivors and nonsurvivors. Pentraxin 3 levels were positively correlated with lung injury score values (p < 0.001) and number of organ failures (p < 0.001). Pentraxin 3 was present in bronchoalveolar lavages fluids (5.03 ng/mL, interquartile range 1.52-8.48 ng/mL) and bronchoalveolar lavages positive to bacterial culture were associated with significantly higher pentraxin 3 values (p < 0.05). The results presented here show that pentraxin 3 is elevated in acute lung injury and acute respiratory distress syndrome and that its levels correlate with parameters of lung injury and systemic involvement. The clinical and pathophysiological significance of pentraxin 3 in acute lung injury and acute respiratory distress syndrome deserves further scrutiny.


Host suitability in soybean cultivars for the reniform nematode, 1999 tests.
Two hundred twenty-six soybean cultivars were tested in greenhouse pot experiments during summer 1999 to determine their suitability as hosts for the reniform nematode, Rotylenchulus reniformis. The cultivars included new entries into the Arkansas and Mississippi soybean variety testing programs and entries submitted by extension nematologists from Auburn University and Louisiana State University. Also included in the R. reniformis tests were the resistant cultivars Forrest and Hartwig, the susceptible control Braxton, and fallow infested soil that served as controls. Total number of eggs + nematodes extracted from the soil and roots per pot, reproductive indices (Pf/Pi) based on the number of nematodes extracted from the soil and roots/initial inoculum level, calculated for each cultivar, and the ratio of the Pf/Pi of each cultivar to the Pf/Pi of Forrest are reported. Cultivars with reproduction not significantly different from Forrest were not suitable hosts, whereas those with greater reproductive indices were considered suitable hosts. One of the 12 cultivars of the relative maturity group (RMG) <=4.4 was not a suitable host. For the 4.5 to 4.9 RMG, 24 of 72 cultivars were not suitable hosts, whereas 9 of 41 cultivars in RMG 5.0 to 5.4 were not suitable hosts. In the 5.5 to 5.9 RMG cultivars, 11 of 66 were not suitable hosts; for the 6.0 or greater RMG, 11 of 35 were not suitable hosts. These data will be useful in the selection of soybean cultivars to use in rotation with cotton or other susceptible crops to help control the reniform nematode.


Probing Nucleation Mechanism of Self-Catalyzed InN Nanostructures.
The nucleation and evolution of InN nanowires in a self-catalyzed growth process have been investigated to probe the microscopic growth mechanism of the self-catalysis and a model is proposed for high pressure growth window at ~760 Torr. In the initial stage of the growth, amorphous InNx microparticles of cone shape in liquid phase form with assistance of an InNx wetting layer on the substrate. InN crystallites form inside the cone and serve as the seeds for one-dimensional growth along the favorable [0001] orientation, resulting in single-crystalline InN nanowire bundles protruding out from the cones. An amorphous InNx sheath around the faucet tip serves as the interface between growing InN nanowires and the incoming vapors of indium and nitrogen and supports continuous growth of InN nanowires in a similar way to the oxide sheath in the oxide-assisted growth of other semiconductor nanowires. Other InN 1D nanostructures, such as belts and tubes, can be obtained by varying the InN crystallites nucleation and initiation process.


Functional Quality-of-Life Outcomes Reported by Men Treated for Localized Prostate Cancer: A Systematic Literature Review.
To systematically evaluate the literature for functional quality-of-life (QOL) outcomes following treatment for localized prostate cancer. . The MEDLINE®, CINAHL®, EMBASE, British Nursing Index, PsycINFO®, and Web of Science™ databases were searched using key words and synonyms for localized prostate cancer treatments. . Of the 2,191 articles screened for relevance and quality, 24 articles were reviewed. Extracted data were tabulated by treatment type and sorted by dysfunction using a data-driven approach. . All treatments caused sexual dysfunction and urinary side effects. Radiation therapy caused bowel dysfunction, which could be long-term or resolved within a few years. Sexual function could take years to return. Urinary incontinence resolved within two years of surgery but worsened following radiation therapy. Fatigue was worse during treatment with adjuvant androgen-deprivation therapy, and some men experienced post-treatment fatigue for several years. . This review identified that QOL outcomes reported by men following different treatments for localized prostate cancer are mostly recorded using standardized health-related QOL outcome measures. Such outcome measures collect data about body system functions but limit understanding of men's QOL following treatment for prostate cancer. Holistic outcome measures are needed to capture data about men's QOL for several years following the completion of treatment for localized prostate cancer. . Nurses need to work with men to facilitate information sharing, identify supportive care needs, and promote self-efficacy, and they should make referrals to specialist services, as appropriate.


Relationship between household socio-economic status and under-five mortality in Rufiji DSS, Tanzania.
Disparities in health outcomes between the poor and the better off are increasingly attracting attention from researchers and policy makers. However, policies aimed at reducing inequity need to be based on evidence of their nature, magnitude, and determinants. The study aims to investigate the relationship between household socio-economic status (SES) and under-five mortality, and to measure health inequality by comparing poorest/least poor quintile mortality rate ratio and the use of a mortality concentration index. It also aims to describe the risk factors associated with under-five mortality at Rufiji Demographic Surveillance Site (RDSS), Tanzania. This analytical cross sectional study included 11,189 children under-five residing in 7,298 households in RDSS in 2005. Principal component analysis was used to construct household SES. Kaplan-Meier survival incidence estimates were used for mortality rates. Health inequality was measured by calculating and comparing mortality rates between the poorest and least poor wealth quintile. We also computed a mortality concentration index. Risk factors of child mortality were assessed using Poisson regression taking into account potential confounders. Under-five mortality was 26.9 per 1,000 person-years [95% confidence interval (CI) (23.7-30.4)]. The poorest were 2.4 times more likely to die compared to the least poor. Our mortality concentration index [-0.16; 95% CI (-0.24, -0.08)] indicated considerable health inequality. Least poor households had a 52% reduced mortality risk [incidence rate ratio (IRR) = 0.48; 95% CI 0.30-0.80]. Furthermore, children with mothers who had attained secondary education had a 70% reduced risk of dying compared to mothers with no education [IRR = 0.30; 95% CI (0.22-0.88)]. Household socio-economic inequality and maternal education were associated with under-five mortality in the RDSS. Targeted interventions to address these factors may contribute towards accelerating the reduction of child mortality in rural Tanzania.


Effects of diabetes and insulin on betaine-homocysteine S-methyltransferase expression in rat liver.
Elevation of plasma homocysteine levels has been recognized as an independent risk factor for the development of cardiovascular disease, a major complication of diabetes. Plasma homocysteine reflects a balance between its synthesis via S-adenosyl-L-methionine-dependent methylation reactions and its removal through the transmethylation and the transsulfuration pathways. Betaine-homocysteine methyltransferase (BHMT, EC 22.214.171.124) is one of the enzymes involved in the remethylation pathway. BHMT, a major zinc metalloenzyme in the liver, catalyzes the transfer of methyl groups from betaine to homocysteine to form dimethylglycine and methionine. We have previously shown that plasma homocysteine levels and the transsulfuration pathway are affected by diabetes. In the present study, we found increased BHMT activity and mRNA levels in livers from streptozotocin-diabetic rats. In the rat hepatoma cell line (H4IIE cells), glucocorticoids (triamcinolone) increased the level and rate of BHMT mRNA synthesis. In the same cell line, insulin decreased the abundance of BHMT mRNA and the rate of de novo mRNA transcription of the gene. Thus the decreased plasma homocysteine in various models of diabetes could be due to enhanced homocysteine removal brought about by a combination of increased transsulfuration of homocysteine to cysteine and increased remethylation of homocysteine to methionine by BHMT.


The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative.
DNA metabarcoding is a technique used to survey biodiversity in many ecological settings, but there are doubts about whether it can provide quantitative results, that is, the proportions of each species in the mixture as opposed to a species list. While there are several experimental studies that report quantitative metabarcoding results, there are a similar number that fail to do so. Here, we provide the rationale to understand under what circumstances the technique can be quantitative. In essence, we simulate a mixture of DNA of S species with a defined initial abundance distribution. In the simulated PCR, each species increases its concentration following a certain amplification efficiency. The final DNA concentration will reflect the initial one when the efficiency is similar for all species; otherwise, the initial and final DNA concentrations would be poorly related. Although there are many known factors that modulate amplification efficiency, we focused on the number of primer-template mismatches, arguably the most important one. We used 15 common primers pairs targeting the mitochondrial COI region and the mitogenomes of ca. 1,200 insect species. The results showed that some primers pairs produced quantitative results under most circumstances, whereas some other primers failed to do so. In conclusion, depending on the primer pair used in the PCR amplification and on the characteristics of the mixture analysed (i.e., high species richness, low evenness), DNA metabarcoding can provide a quantitative estimate of the relative abundances of different species.


Genetic Variation of X-STRs in the Wichí Population from Chaco Province, Argentina.
The Wichí people from Chaco Province inhabit the region called Impenetrable Chaqueño, where the climatic conditions are extreme. Besides scarce communication with the main urban centers, the cultural patterns of the Wichí cause these communities to live in certain degree of isolation. The effect of this situation is an increased genetic differentiation from other populations, as it was observed through autosomal and Y chromosome markers. However, the genetic variation of X chromosome has not yet been fully analyzed. The patterns of allele distribution of different markers of X chromosome can be highly informative in comparative studies, because its special features make this chromosome a potential source to uncover ethnic differences. The aim of this study was to assess the variation of X chromosome present in the Wichí population living around Misión Nueva Pompeya, in Chaco Province, and to identify particular variation of X-repetitive markers (short tandem repeats, X-STRs) in Chaco Amerindians. We genotyped Wichí for 10 noncoding X-STRs and compared them with a Mocoví population and with published data on individuals coming from European immigration. We found high homozygote proportion and linkage disequilibrium values for the X chromosome in the Wichí population. The data showed the Wichí to be more distant from nonnative people than was the Mocoví population analyzed for comparison. We also found certain differences with Mocoví, possibly due to a higher genetic flow of the latter with nonnatives. A process of genetic drift seems to be enhanced by the social behavior of the Wichí, since they live apart from other native and nonnative groups. The geographic isolation and the extreme environmental conditions can also be considered as major factors contributing to the population differentiation. Although we found no new allele or undescribed variation, the whole pattern of variation for these markers gives the Wichí a particular population identity.


Disease progression in women with X-linked adrenoleukodystrophy is slow.
Over 80% of women with X-linked adrenoleukodystrophy (ALD) develop spinal cord disease in adulthood for which treatment is supportive only. For future clinical trials quantitative data on disease progression rates are essential. Moreover, diagnosis can be challenging in ALD women, as the most important diagnostic biomarker is normal in 15-20%. Better biomarkers are needed. The purpose of this single centre cross-sectional follow-up study in women with ALD was to assess whether Expanded Disability Status Scale (EDSS), AMC Linear Disability Scale (ALDS) and Short Form (36) Health Survey (SF-36) can detect disease progression and to model the effect of age and duration of symptoms on the rate of progression. Moreover, we performed a pilot study to assess if a semi-targeted lipidomics approach can identify possible new diagnostic biomarkers. In this study 46 women (baseline clinical data published by our group previously) were invited for a follow-up visit. Newly identified women at our center were also recruited. We analysed 65 baseline and 34 follow-up assessments. Median time between baseline and follow-up was 7.8 years (range 6.4-8.7). Mean age at baseline was 49.2 ± 14.2 years, at follow-up 55.4 ± 10.1. EDSS increased significantly (+ 0.08 points/year), but the other outcome measures did not. Increasing age and duration of symptoms were associated with more disability. For the pilot study we analysed plasma of 20 ALD women and 10 controls with ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry, which identified 100 potential biomarker ratios with strong differentiating properties and non-overlapping data distributions between ALD women and controls. Progression of spinal cord disease can be detected with EDSS, but not with ALDS or SF-36 after a follow-up period of almost 8 years. Moreover, age and the duration of symptoms seem positively associated with the rate of progression. Although a significant progression was measurable, it was below the rate generally conceived as clinically relevant. Therefore, EDSS, ALDS and SF-36 are not suitable as primary outcome measures in clinical trials for spinal cord disease in ALD women. In addition, a semi-targeted lipidomics approach can identify possible new diagnostic biomarkers for women with ALD.


Preemptive CD20+ B cell depletion attenuates cardiac allograft vasculopathy in cyclosporine-treated monkeys.
Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (alphaCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with alphaCD20 or CsA alone. In animals treated with both alphaCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic.


Nutrient regulation of insulin secretion and action.
Pancreatic β-cell function is of critical importance in the regulation of fuel homoeostasis, and metabolic dysregulation is a hallmark of diabetes mellitus (DM). The β-cell is an intricately designed cell type that couples metabolism of dietary sources of carbohydrates, amino acids and lipids to insulin secretory mechanisms, such that insulin release occurs at appropriate times to ensure efficient nutrient uptake and storage by target tissues. However, chronic exposure to high nutrient concentrations results in altered metabolism that impacts negatively on insulin exocytosis, insulin action and may ultimately lead to development of DM. Reduced action of insulin in target tissues is associated with impairment of insulin signalling and contributes to insulin resistance (IR), a condition often associated with obesity and a major risk factor for DM. The altered metabolism of nutrients by insulin-sensitive target tissues (muscle, adipose tissue and liver) can result in high circulating levels of glucose and various lipids, which further impact on pancreatic β-cell function, IR and progression of the metabolic syndrome. Here, we have considered the role played by the major nutrient groups, carbohydrates, amino acids and lipids, in mediating β-cell insulin secretion, while also exploring the interplay between amino acids and insulin action in muscle. We also focus on the effects of altered lipid metabolism in adipose tissue and liver resulting from activation of inflammatory processes commonly observed in DM pathophysiology. The aim of this review is to describe commonalities and differences in metabolism related to insulin secretion and action, pertinent to the development of DM.


Clustered and missing data in the US National Trauma Data Bank: implications for analysis.
Injury researchers are increasingly using the US National Trauma Data Bank (NTDB). However, there are some methodological issues that might threaten the validity of studies that use this database for injury research. Two methodological issues were evaluated: clustering of patients within trauma centers and missing data. To illustrate how these issues might affect the results of a study, the following four analytical approaches that evaluated the association between patients' blood alcohol concentration (BAC) in the emergency department (ED), patients' resource utilization, and ED or hospital disposition were compared: (A) deleting subjects with missing BAC and ignoring clustering of patients within trauma centers; (B) deleting subjects with missing BAC while taking into account clustering; (C) using imputed values for patients' BAC and ignoring the clustering issue; (D) using the imputed data while taking into account clustering. Adjustment for clustering of patients within trauma centers increased the CIs in models B and D. The results of the analyses based on imputed data showed that estimates based on complete case analysis were biased. For example, the odds ratio for the use of a head CT scan fell from 1.84 (95% CI 1.49 to 2.28) in approach B to 1.26 (95% CI 0.98 to 1.64) in approach D. Excluding patients with missing values for BAC in studies that evaluate the association between this variable and patients' resource utilization and ED or hospital disposition, using the NTDB, led to biased estimates. Furthermore, ignoring the clustering design led to artificially narrow CIs.


Correlates of alcohol abuse/dependence in early-onset alcohol-using women.
Early-onset alcohol use is associated with increased vulnerability to subsequent alcohol abuse and dependence. However, not all early-onset alcohol users develop alcohol use disorders (AUDs). Using a sample of young women from the United States, we identify correlates that contribute to a greater likelihood of AUDs in early-onset alcohol users. Using interview and questionnaire data on participants of the Missouri Adolescent Female Twin Study (MOAFTS), we examine whether measures from domains including sociodemographic, pubertal development, religiosity, educational achievement, adverse life events, internalizing disorders, externalizing disorders, and family history and discipline were associated with development of AUDs in 1,158 women who had their first drink of alcohol prior to age 16. Early-onset drinkers were 3.6 times more likely to meet criteria for AUDs than later onset drinkers. While univariate analyses revealed that a host of correlates were associated with likelihood of AUDs in early-onset drinkers, multivariate analyses suggested that, even after accounting for a particularly early age of onset of drinking, those with a history of physical abuse, cotwin alcohol problems, conduct disorder, regular smoking, older peers, and peer substance use were considerably more likely to meet criteria for AUDs than early-onset drinkers without a lifetime history of these correlates. The progression from first drink to AUDs is complex, and while early age at first drink is a potent risk factor, other aspects of psychopathology, family history, conduct problems, and peer affiliations can exacerbate or alleviate the risk of AUDs in these young female drinkers.


Fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy is highly effective treatment for relapsed patients with CLL.
Optimal management of patients with relapsed/refractory chronic lymphocytic leukemia (CLL) is dictated by patient characteristics, prior therapy, and response to prior therapy. We report the final analysis of combined fludarabine, cyclophosphamide, and rituximab (FCR) for previously treated patients with CLL and identify patients who benefit most from this therapy. We explore efficacy of FCR in patients beyond first relapse, patients with prior exposure to fludarabine and alkylating agent combinations, and patients with prior exposure to rituximab. The FCR regimen was administered to 284 previously treated patients with CLL. Patients were assessed for response and progression by 1996 National Cancer Institute-Working Group (NCI-WG) criteria for CLL and followed for survival. The overall response rate was 74%, with 30% complete remission. The estimated median overall survival was 47 months and median progression-free survival for all patients was 21 months. Subgroup analyses indicated that the following patients were most suitable for FCR treatment: patients with up to 3 prior treatments, fludarabine-sensitive patients irrespective of prior rituximab exposure, and patients without chromosome 17 abnormalities. FCR is an active and well-tolerated therapy for patients with relapsed CLL. The addition of rituximab to FC improved quality and durability of response in this patient population.


Seroprevalence of hepatitis E virus among blood donors on Corsica, France, 2017.
BackgroundHepatitis E virus (HEV) is an emerging zoonotic pathogen and an important cause of acute viral hepatitis in European countries. Corsica Island has been previously identified as a hyperendemic area for HEV.AimOur aim was to characterise the prevalence and titres of IgG antibodies to HEV among blood donors on Corsica and establish a model of the annual force of infection.MethodsBetween September 2017 and January 2018, 2,705 blood donations were tested for anti-HEV IgG using the Wantai HEV IgG enzyme immunoassay.ResultsThe overall seroprevalence was 56.1%. In multivariate analysis, seroprevalence was higher in men than in women (60.0% vs 52.2%; p < 0.01), increased with age and was significantly higher among donors born on Corsica (60.6% vs 53.2%; p < 0.01). No significant difference was observed between the five districts of the island. IgG anti-HEV titres were mostly low (70% of positive donors had titres < 3 IU/mL). In Corsican natives, increasing seroprevalence by age could be explained by models capturing a loss of immunity (annual probability of infection: 4.5%; duration of immunity: 55 years) or by age-specific probabilities of infection (3.8% for children, 1.3% for adults).ConclusionWe confirmed the high HEV seroprevalence on Corsica and identified three aspects that should be further explored: (i) the epidemiology in those younger than 18 years, (ii) common sources of contamination, in particular drinking water, that may explain the wide exposure of the population, and (iii) the actual protection afforded by the low IgG titres observed and the potential susceptibility to secondary HEV infection.


Identification and characterization of a unique role for EDB fibronectin in phagocytosis.
Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was mediated by αvβ3 integrin as shown using inhibitors or cells from β3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for β3 integrin in bacterial phagocytosis in mammals. • Fibronectin containing an extra domain called EDB is released in bacterial meningitis. • EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin. • The enhancement is mediated by activation of αvβ3 integrin in the presence of EDB.


Amoebic grazing of freshwater Synechococcus strains rich in phycocyanin.
Fifteen strains of naked amoebae were presented with 19 strains of Synechococcus on an agar surface. After 14 days of incubation, each of the 285 combinations yielded one of three responses. 42.1% of combinations showed clearing (digestion) of the Synechococcus (C), 56.5% of combinations showed no clearing of the Synechococcus (N) while 1.4% of combinations showed partial clearing of the Synechococcus (P). In general, the Synechococcus strains showed variability in their susceptibility to digestion by the amoebae and the amoebae showed variability in their ability to digest the Synechococcus strains. There was no evidence for amoebae actively selecting profitable prey and equivalent-sized Synechococcus strains were ingested at the same rate, irrespective of their fate. There was some evidence of 'size-selective' grazing in that amoebae ingested the smaller Synechococcus strains at higher rates than the larger strains. However, there was no correlation between prey size and their ultimate fate. These data suggest that amoebae are not selective with regard to the ingestion of synechococci, but that 'selection' occurs at the digestion stage, i.e. whether the synechococci are digested or not.


Prevention and treatment of acute kidney injury in patients undergoing cardiac surgery: a systematic review.
Acute kidney injury (AKI) is common in patients undergoing cardiac surgery and is associated with a high rate of death, long-term sequelae and healthcare costs. We conducted a systematic review of randomized controlled trials for strategies to prevent or treat AKI in cardiac surgery. We screened Medline, Scopus, Cochrane Renal Library, and Google Scholar for randomized controlled trails in cardiac surgery for prevention or treatment of AKI in adults. We identified 70 studies that contained a total of 5,554 participants published until November 2008. Most studies were small in sample size, were single-center, focused on preventive strategies, and displayed wide variation in AKI definitions. Only 26% were assessed to be of high quality according to the Jadad criteria. The types of strategies with possible protective efficacy were dopaminergic agents, vasodilators, anti-inflammatory agents, and pump/perfusion strategies. When analyzed separately, dopamine and N-acetylcysteine did not reduce the risk for AKI. This summary of all the literature on prevention and treatment strategies for AKI in cardiac surgery highlights the need for better information. The results advocate large, good-quality, multicenter studies to determine whether promising interventions reliably reduce rates of acute renal replacement therapy and mortality in the cardiac surgery setting.


Design and physicochemical stability studies of paediatric oral formulations of sildenafil.
Personalized medicine is a challenging research area in paediatric treatments. Elaborating new paediatric formulations when no commercial forms are available is a common practice in pharmacy laboratories; among these, oral liquid formulations are the most common. But due to the lack of specialized equipment, frequently studies to assure the efficiency and safety of the final medicine cannot be carried out. Thus the purpose of this work was the development, characterization and stability evaluation of two oral formulations of sildenafil for the treatment of neonatal persistent pulmonary hypertension. After the establishment of a standard operating procedure (SOP) and elaboration, the physicochemical stability parameters appearance, pH, particle size, rheological behaviour and drug content of formulations were evaluated at three different temperatures for 90 days. Equally, prediction of long term stability, as well as, microbiological stability was performed. Formulations resulted in a suspension and a solution slightly coloured exhibiting fruity odour. Formulation I (suspension) exhibited the best physicochemical properties including Newtonian behaviour and uniformity of API content above 90% to assure an exact dosification process.


Interferometric ellipsometer.
We describe an interferometric ellipsometer that, in contrast to previous designs, requires a single reflection from a sample surface so that tanpsi and Delta are measured directly. A reference beam is created in one arm of a modified Mach-Zehnder interferometer such that the p and s polarizations have a common phase and fixed relative amplitude, irrespective of the sample. This beam is combined interferometrically with the measurement beam. The output beam is spatially separated into its polarization components and temporal fringes created at the photodetectors via mechanical scanning of one of the mirrors. Measurements made on a reference SiO(2) film are in excellent agreement with the calibration certificate while those made on a glass surface demonstrate measurement capabilities for low reflectivity samples. Estimates of the noise performance indicate a precision, in air, of approximately 41 pm.


Distinct dependence on size of Pt and Rh nanoclusters on graphene/Pt(111) in the decomposition of methanol-d4.
Pt and Rh nanoclusters, grown on deposition of Pt and Rh vapors onto graphene/Pt(111), show separate reactivity toward the decomposition of methanol-d4. The Pt (Rh) clusters had a mean diameter 2.0-3.5 nm (2.1-4.0 nm) and height 0.45-0.94 nm (0.41-0.9 nm) evolving with the coverage; they were structurally ordered, having an fcc phase and growing in (111) orientation, and had lattice constants similar to their bulk values. Methanol-d4 on the Pt clusters did not decompose but desorbed mostly, disparate from that on Pt(111) surface; the disparity arose as the adsorption energies of methanol-d4 on most surface sites of the Pt clusters became smaller than their single crystal counterpart. This size effect, nevertheless, did not apply on the Rh clusters, despite their similar atomic stacking; the Rh clusters showed a reactivity similar to that of the Rh(111) surface because the adsorption energies of methanol-d4 on both Rh clusters and Rh(111) are comparable. The distinct size dependence was rationalized through their electronic structures and charge distribution of Fukui function mapping. Our results suggest that reactive transition metals do not necessarily become more reactive while they are scaled down to nanoscale; their reactivity evolves with their size in a manner largely dependent on their electronic nature.


Food insecurity is associated with anxiety, stress, and symptoms of posttraumatic stress disorder in a cohort of women with or at risk of HIV in the United States.
Food insecurity, which disproportionately affects marginalized women in the United States, is associated with depressive symptoms. Few studies have examined relations of food insecurity with other mental health outcomes. The aim of this study was to investigate the associations of food insecurity with symptoms of generalized anxiety disorder (GAD), stress, and posttraumatic stress disorder (PTSD) in the Women's Interagency HIV Study (WIHS), a prospective cohort study of women with or at risk of HIV in the United States. Participants were 2553 women with or at risk of HIV, predominantly African American/black (71.6%). Structured questionnaires were conducted during April 2013-March 2016 every 6 mo. Food security (FS) was the primary predictor, measured using the Household Food Security Survey Module. We measured longitudinal outcomes for GAD (GAD-7 score and a binary GAD-7 screener for moderate-to-severe GAD). Only cross-sectional data were available for outcomes measuring perceived stress (PSS-10 score) and PTSD (PCL-C score and a binary PCL-C screener for PTSD). We examined associations of FS with the outcomes through use of multivariable linear and logistic regression, including lagged associations with GAD outcomes. After adjusting for sociodemographic and health-related factors including HIV serostatus, current marginal, low, and very low FS were associated with increasingly higher GAD-7 scores, and with 1.41 (95% CI: 1.10, 1.80; P < 0.01), 2.03 (95% CI: 1.59, 2.61; P < 0.001), and 3.23 (95% CI: 2.43, 4.29; P < 0.001) times higher odds of screening positive for moderate-to-severe GAD, respectively. Low and very low FS at the previous visit (6 mo earlier) were independently associated with GAD outcomes at current visit. Associations of FS with PSS-10 and PCL-C scores exhibited similar dose-response relations. Very low FS was associated with 1.93 (95% CI: 1.15, 3.24; P < 0.05) times higher odds of screening positive for PTSD. Food insecurity may be associated with a range of poor mental health outcomes among women in the United States with or at risk of HIV.


Slow relaxation of the magnetization observed in mononuclear Ln-radical compounds with D4d geometry configurations.
The combination of LnIII ions (GdIII, TbIII or DyIII) and a pyrazole nitronyl nitroxide radical results in three isomorphous complexes, namely, [Ln(hfac)3(NIT-Pyz)]2 (Ln = Gd(1), Tb(2), Dy(3); hfac = hexafluoroacetylacetone; NIT-Pyz = 2-{3-pyrazolyl}-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide). Single crystal X-ray diffraction studies revealed that all of them are composed of two crystallographically independent mononuclear systems, in which the central LnIII ions are coordinated by three hfac and one bidentate chelating NIT-Pyz radical. The central LnIII ions are all in square antiprism geometry (D4d) polyhedron configurations. Based on the spin Hamiltonian calculations, there exist antiferromagnetic couplings in the GdIII-NIT radical system in complex 1. Complexes 2 and 3 show frequency-dependent out-of-phase signals in a zero field indicating single-molecule magnetic behavior. Moreover, Tb's complex (2) shows a single thermal relaxation process with an energy barrier of 26 K. For Dy's complex (3), the Orbach and Raman processes both contribute to the magnetic relaxation behaviors.


Analogy, higher order thinking, and education.
Analogical reasoning, the ability to understand phenomena as systems of structured relationships that can be aligned, compared, and mapped together, plays a fundamental role in the technology rich, increasingly globalized educational climate of the 21st century. Flexible, conceptual thinking is prioritized in this view of education, and schools are emphasizing 'higher order thinking', rather than memorization of a cannon of key topics. The lack of a cognitively grounded definition for higher order thinking, however, has led to a field of research and practice with little coherence across domains or connection to the large body of cognitive science research on thinking. We review literature on analogy and disciplinary higher order thinking to propose that relational reasoning can be productively considered the cognitive underpinning of higher order thinking. We highlight the utility of this framework for developing insights into practice through a review of mathematics, science, and history educational contexts. In these disciplines, analogy is essential to developing expert-like disciplinary knowledge in which concepts are understood to be systems of relationships that can be connected and flexibly manipulated. At the same time, analogies in education require explicit support to ensure that learners notice the relevance of relational thinking, have adequate processing resources available to mentally hold and manipulate relations, and are able to recognize both the similarities and differences when drawing analogies between systems of relationships.


Microbial enzymes catalyzing keratin degradation: Classification, structure, function.
Keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. It is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. Keratin is packed by disulfide bonds and hydrogen bonds. Based on the secondary structure, keratin can be classified into α-keratin and β-keratin. Keratinases (EC 3.4.-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. Currently, the known keratinolytic enzymes belong to at least 14 different protease families: S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, M55 (MEROPS database). The various keratinolytic enzymes act via endo-attack (proteases in families S1, S8, S16, M4, M16, M36), exo-attack (proteases in families S9, S10, M14, M28, M38, M55) or by action only on oligopeptides (proteases in families M3, M32), respectively. Other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. This review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. We also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications.


Treatment and outcomes of Ewing sarcoma in Turkish adults: a single centre experience.
Ewing sarcoma is a small round cell tumor arising from soft tissue and bone that predominantly affects children and adolescents. The most unfavorable prognostic factor is the presence of distant metastasis at the time of diagnosis. The records of 26 Ewing sarcoma patients (14 male, 12 female) were re-evaluated retrospectively. The median age was 26.5 (19-42) years. Eight patients (31%) showed a primary tumor in their extremities, 8 (31%) in the thorax, 4 (15%) at the vertebra, 4 (15%) in the head and neck, and 2 (8%) in the abdomen. Five patients (19%) had distant metastasis at diagnosis. The median progression-free survival was 72 months and 10 months in localized and metastatic disease, respectively (p=0.005). The overall survival rate was 19 months in metastatic disease, and the 5-year overall survival rate was 64% in localized disease (p=0.006). Patients who had localized disease in the extremities and were under age 30 had a favorable prognosis. Although Ewing sarcoma is a tumor affecting children and adolescents, it may be seen in adults, where the prognosis is generally worse. Although it is a highly malignant tumor, it is possible to achieve improved survival with combined modality treatments.


Alkylphospholipids - a promising class of chemotherapeutic agents with a broad pharmacological spectrum.
Since when alkylphospholipds (ALPs) were discovered and, even further after miltefosine's approval for the treatment of cutaneous metastasis of breast cancer and leishmaniasis, their activity against many other diseases have been extensively studied. This review aims to provide a summary of the alkylphospholipids' applications investigated so far. The mechanism of action of ALPs is not fully understood, however it is believed that they interfere with lipid homeostasis leading to cell apoptosis. Due to ALPs cytotoxic activity, this class of molecules has shown to be effective against many diseases and conditions. Besides the approval of miltefosine for application in cutaneous metastasis of breast cancer and visceral and cutaneous leishmaniasis, several other analogs have proved efficacy and are promising as less toxic alternatives. ALPs have also shown in vitro and in vivo activity against Trypanosoma spp., amoebae, Tricomonas vaginalis, Schistosoma mansoni, HIV, and some fungi and bacteria species. The use of ALPs for intraocular lens coating is also under investigation. In addition, a clinical trial comparing miltefosine with usual treatments to spontaneous urticaria is also being conducted. Alkylphospholipids present such a broad pharmacological spectrum that justifies the need for further investigations of the drug class mechanisms of action, as well as the search for new analogs with improved activity and toxicological profiles.


Chiropractic management of a veteran with lower back pain associated with diffuse idiopathic skeletal hypertrophy and degenerative disk disease.
The purpose of this article is to report the response of chiropractic care of a geriatric veteran with degenerative disk disease and diffuse idiopathic skeletal hyperostosis. A 74-year-old man presented with low back pain (LBP) and loss of feeling in his lower extremities for 3 months. The LBP was of insidious onset with a 10/10 pain rating on the numeric pain scale (NPS) and history of degenerative disk disease and diffuse idiopathic skeletal hypertrophy. Oswestry questionnaire was 44% and health status questionnaire was 52%, which were below average for his age. The patient presented with antalgia and severe difficulty with ambulation and thus used a walker. Chiropractic care included Activator Methods protocol. Two weeks into treatment, he reported no back pain; and after 4 treatments, he was able to walk with a cane instead of a walker. The NPS decreased from a 10/10 to a 0/10, and his Revised Oswestry score decreased from 44/100 to 13.3/100. His Health Status Questionnaire score increased 25 points to 77/100, bringing him from below average for his age to above average for his age. Follow-up with the patient at approximately 1 year and 9 months showed an Oswestry score of 10/100 and a Health Status Questionnaire score of 67/100, still above average for his age. The findings in this case study showed that Activator-assisted spinal manipulative therapy had positive subjective and objective results for LBP and ambulation in a geriatric veteran with degenerative disk disease and diffuse idiopathic skeletal hyperostosis.


Interictal amylin levels in chronic migraine patients: A case-control study.
Recently, amylin and its receptors were found in different structures involved in migraine pathophysiology. Here, we evaluate interictal concentrations of amylin and calcitonin gene-related peptide in peripheral blood as biomarkers for chronic migraine. We prospectively recruited patients with episodic migraine, chronic migraine and healthy controls. Interictal amylin and calcitonin gene-related peptide levels were assessed in blood samples using enzyme linked immunosorbent assay. We assessed plasma samples from 58 patients with episodic migraine (mean age 37.71 ± 10.47, 87.9% female), 191 with chronic migraine (mean age 46.03 ± 11.93, 95% female), and on 68 healthy controls (mean age 43.58 ± 11.08 years, 86% female). Body mass index was 25.94 ± 4.53 kg/m2 for migraine patients and 25.13 ± 4.92 kg/m2 for healthy controls (p = 0.0683). Interictal plasma amylin levels were higher in chronic migraine patients (47.1 pg/mL) than in the episodic migraine patients (28.84 pg/mL, p < 0.0001) and healthy controls (24.74 pg/mL, p < 0.0001). Plasma calcitonin gene-related peptide levels were increased (20.01 pg/mL) in chronic migraine patients when compared to healthy controls (11.37 pg/mL, p = 0.0016), but not to episodic migraine patients (18.89 pg/mL, p = 0.4369). Applying a cut-off concentration of 39.68 pg/mL plasma amylin, the sensitivity to differentiate chronic migraine from healthy controls was 57.6% and the specificity was 88.2%. Variables such as age, analgesic overuse, depression, allodynia, use of preventive medication or a history of aura did not influence the plasma concentrations of amylin or calcitonin gene-related peptide. Interictal plasma amylin levels are higher in patients with chronic migraine and may serve as a diagnostic biomarker for chronic migraine.


rSNPBase: a database for curated regulatory SNPs.
In recent years, human regulatory SNPs (rSNPs) have been widely studied. Here, we present database rSNPBase, freely available at http://rsnp.psych.ac.cn/, to provide curated rSNPs that analyses the regulatory features of all SNPs in the human genome with reference to experimentally supported regulatory elements. In contrast with previous SNP functional annotation databases, rSNPBase is characterized by several unique features. (i) To improve reliability, all SNPs in rSNPBase are annotated with reference to experimentally supported regulatory elements. (ii) rSNPBase focuses on rSNPs involved in a wide range of regulation types, including proximal and distal transcriptional regulation and post-transcriptional regulation, and identifies their potentially regulated genes. (iii) Linkage disequilibrium (LD) correlations between SNPs were analysed so that the regulatory feature is annotated to SNP-set rather than a single SNP. (iv) rSNPBase provides the spatio-temporal labels and experimental eQTL labels for SNPs. In summary, rSNPBase provides more reliable, comprehensive and user-friendly regulatory annotations on rSNPs and will assist researchers in selecting candidate SNPs for further genetic studies and in exploring causal SNPs for in-depth molecular mechanisms of complex phenotypes.


Zoonotic infections in Europe in 2007: a summary of the EFSA-ECDC annual report.
The European Food Safety Authority and the European Centre for Disease Prevention and Control have just published their Community Zoonoses Report for 2007, analysing the occurrence of infectious diseases transmittable from animals to humans. Campylobacter infections still topped the list of zoonotic diseases in the European Union and the number of Salmonella infections in humans decreased for the fourth year in a row. Cases of listeriosis remained at the same level as in 2006, but due to the severity of the disease, more studies on transmission routes are warranted. The report highlights the importance of continued co-operation between veterinarians and public health specialists, both at the EU level and within Member States.


Nonlinear response properties of ultralarge hyperpolarizability twisted pi-system donor-acceptor chromophores. Dramatic environmental effects on response.
State-average complete active space self-consistent field (SA-CASSCF) calculations are performed on the energetically lowest two electronic states of a novel alkyl-substituted 4-quinopyran twisted pi-system electro-optic chromophore. In the gas phase, the ground-state electronic configuration is diradicaloid (D), and the first excited state is zwitterionic (Z). When an external dipolar field is applied to simulate polar solvation, the relative energies of D and Z are dramatically perturbed. At sufficient field strengths, the relative ordering of the states is inverted so that Z becomes the ground state. As the energy difference between the D and Z states falls, the magnitudes of the longitudinal static polarizability (alpha) and hyperpolarizability (beta) increase appreciably--in certain cases, by 2 orders of magnitude. These computational results are interpreted and supported by qualitative state correlation diagrams constructed from qualitative molecular orbital theory and are in agreement with recent experimental results on twisted pi-system electro-optic chromophores (Kang, H. et al. J. Am. Chem. Soc. 2007, 129, 3267). The computational results also suggest that changing the environmental polarity is a promising strategy for tuning alpha and beta in such types of chromophores, which experimentally exhibit large nonlinear optical response.


Association between birth weight and refractive error in adulthood: a Mendelian randomisation study.
Pathological myopia is one of the leading causes of blindness globally. Lower birth weight (BW) within the normal range has been reported to increase the risk of myopia, although findings conflict. We sought to estimate the causal effect of BW on refractive error using Mendelian randomisation (MR), under the assumption of a linear relationship. Genetic variants associated with BW were identified from meta-analysis of a genome-wide association study (GWAS) for self-reported BW in 162 039 UK Biobank participants and a published Early Growth Genetics (EGG) consortium GWAS (n=26 836). We performed a one-sample MR analysis in 39 658 unrelated, adult UK Biobank participants (independent of the GWAS sample) using an allele score for BW as instrumental variable. A two-sample MR sensitivity analysis and conventional ordinary least squares (OLS) regression analyses were also undertaken. In OLS analysis, BW showed a small, positive association with refractive error: +0.04 D per SD increase in BW (95% CI 0.02 to 0.07; p=0.002). The one-sample MR-estimated causal effect of BW on refractive error was higher, at +0.28 D per SD increase in BW (95% CI 0.05 to 0.52, p=0.02). A two-sample MR analysis provided similar causal effect estimates, with minimal evidence of directional pleiotropy. Our study suggests lower BW within the normal range is causally associated with a more myopic refractive error. However, the impact of the causal effect was modest (range 1.00 D covering approximately 95% of the population).


G-->A hypermutation in protease and reverse transcriptase regions of human immunodeficiency virus type 1 residing in resting CD4+ T cells in vivo.
In vitro studies have shown that the host cytidine deaminase APOBEC3G causes lethal hypermutation in human immunodeficiency virus type 1 reverse transcripts unless its incorporation into virions is blocked by Vif. By examining stably archived sequences in resting CD4+ T cells, we show that hypermutation occurs in most if not all infected individuals. Hypermutated sequences comprised >9% of archived species in resting CD4+ T cells but were not found in plasma virus. Mutations occurred in predicted contexts, with notable hotspots. Thus, defects in Vif function in vivo give rise to hypermutated viral genomes that can be integrated but do not produce progeny viruses.


Characterization of an IL-2 mimetic with therapeutic potential.
Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.


An anomalous basaltic meteorite from the innermost main belt.
Triangulated observations of fireballs allow us to determine orbits and fall positions for meteorites. The great majority of basaltic meteorites are derived from the asteroid 4 Vesta. We report on a recent fall that has orbital properties and an oxygen isotope composition that suggest a distinct parent body. Although its orbit was almost entirely contained within Earth's orbit, modeling indicates that it originated from the innermost main belt. Because the meteorite parent body would likely be classified as a V-type asteroid, V-type precursors for basaltic meteorites unrelated to Vesta may reside in the inner main belt. This starting location is in agreement with predictions of a planetesimal evolution model that postulates the formation of differentiated asteroids in the terrestrial planet region, with surviving fragments concentrated in the innermost main belt.


Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel.
Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the alpha- and beta-connexin subgroups but not the gamma-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.


Navigation by light polarization in clear and turbid waters.
Certain terrestrial animals use sky polarization for navigation. Certain aquatic species have also been shown to orient according to a polarization stimulus, but the correlation between underwater polarization and Sun position and hence the ability to use underwater polarization as a compass for navigation is still under debate. To examine this issue, we use theoretical equations for per cent polarization and electric vector (e-vector) orientation that account for the position of the Sun, refraction at the air-water interface and Rayleigh single scattering. The polarization patterns predicted by these theoretical equations are compared with measurements conducted in clear and semi-turbid coastal sea waters at 2 m and 5 m depth over sea floors of 6 m and 28 m depth. We find that the per cent polarization is correlated with the Sun's elevation only in clear waters. We furthermore find that the maximum value of the e-vector orientation angle equals the angle of refraction only in clear waters, in the horizontal viewing direction, over the deeper sea floor. We conclude that navigation by use of underwater polarization is possible under restricted conditions, i.e. in clear waters, primarily near the horizontal viewing direction, and in locations where the sea floor has limited effects on the light's polarization.


Immunological heterogeneity of carcinoembryonic antigen: purification from meconium of an antigen related to carcinoembryonic antigen.
Two antigens cross-reactive with carcinoembryonic antigen (CEA) and distinct from the nonspecific cross-reacting antigen were identified in meconium by double immunodiffusion with a conventional goat anti-CEA antiserum. These two antigens together competitively inhibited cross-reacting antibodies against them in CEA radioimmunoassay and contributed to the measurement of meconium CEA levels which averaged 6 times higher than that determined with anti-CEA specific antibody. A purification method for one of these antigens, tentatively designated meconium antigen, is described and uses a combination of ethanol fractionation, ion-exchange and molecular sieve chromatography, and adsorption to an immunoadsorbent containing a cross-reactive murine monoclonal antibody to CEA. Preliminary characterization of the purified meconium antigen showed it to be a glycoprotein, migrating as an alpha-globulin and having a molecular size similar to that of CEA (Mr 185,000 versus 200,000). Antigenically, it lacks at least one determinant present on CEA and differs further from CEA by being weakly reactive with concanavalin A and resistant to proteolytic digestion with Pronase E. Although these properties of meconium antigen suggest that it may be nonspecific cross-reacting antigen 2, additional chemical and antigenic studies are required to establish its relationship to CEA and other CEA-related antigens in meconium.


Accuracy of burn size assessment prior to arrival in Dutch burn centres and its consequences in children: a nationwide evaluation.
Total body surface area (TBSA) burned, expressed as percentage is one of the most important aspects of the initial care of a burn victim. It determines whether transfer to a burn centre is necessary as well as the need for, and amount of, intravenous fluid resuscitation. Numerous studies, however, have highlighted inaccuracies in TBSA assessment. Therefore, the differences in burn size estimates between referrers and burn centre's in children and its consequences in terms of transfer and intravenous fluid resuscitation were investigated. This study involved two time periods from January 2002 until March 2004 and January 2007 until August 2008. All referred children admitted to a Dutch Burn centre within 24h post burn were eligible. Data were obtained from patient records retrospectively and in part prospectively. A total of 323 and 299 children were included in periods 1 and 2, respectively. Referring physicians overestimated burn size with a factor two (mean difference: 6% TBSA ± 5.5). About one in five children was referred to a burn centre without fulfilling the criteria for referral with regard to burn size (assessed by burn specialists) special localisation or inhalation trauma. Proportions of children receiving intravenous fluid resuscitation regardless of indication increased from 33% to 49% (p<0.01). The received volumes tended to be higher than necessary. Referring physicians overestimate burn size in children admitted to Dutch burn centres. This has little negative consequences, however, in terms of unindicated transfers to a burn centre or unnecessary fluid resuscitation.


Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 protein.
Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to approximately 3.2 A resolution. They belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a=85.64, c=292.74 A. Structure determination is ongoing.


SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer.
SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.