Care Groups II: A Summary of the Child Survival Outcomes Achieved Using Volunteer Community Health Workers in Resource-Constrained Settings.
The Care Group approach, described in detail in a companion paper in this journal, uses volunteers to convey health promotion messages to their neighbors. This article summarizes the available evidence on the effectiveness of the Care Group approach, drawing on articles published in the peer-reviewed literature as well as data from unpublished but publicly available project evaluations and summary analyses of these evaluations. When implemented by strong international NGOs with adequate funding, Care Groups have been remarkably effective in increasing population coverage of key child survival interventions. There is strong evidence that Care Groups can reduce childhood undernutrition and reduce the prevalence of diarrhea. Finally, evidence from multiple sources, comprising independent assessments of mortality impact, vital events collected by Care Group Volunteers themselves, and analyses using the Lives Saved Tool (LiST), that Care Groups are effective in reducing under-5 mortality. For example, the average decline in under-5 mortality, estimated using LiST, among 8 Care Group projects was 32%. In comparison, among 12 non-Care Group child survival projects, the under-5 mortality declined, on average, by an estimated 11%. Care Group projects cost in the range of US$3-$8 per beneficiary per year. The cost per life saved is in the range of $441-$3,773, and the cost per disability-adjusted life year (DALY) averted is in the range of $15-$126. The Care Group approach, when implemented as described, appears to be highly cost-effective based on internationally accepted criteria. Care Groups represent an important and promising innovative, low-cost approach to increasing the coverage of key child survival interventions in high-mortality, resource-constrained settings. Next steps include further specifying the adjustments needed in government health systems to successfully incorporate the Care Group approach, testing the feasibility of these adjustments and of the effectiveness of Care Groups in pilot programs in government health systems, and finally assessing effectiveness at scale under routine field conditions in government health programs.


ReFeaFi: Genome-wide prediction of regulatory elements driving transcription initiation.
Regulatory elements control gene expression through transcription initiation (promoters) and by enhancing transcription at distant regions (enhancers). Accurate identification of regulatory elements is fundamental for annotating genomes and understanding gene expression patterns. While there are many attempts to develop computational promoter and enhancer identification methods, reliable tools to analyze long genomic sequences are still lacking. Prediction methods often perform poorly on the genome-wide scale because the number of negatives is much higher than that in the training sets. To address this issue, we propose a dynamic negative set updating scheme with a two-model approach, using one model for scanning the genome and the other one for testing candidate positions. The developed method achieves good genome-level performance and maintains robust performance when applied to other vertebrate species, without re-training. Moreover, the unannotated predicted regulatory regions made on the human genome are enriched for disease-associated variants, suggesting them to be potentially true regulatory elements rather than false positives. We validated high scoring "false positive" predictions using reporter assay and all tested candidates were successfully validated, demonstrating the ability of our method to discover novel human regulatory regions.


Cumulative culture in the laboratory: methodological and theoretical challenges.
In the last decade, cultural transmission experiments (transmission chains, replacement, closed groups and seeded groups) have become important experimental tools in investigating cultural evolution. However, these methods face important challenges, especially regarding the operationalization of theoretical claims. In this review, we focus on the study of cumulative cultural evolution, the process by which traditions are gradually modified and, for technological traditions in particular, improved upon over time. We identify several mismatches between theoretical definitions of cumulative culture and their implementation in cultural transmission experiments. We argue that observed performance increase can be the result of participants learning faster in a group context rather than effectively leading to a cumulative effect. We also show that in laboratory experiments, participants are asked to complete quite simple tasks, which can undermine the evidential value of the diagnostic criterion traditionally used for cumulative culture (i.e. that cumulative culture is a process that produces solutions that no single individual could have invented on their own). We show that the use of unidimensional metrics of cumulativeness drastically curtail the variation that may be observed, which raises specific issues in the interpretation of the experimental evidence. We suggest several solutions to these mismatches (learning times, task complexity and variation) and develop the use of design spaces in experimentally investigating old and new questions about cumulative culture.


The effect of artificial lipid envelopment of Adenovirus 5 (Ad5) on liver de-targeting and hepatotoxicity.
Human Adenovirus type 5 (Ad5) has been extensively explored in clinical gene therapy, but its immunogenicity dramatically affects the kinetics and toxicity profile of the vector. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid to develop safer hybrid vectors. Here, we studied the interaction of enveloped Ad in cationic (DOTAP:Chol) or anionic (DOPE:CHEMS) lipid bilayers with different blood components. When Ad was enveloped by cationic lipids, significantly high levels of viral uptake in HepG2 cultured cells were achieved, independent of blood coagulation factors present. In vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration in BALB/c mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction significantly increasing virus lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. ALT/AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. This study suggests that artificial envelopes for Ad significantly alter the interactions with blood components and can divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity.


Left-right asymmetry in vertebrate development.
Externally the vertebrate body plan presents a bilateral symmetry in relation to the midline. However, inside the body the distribution of the visceral organs follows a very particular pattern that is not symmetrical in relation to the midline. The last 10 years have seen remarkable advances in our understanding of how the internal asymmetries typical of the vertebrate body are established and controlled. The use of different development models has permitted to uncover fascinating ways of creating asymmetry, like the activity of the nodal cilia. A host of studies has also unravelled the involvement of many genes in the left right patterning pathway. Based on this knowledge the genetic basis of human laterality defects are beginning to be revealed. It is a major challenge now to understand how all these genes control left right development as well as the complex set of interactions established between them.


Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa.
The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. We prospectively studied children presenting with suspected severe infection to 2 urban hospitals in The Gambia, between January 2013 and September 2015. Demographic and anthropometric data, clinical features, management, and blood culture results were documented. Urine screens for antibiotic activity were performed in a subset of participants. Of 411 children enrolled (median age, 29 months; interquartile range, 11-82), 79.5% (325 of 409) reported prehospital antibiotic use. Antimicrobial activity by urinary screen for antibiotic activity was detected in 70.8% (n = 80 of 113). Sixty-six bacterial pathogens were identified in 65 (15.8%) participants and Staphylococcus aureus predominated. Gram-positive organisms were more commonly identified than Gram-negative (P < .01). Antibiotic resistance against first-line antimicrobials (ampicillin and gentamicin) was common among Gram-negative bacteria (39%; range, 25%-100%). Factors significantly associated with bacteremia included the following: gender, hydration status, musculoskeletal examination findings, admission to the Medical Research Council The Gambia at London School of Hygiene & Tropical Medicine hospital, and meeting sepsis criteria. Those associated with increased mortality were presence of a comorbidity, clinical pallor, tachypnea, and altered consciousness. Tachycardia was associated with reduced mortality. The bacteremia rate in children with suspected childhood life-threatening infectious diseases in The Gambia is high. The pattern of pathogen prevalence and antimicrobial resistance has changed over time compared with previous studies illustrating the importance of robust bacterial surveillance programs in resource-limited settings.


Assessment of the required performance and the development of corresponding program decision rules for neglected tropical diseases diagnostic tests: Monitoring and evaluation of soil-transmitted helminthiasis control programs as a case study.
Recently, the World Health Organization established the Diagnostic Technical Advisory Group to identify and prioritize diagnostic needs for neglected tropical diseases, and to ultimately describe the minimal and ideal characteristics for new diagnostic tests (the so-called target product profiles (TPPs)). We developed two generic frameworks: one to explore and determine the required sensitivity (probability to correctly detect diseased persons) and specificity (probability to correctly detect persons free of disease), and another one to determine the corresponding samples sizes and the decision rules based on a multi-category lot quality assurance sampling (MC-LQAS) approach that accounts for imperfect tests. We applied both frameworks for monitoring and evaluation of soil-transmitted helminthiasis control programs. Our study indicates that specificity rather than sensitivity will become more important when the program approaches the endgame of elimination and that the requirements for both parameters are inversely correlated, resulting in multiple combinations of sensitivity and specificity that allow for reliable decision making. The MC-LQAS framework highlighted that improving diagnostic performance results in a smaller sample size for the same level of program decision making. In other words, the additional costs per diagnostic tests with improved diagnostic performance may be compensated by lower operational costs in the field. Based on our results we proposed the required minimal and ideal diagnostic sensitivity and specificity for diagnostic tests applied in monitoring and evaluating of soil-transmitted helminthiasis control programs.


Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles.
To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined 'Penner' serotypes for this species. Field strains (n = 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established 'Penner' scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of 'low risk' to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77.6% S-level. Most other serotypes did not form homogeneous clusters. High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. The genetic relatedness among all 'Penner' serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.


Nuclear receptor Rev-erbalpha is a critical lithium-sensitive component of the circadian clock.
Lithium is commonly used to treat bipolar disorder, which is associated with altered circadian rhythm. Lithium is a potent inhibitor of glycogen synthase kinase 3 (GSK3), which regulates circadian rhythm in several organisms. In experiments with cultured cells, we show here that GSK3beta phosphorylates and stabilizes the orphan nuclear receptor Rev-erbalpha, a negative component of the circadian clock. Lithium treatment of cells leads to rapid proteasomal degradation of Rev-erbalpha and activation of clock gene Bmal1. A form of Rev-erbalpha that is insensitive to lithium interferes with the expression of circadian genes. Control of Rev-erbalpha protein stability is thus a critical component of the peripheral clock and a biological target of lithium therapy.


Neuropsychiatric symptoms and comorbidity: Associations with dementia progression rate in a memory clinic cohort.
Neuropsychiatric symptoms (NPS) are associated with dementia severity and progression rate. NPS clusters have different neurobiological underpinnings; therefore, their effect on dementia progression may differ. Furthermore, little is known about whether individual comorbidities affect progression rate. We investigated the effect of NPS clusters and individual comorbidities on dementia progression. A memory clinic cohort with all-cause dementia (N = 442) was followed for up to 3 years from diagnosis. Previously, we found trajectory groups of dementia progression in this cohort: one with slow progression and two with rapid progression. In the present study, using principal component analysis, three symptom clusters of NPS were identified on the Neuropsychiatric Inventory Questionnaire (NPI-Q): agitation, affective and psychosis symptom clusters. Data regarding comorbidity were collected by linkage to the Norwegian Patient Registry. Multinomial logistic regression was applied to explore the association between NPS clusters and comorbidity with trajectory-group membership. Adjusted for demographics, dementia aetiology, comorbidity and cognition, we found that, at the time of dementia diagnosis, for every point within the psychosis symptom cluster of the NPI-Q, the risk of rapid progression increased by 53%; for every point within the affective symptom cluster, the risk of rapid progression increased by 29%. A previous diagnosis of mental and behavioural disorders (excluding dementia) decreased the risk of rapid dementia progression by 65%. Psychosis and affective symptom clusters at the time of diagnosis were associated with rapid progression of dementia. Previous diagnoses of mental and behavioural disorders (excluding dementia) were associated with slow progression.


Ubiquitination of RIP is required for tumor necrosis factor alpha-induced NF-kappaB activation.
Stimulation of cells with tumor necrosis factor (TNFalpha) triggers a recruitment of various signaling molecules, such as RIP, to the TNFalpha receptor 1 complex, leading to activation of NF-kappaB. Previous studies indicate that RIP plays an essential role for TNFalpha-induced NF-kappaB activation, but the molecular mechanism by which RIP mediates TNFalpha signals to activate NF-kappaB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-kappaB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFalpha receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation.


Evolutionary constraints permeate large metabolic networks.
Metabolic networks show great evolutionary plasticity, because they can differ substantially even among closely related prokaryotes. Any one metabolic network can also effectively compensate for the blockage of individual reactions by rerouting metabolic flux through other pathways. These observations, together with the continual discovery of new microbial metabolic pathways and enzymes, raise the possibility that metabolic networks are only weakly constrained in changing their complement of enzymatic reactions. To ask whether this is the case, I characterized pairwise and higher-order associations in the co-occurrence of genes encoding metabolic enzymes in more than 200 completely sequenced representatives of prokaryotic genera. The majority of reactions show constrained evolution. Specifically, genes encoding most reactions tend to co-occur with genes encoding other reaction(s). Constrained reaction pairs occur in small sets whose number is substantially greater than expected by chance alone. Most such sets are associated with single biochemical pathways. The respective genes are not always tightly linked, which renders horizontal co-transfer of constrained reaction sets an unlikely sole cause for these patterns of association. Even a limited number of available genomes suffices to show that metabolic network evolution is highly constrained by reaction combinations that are favored by natural selection. With increasing numbers of completely sequenced genomes, an evolutionary constraint-based approach may enable a detailed characterization of co-evolving metabolic modules.


Acidic fibroblast growth factor modulates Staphylococcus aureus adherence to human endothelial cells.
Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo.


Effects of angiopeptin on transplant arteriosclerosis in the rat.
The influence of the somatostatin analogue angiopeptin on transplant arteriosclerosis was investigated using two aortic transplantation rat models. One was characterized by ischemia/reperfusion-induced changes in syngeneic transplants while immunologically induced changes dominated in the other allogeneic model. Angiopeptin, 100 micrograms/kg per day, was administered continuously until the sacrifice of the rats after 8 weeks. No additional immunosuppression was used in either model. An image analysis system was used to quantify the intimal and medial thicknesses of the grafts. In the syngeneic grafts, the intimal thickness was less than 50% of that of control grafts (P < 0.05), but no difference was seen in the allogeneic model. The expression of selected cells, TGF-beta s, and PDGF and PDGF alpha-receptors was detected immunohistochemically and displayed a similar picture in control and angiopeptin-treated grafts in both models. We conclude that angiopeptin has no clear immunosuppressive properties but may counteract ischemia-induced transplant arteriosclerosis.


Increased Arctic influence on the midlatitude flow during Scandinavian Blocking episodes.
Recent studies have suggested that Arctic teleconnections affect the weather of the midlatitudes on time-scales relevant for medium-range weather forecasting. In this study, we use several numerical experimentation approaches with a state-of-the-art global operational numerical weather prediction system to investigate this idea further. Focusing on boreal winter, we investigate whether the influence of the Arctic on midlatitude weather, and the impact of the current Arctic observing system on the skill of medium-range weather forecasts in the midlatitudes is more pronounced in certain flow regimes. Using so-called Observing System Experiments, we demonstrate that removing in situ or satellite observations from the data assimilation system, used to create the initial conditions for the forecasts, deteriorates midlatitude synoptic forecast skill in the medium-range, particularly over northern Asia. This deterioration is largest during Scandinavian Blocking episodes, during which: (a) error growth is enhanced in the European-Arctic, as a result of increased baroclinicity in the region, and (b) high-amplitude planetary waves allow errors to propagate from the Arctic into midlatitudes. The important role played by Scandinavian Blocking, in modulating the influence of the Arctic on midlatitudes, is also corroborated in relaxation experiments, and through a diagnostic analysis of the ERA5 reanalysis and reforecasts.


Pentoxifylline is as effective as leukocyte depletion for modulating pulmonary reperfusion injury.
Previous studies have suggested the amelioration of lung reperfusion injury when initial reperfusion is undertaken with leukocyte-depleted blood. Pharmacologic agents, such as pentoxifylline, are also effective, but no previous studies have demonstrated which is superior. We investigated these agents in a porcine model of left single-lung transplantation. Donor lungs were preserved with modified Euro-Collins solution for a mean ischemic time of 18.6 hours. Gas exchange, pulmonary vascular resistance, neutrophil elastase level, and free radical release (measured on the basis of malonaldehyde levels) were assessed over a 12-hour period. Group A (n = 5) was a control group with no interventions added. Group B was reperfused through an extracorporeal circuit incorporating a leukocyte-depleting filter for 30 minutes before conventional blood flow was restored. Group C was reperfused with the addition of intravenous pentoxifylline (2 mg x kg(-1) x h(-1)). Groups B and C were similar in terms of oxygenation, pulmonary vascular resistance, and free radical release. Group B displayed increased levels of neutrophil elastase. Both groups were superior with regard to these outcome measures compared with control group A. Pentoxifylline, when administered to recipient animals, attenuates reperfusion injury to a degree similar to that seen with leukocyte-depleted reperfusion. This technique is simple, safe, and as effective as using a more complex extracorporeal circuit incorporating a leukocyte-depleting filter to ameliorate acute lung injury.


Effect of safranal, a constituent of saffron, on olanzapine (an atypical antipsychotic) induced metabolic disorders in rat.
Olanzapine, an atypical antipsychotic, causes weight gain and metabolic disorders in humans. Safranal, one of the active components of Crocus sativus (saffron), has been shown to have anti-obesity, lipid and blood pressure lowering and anti-diabetes effects. In this investigation, the effect of safranal on metabolic disorders induced by olanzapine was studied. Fourty-two female Wistar rats were divided into 7 groups of 6 animals. The two groups were selected as controls, which received olanzapine and safranal solvents, respectively. The third group treated by olanzapine 5 mg/kg. Groups 4, 5 and 6 treated by olanzapine 5 mg/kg plus safranal (2.5, 5 and 10 mg/kg) and the last group received safranal 10 mg/kg. The injections were performed intraperitoneally for 14 days and on the 15th day the rats were killed and their serum were collected to measure metabolic factors including glucose, insulin, triglyceride, total cholesterol and HDL cholesterol. Leptin level in plasma was also measured. Mean systolic blood pressure was measured using tail cuff method at the end of study. The rats were weighed every other day and amount of food consumed was measured daily. Olanzapine significantly elevated body weight, food intake, fasting blood glucose, TG, leptin, and mean systolic blood pressure (MSBP). It also significantly decreased HDL cholesterol blood level. Safranal significantly improved all these complications at three doses. Based on the results of this study, safranal is thought to be used as an effective combination in controlling metabolic complications caused by olanzapine.


Prediction of febrile seizures in siblings: a practical approach.
To quantify the risk of febrile seizures (FS) in relatives of children with FS and to predict the risk of FS in siblings, we calculated cumulative risks of FS in first degree relatives of 129 children with FS. The study was conducted as a prospective follow up study of FS recurrences at the outpatient clinic of the Sophia Children's Hospital in Rotterdam. Thirteen parents and 12 siblings had experienced FS, accounting for a 6-year cumulative risk of 7%. The risk of FS was increased in relatives of children with recurrent FS (12%). The risk of FS in siblings (10%) in our study was more than twice the average risk in a similar population (4%). A positive FS history in a parent, young age at onset in the proband, and recurrences in the proband were selected in a multivariable prediction model. If two or more of these risk factors were present, the risk of West European siblings to develop FS was 46% (hazard ratio 5.4). The cumulative risk of FS in siblings of children with FS is increased. The age attained risk of FS can be estimated using a practical model incorporating three readily available risk factors.


Evaluating fine motor coordination in children who are not ready for handwriting: which test should we take?
When children are not ready to write, assessment of fine motor coordination may be indicated. The purpose of this study was to evaluate which fine motor test, the Nine-Hole Peg Test (9-HPT) or the newly developed Timed Test of In-Hand Manipulation (Timed-TIHM), correlates best with handwriting readiness as measured by the Writing Readiness Inventory Tool In Context-Task Performance (WRITIC-TP). From the 119 participating children, 43 were poor performers. Convergent validity of the 9-HPT and Timed-TIHM with WRITIC-TP was determined, and test-retest reliability of the Timed-TIHM was examined in 59 children. The results showed that correlations of the 9-HPT and Timed-TIHM with the WRITIC-TP were similar (rs = -0.40). The 9-HPT and the complex rotation subtask of the Timed-TIHM had a low correlation with the WRITIC-TP in poor performers (rs = -0.30 and -0.32 respectively). Test-retest reliability of the Timed-TIHM was significant (Intraclass Correlation Coefficient = 0.71). Neither of these two fine motor tests is appeared superior. They both relate to different aspects of fine motor performance. One of the limitations of the methodology was unequal numbers of children in subgroups. It is recommended that further research is indicated to evaluate the relation between development of fine motor coordination and handwriting proficiency, on the Timed-TIHM in different age groups.


Paraneoplastic syndromes: an approach to diagnosis and treatment.
Recent medical advances have improved the understanding, diagnosis, and treatment of paraneoplastic syndromes. These disorders arise from tumor secretion of hormones, peptides, or cytokines or from immune cross-reactivity between malignant and normal tissues. Paraneoplastic syndromes may affect diverse organ systems, most notably the endocrine, neurologic, dermatologic, rheumatologic, and hematologic systems. The most commonly associated malignancies include small cell lung cancer, breast cancer, gynecologic tumors, and hematologic malignancies. In some instances, the timely diagnosis of these conditions may lead to detection of an otherwise clinically occult tumor at an early and highly treatable stage. Because paraneoplastic syndromes often cause considerable morbidity, effective treatment can improve patient quality of life, enhance the delivery of cancer therapy, and prolong survival. Treatments include addressing the underlying malignancy, immunosuppression (for neurologic, dermatologic, and rheumatologic paraneoplastic syndromes), and correction of electrolyte and hormonal derangements (for endocrine paraneoplastic syndromes). This review focuses on the diagnosis and treatment of paraneoplastic syndromes, with emphasis on those most frequently encountered clinically. Initial literature searches for this review were conducted using PubMed and the keyword paraneoplastic in conjunction with keywords such as malignancy, SIADH, and limbic encephalitis, depending on the particular topic. Date limitations typically were not used, but preference was given to recent articles when possible.


The influence of processing factors and non-atopy-related maternal and neonate characteristics on yield and cytokine responses of cord blood mononuclear cells.
Several studies have evaluated the associations between cord blood cellular responses and atopic diseases in children, but the results of these studies are inconsistent. Variations in blood processing factors and maternal and infant characteristics are typically not accounted for and may contribute to these inconsistencies. Cord blood samples were obtained from 287 subjects participating in the Childhood Origins of ASThma project, a prospective study of children at high risk for the development of asthma/allergies. Mononuclear cells were stimulated with phytohaemagglutinin (PHA), phorbal myristate acetate/ionomycin or a suspension of killed staphylococcus, and IFN-gamma, IL-10 and IL-13 were quantitated by ELISA. Cell yields and cytokine production were related to processing factors and maternal and infant characteristics. The strongest relationships between independent variables and cell yield or cytokine responses occurred with the season of birth. The highest median cell yields were seen in fall, and the lowest in summer (difference of 47%, P=0.0027). Furthermore, PHA-induced IL-5 and IL-13 responses were approximately 50% higher in spring and summer than in fall or winter (P<0.0001). Clots in the cord blood samples were associated with a reduced median cell yield (42% reduction, P<0.0001), and an increased PHA-induced IL-10 secretion (27% increase, P=0.004). These data suggest that season of collection, and to a lesser extent clotting in samples, affect cord blood mononuclear cell yield and cytokine responses. Careful documentation and analysis of processing and environmental variables are important in understanding biological relationships with cytokine responses, and also lead to greater comparability among studies using these techniques.


Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.
The mammalian hair follicle is a complex 'mini-organ' thought to form only during development; loss of an adult follicle is considered permanent. However, the possibility that hair follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair follicles form de novo in genetically normal adult mice. The regenerated hair follicles establish a stem cell population, express known molecular markers of follicle differentiation, produce a hair shaft and progress through all stages of the hair follicle cycle. Lineage analysis demonstrated that the nascent follicles arise from epithelial cells outside of the hair follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.


[Use of antimicrobial agents in periodontology].
With mechanical cleaning, it is never possible to remove all bacteria from tooth surfaces, in, for example, furcation sites and at the bottom of the pocket. Supporting non-surgical periodontal treatment with the use of antimicrobial agents is, therefore, an obvious approach in order to achieve a better treatment outcome.. The combination of amoxicillin and metronidazole has been thoroughly investigated as a support in periodontal treatment and deserves a place among the tools the dental care professional has for effective treatment. However, there is at present no strict indication in which clinical situations antibiotics can be successfully prescribed. Considering responsible use of antimicrobials by healthcare professionals (antimicrobial stewardship) the decision to support periodontal treatment with antibiotics should be in the hands of those authorised to provide a prescription. Additional rinsing with chlorhexidine twice daily during periodontal treatment and the subsequent two weeks (6-8 weeks) can be considered in order to enhance the effect of clinical treatment.


TPC2 proteins mediate nicotinic acid adenine dinucleotide phosphate (NAADP)- and agonist-evoked contractions of smooth muscle.
Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca(2+) from sarcoplasmic reticulum. However, the additional Ca(2+)-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca(2+)-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2(-/-) mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.


Relationship between Dietary Approaches to Stop Hypertension score and Alternative Healthy Eating Index score with plasma asymmetrical dimethylarginine levels in patients referring for coronary angiography.
Asymmetrical dimethyl arginine (ADMA) is a competitive inhibitor in the production of nitric oxide (NO) from arginine and NO plays an important role in the preservation of vascular dilation. Elevated ADMA is a strong predictive factor for coronary artery disease (CAD). Dietary Approaches to Stop Hypertension (DASH) and Alternative Healthy Eating Index (AHEI) patterns contain factors that may influence plasma ADMA levels. The present study examined the association between the DASH score and AHEI score with plasma ADMA concentration in people with suspected heart disease selected for coronary angiography. This cross-sectional study was conducted in 148 people aged 40-80 years who were referred for coronary angiography. The DASH diet score and AHEI score were calculated for each individual based on food groups. Plasma ADMA levels were measured by high-performance liquid chromatography. ADMA concentrations were higher in the CAD group compared to the non-CAD group [0.98 (0.37) μmol L(-1) compared to 0.84 (0.42) μmol L(-1) ; P = 0.02]. There was a significant negative association between the quartile DASH score and ADMA concentration (standardised β = -0.172, P = 0.038). The ADMA concentration was lower in patients who were at the highest quartile of DASH score compared to patients with the lowest quintile score. The ADMA concentration and quartiles of AHEI score were not significantly associated. Higher scores of the DASH diet are associated with lower plasma ADMA levels and with reduced coronary artery stenosis.


The influence of biomass energy consumption on CO2 emissions: a wavelet coherence approach.
In terms of today, one may argue, throughout observations from energy literature papers, that (i) one of the main contributors of the global warming is carbon dioxide emissions, (ii) the fossil fuel energy usage greatly contributes to the carbon dioxide emissions, and (iii) the simulations from energy models attract the attention of policy makers to renewable energy as alternative energy source to mitigate the carbon dioxide emissions. Although there appears to be intensive renewable energy works in the related literature regarding renewables' efficiency/impact on environmental quality, a researcher might still need to follow further studies to review the significance of renewables in the environment since (i) the existing seminal papers employ time series models and/or panel data models or some other statistical observation to detect the role of renewables in the environment and (ii) existing papers consider mostly aggregated renewable energy source rather than examining the major component(s) of aggregated renewables. This paper attempted to examine clearly the impact of biomass on carbon dioxide emissions in detail through time series and frequency analyses. Hence, the paper follows wavelet coherence analyses. The data covers the US monthly observations ranging from 1984:1 to 2015 for the variables of total energy carbon dioxide emissions, biomass energy consumption, coal consumption, petroleum consumption, and natural gas consumption. The paper thus, throughout wavelet coherence and wavelet partial coherence analyses, observes frequency properties as well as time series properties of relevant variables to reveal the possible significant influence of biomass usage on the emissions in the USA in both the short-term and the long-term cycles. The paper also reveals, finally, that the biomass consumption mitigates CO2 emissions in the long run cycles after the year 2005 in the USA.


Guinea pig model of infectious disease - viral infections.
The guinea pig model of disease has been considered synonymous with the experimental laboratory animal since the nineteenth century. Recently we have reviewed the use of this species in models of bacterial infectious disease. The present review extends to viral diseases for which the guinea pig is less frequently considered the relevant animal model. The use of the guinea pig as a laboratory animal, aspects of immunology, viral pathogens and host-pathogen models are discussed. As a small and relatively inexpensive model for infection and immunity the guinea pig has a significant future but there are substantial requirements for development of validated quantitative analytical methods for immunological and disease biomarkers if it is to reach its potential.


Propofol-mediated Unconsciousness Disrupts Progression of Sensory Signals through the Cortical Hierarchy.
A critical component of anesthesia is the loss of sensory perception. Propofol is the most widely used drug for general anesthesia, but the neural mechanisms of how and when it disrupts sensory processing are not fully understood. We analyzed local field potential and spiking recorded from Utah arrays in auditory cortex, associative cortex, and cognitive cortex of nonhuman primates before and during propofol-mediated unconsciousness. Sensory stimuli elicited robust and decodable stimulus responses and triggered periods of stimulus-related synchronization between brain areas in the local field potential of awake animals. By contrast, propofol-mediated unconsciousness eliminated stimulus-related synchrony and drastically weakened stimulus responses and information in all brain areas except for auditory cortex, where responses and information persisted. However, we found stimuli occurring during spiking up states triggered weaker spiking responses than in awake animals in auditory cortex, and little or no spiking responses in higher order areas. These results suggest that propofol's effect on sensory processing is not just because of asynchronous down states. Rather, both down states and up states reflect disrupted dynamics.


Protein-protein interaction inhibition (2P2I): fewer and fewer undruggable targets.
Low molecular weight inhibitors of Protein-Protein Interactions (PPI's) have been identified for a number of different systems indicating the viability of the computer-aided drug design approaches. However, pathways undertaken by researchers in pharmaceutical companies or in academic laboratories are not always clearly defined and the protocols that allow the identification of lead compounds often remain blurry. We will enumerate in this review the main approaches carried out to identify and validate PPI's inhibitors. Emphasis will be placed, in a first part, on issues of particular significance to PPI's such as the problem of identification and validation of interacting sites and on the methods that allow assessing the 3D structure of a targeted complex. On the second part of this review, we will define approaches that allow a rapid identification of hits capable of inhibiting PPI's. We will highlight the problem of the scoring functions and demonstrate that the majority of the functions available to researchers are not especially relevant for PPI's. We will define why consensus scoring can be an alternative and we will propose GFscore, a non linear ranked-by number consensus scoring function as a solution to this problem. We will finally discuss the actual challenges that still remain, particularly the problem of the treatment of the receptor flexibility and of the water molecules at the interface of the Protein-Protein complexes.


A Cervical Hemi-Contusion Spinal Cord Injury Model for the Investigation of Novel Therapeutics Targeting Proximal and Distal Forelimb Functional Recovery.
Cervical spinal cord contusion is the most common human spinal cord injury, yet few rodent models replicate the pathophysiological and functional sequela of this injury. Here, we modified an electromechanical injury device and characterized the behavioral and histological changes occurring in response to a lateralized C4 contusion injury in rats. A key feature of the model includes a non-injurious touch phase where the spinal cord surface is dimpled with a consistent starting force. Animals were either left intact as a control, received a non-injury-producing touch on the surface of the cord ("sham"), or received a 0.6 mm or a 0.8 mm displacement injury. Rats were then tested on the forelimb asymmetry use test, CatWalk, and the Irvine, Beatties, and Bresnahan (IBB) cereal manipulation task to assess proximal and distal upper limb function for 12 weeks. Injuries of moderate (0.6 mm) and large (0.8 mm) displacement showed consistent differences in forelimb asymmetry, metrics of the CatWalk, and sub-scores of the IBB. Overall findings indicated long lasting proximal and distal upper limb deficits following 0.8 mm injury but transient proximal with prolonged distal limb deficits following 0.6 mm injury. Significant differences in loss of ipsilateral unmyelinated and myelinated white matter was detected between injury severities. Demyelination was primarily localized to the dorsolateral region of the hemicord and extended further rostral following 0.8 mm injury. These findings establish the C4 hemi-contusion injury as a consistent, graded model for testing novel treatments targeting forelimb functional recovery.


Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics.
The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.


Trabectedin and olaparib in patients with advanced and non-resectable bone and soft-tissue sarcomas (TOMAS): an open-label, phase 1b study from the Italian Sarcoma Group.
Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675-1·3 mg/m2 every 3 weeks; olaparib 100-300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2-6; range 1-17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5-23). Considering all dose levels, the most common grade 3-4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6-27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.


A Case Report of Guillain-Barre Syndrome in an Eleven-Month Infant.
Guillain-Barre syndrome (GBS) is an acute immune-mediated progressive predominantly motor symmetric polyradiculoneuropathy which causes demyelination and leads to weakness, ataxia and areflexia. There are a variety of forms of the syndrome; and despite being the most common cause of acute flaccid paralysis in children, it has a low incidence under 18 years old, and it is even rarer in children less than 2 years of age. Very few cases have been reported under 12 months of age. We describe a case of an 11-month-old male infant presenting with weakness and inability to ambulate who was diagnosed with GBS.


Limits on the neutrino magnetic moment using 1496 days of Super-Kamiokande-I solar neutrino data.
A search for a nonzero neutrino magnetic moment has been conducted using 1496 live days of solar neutrino data from Super-Kamiokande-I. Specifically, we searched for distortions to the energy spectrum of recoil electrons arising from magnetic scattering due to a nonzero neutrino magnetic moment. In the absence of a clear signal, we found micro(nu)</=(3.6x10(-10))micro(B) at 90% C.L. by fitting to the Super-Kamiokande day-night spectra. The fitting took into account the effect of neutrino oscillation on the shapes of energy spectra. With additional information from other solar neutrino and KamLAND experiments constraining the oscillation region, a limit of micro(nu)</=(1.1x10(-10))micro(B) at 90% C.L. was obtained.


Acute myeloid leukemia in the elderly:--159 Nagoya case studies--Nagoya Cooperative Study Group for Elderly Leukemia.
To obtain background information on elderly acute myeloid leukemia (AML), unselected data covering 159 patients aged 60 years or over with AML from 14 hospitals in Nagoya, Japan was analyzed retrospectively. Among these patients, 119 had de novo acute AML, 32 had AML which evolved from myelodysplastic syndrome (MDS-AML), and 8 had other types of leukemia. The survey showed that MDS-AML tended to be more prevalent in patients aged 70 years and older and that MDS-AML showed a significantly more severe degree of leukopenia and anemia than de novo AML. MDS-AML also showed a significantly lower complete remission (CR) rate than that of de novo AML [6.9% (2/29) vs 58.3% (67/11), P < 0.01] and significantly shorter survival times than those of de novo AML [median: 3.6 months vs 9.6 months, P < 0.01 (generalized Wilcoxon test; GW]. In de novo AML, the proportion of patients treated with conventional therapy (CT group) decreased significantly, and that of those with attenuated therapy (AT group) increased significantly as age elevated (P < 0.01). The CT group showed a significantly higher CR rate (65.4% vs 41.2%, P < 0.05) and a significantly longer survival period than those of the AT group [median: 11.6 months vs 4.8 months, P < 0.05 (GW)]. Overall survival rates of the older age groups became significantly shorter with aging [P < 0.01 (GW)].


Factors Influencing Prognosis After Initial Inadequate Excision (IIE) for Soft Tissue Sarcoma.
Purpose. The influence of initial inadequate excision (IIE) of soft tissue sarcoma (STS) on local control and overall survival is not well established. It is generally believed that an IIE may have a negative impact on both, despite subsequent treatment by radical surgery and radiotherapy. However, data on local recurrence-free survival/overall survival are conflicting and there are no data on the effect of IIE on overall survival.Patients and methods. A retrospective analysis was made of 86 patients with soft tissue sarcoma of the extremities and trunk after an IIE had been performed due to inappropriate work-up. The minimal follow-up was 7 years. Specimens of the subsequent radical resection were evaluated for residual tumor, grade of tumor and complications of IIE. Endpoints were recurrence-free survival and overall survival.Results. Specimens of the subsequent radical resection showed residual tumor in 66 patients (77%). The most common complication after IIE was hematoma. In both univariate and multivariate analyses, grade II/III tumors and complications after IIE are significant negative prognostic factors for local recurrence-free survival (P = 0.008 and P = 0.002, respectively, in the Cox model). For this survival, three prognostic groups could be formed based on grade, or presence or absence of complications. Adjuvant radiotherapy did not change the rate of local recurrence-free survival. For overall survival, only tumor grade is a significant factor (log-rank test).Conclusion. This retrospective study shows that complications associated with an IIE have a significant negative effect on local control, but not on overall survival, because IIE is often the result of inappropriate work-up before surgery. For better diagnosis and therapy STS should be treated in specialized centers.


miR-9: a versatile regulator of neurogenesis.
Soon after its discovery, microRNA-9 (miR-9) attracted the attention of neurobiologists, since it is one of the most highly expressed microRNAs in the developing and adult vertebrate brain. Functional analyses in different vertebrate species have revealed a prominent role of this microRNA in balancing proliferation in embryonic neural progenitor populations. Key transcriptional regulators such as FoxG1, Hes1 or Tlx, were identified as direct targets of miR-9, placing it at the core of the gene network controlling the progenitor state. Recent data also suggest that this function could extend to adult neural stem cells. Other studies point to a role of miR-9 in differentiated neurons. Moreover miR-9 has been implicated in human brain pathologies, either displaying a protective role, such as in Progeria, or participating in disease progression in brain cancers. Altogether functional studies highlight a prominent feature of this highly conserved microRNA, its functional versatility, both along its evolutionary history and across cellular contexts.


[Efficacy of eculizumab in a case of pregnancy-associated aHUS].
Pregnancy-associated thrombotic microangiopathy (TMA) is a rare condition, but it is burdened by a significant perinatal and maternal morbidity as well as mortality. We describe the case of a 33-year-old woman, who developed a TMA at the 36th week of gestation characterized by increased LDH, haptoglobin consumption, schistocytes, thrombocytopenia and acute renal failure requiring dialysis. There were not gestational hypertension nor proteinuria until the day of hospitalization. ADAMTS 13 deficiency was ruled out and the patient did not have diarrhea. She was initially treated with caesarean section, plasma infusion and plasmapheresis with no benefit. Five days after the onset of TMA, a temptative diagnosis of atypical uremic syndrome (aHUS) was made and the patient was switched to eculizumab. Antibiotic prophylaxis and anti-meningococcal A,B, C, W135 and Y vaccination was performed. TMA rapidly resolved and renal function completely recovered. The newborn had a normal perinatal course. A complement dysregulation was ruled out by testing for mutations on CFH, CFHR3-R1, CFI, MCP, CFB, C3 and for anti CFH antibodies. In conclusion the differential diagnosis of aHUS with HELLP syndrome is often not straightforward. The severity and persistence of TMA, the high mortality associated to peripartum TMA and the risk for irreversible kidney failure require an early therapeutic decision as to the use of eculizumab.


Family aggregation of mental disorders in the nationwide Danish three generation study.
The study of familial aggregation of major mental disorders in a national population. Within a Danish register-based cohort study, aggregation of mental disorders was analysed in all case-probands with first psychiatric contact before the age of 19 years in the time period between 1 April 1969 and 29 June 2004 followed up until the age of 35 years, their first-degree relatives, and a matched group of control-probands including their first-degree relatives. Hazard rate ratios were significantly elevated for cases as compared to controls for all diagnoses among probands, parents, and siblings. Among children of the probands, these ratios were significantly elevated for neurotic (anxiety) disorders, mental retardation, developmental disorders, behavioural and emotional disorders of childhood and adolescence, and miscellaneous disorders. Family aggregation of any diagnosis was significantly higher in probands with substance use disorder, schizophrenia, affective disorders, neurotic (anxiety) disorders, and miscellaneous disorders. There was specificity of familial transmission for affective and neurotic (anxiety) disorders. This large nationwide study found some differential patterns of familial aggregation of major mental disorders.


Cognitive therapy with depressed inpatients: specific effects on dysfunctional cognitions.
Specificity of cognitive change following cognitive therapy for depression was assessed in 39 depressed inpatients who completed either a standard inpatient treatment (pharmacotherapy and milieu management) or the standard treatment plus cognitive therapy. Following treatment, patients in both groups endorsed fewer dysfunctional cognitions on 2 of 4 measures of cognitive distortion. Compared with patients receiving only the standard treatment, patients also receiving cognitive therapy reported less hopelessness and fewer cognitive biases at posttreatment and 6- and 12-month follow-up assessments and fewer dysfunctional attitudes at the 6-month follow-up. Treatment effects for dysfunctional cognitions were found even though the treatment groups did not differ in depression severity, suggesting that results did not reflect state-dependent differences between treatments secondary to difference in depression.


Coronary artery disease severity and aortic stenosis: clinical outcomes according to SYNTAX score in patients undergoing transcatheter aortic valve implantation.
The aim of this study was to evaluate whether coronary artery disease (CAD) severity exerts a gradient of risk in patients with aortic stenosis (AS) undergoing transcatheter aortic valve implantation (TAVI). A total of 445 patients with severe AS undergoing TAVI were included into a prospective registry between 2007 and 2012. The preoperative SYNTAX score (SS) was determined from baseline coronary angiograms. In case of revascularization prior to TAVI, residual SS (rSS) was also determined. Clinical outcomes were compared between patients without CAD (n = 158), patients with low SS (0-22, n = 207), and patients with high SS (SS > 22, n = 80). The pre-specified primary endpoint was the composite of cardiovascular death, stroke, or myocardial infarction (MI). At 1 year, CAD severity was associated with higher rates of the primary endpoint (no CAD: 12.5%, low SS: 16.1%, high SS: 29.6%; P = 0.016). This was driven by differences in cardiovascular mortality (no CAD: 8.6%, low SS: 13.6%, high SS: 20.4%; P = 0.029), whereas the risk of stroke (no CAD: 5.1%, low SS: 3.3%, high SS: 6.7%; P = 0.79) and MI (no CAD: 1.5%, low SS: 1.1%, high SS: 4.0%; P = 0.54) was similar across the three groups. Patients with high SS received less complete revascularization as indicated by a higher rSS (21.2 ± 12.0 vs. 4.0 ± 4.4, P < 0.001) compared with patients with low SS. High rSS tertile (> 14) was associated with higher rates of the primary endpoint at 1 year (no CAD:12.5%, low rSS: 16.5%, high rSS: 26.3%, P = 0.043). Severity of CAD appears to be associated with impaired clinical outcomes at 1 year after TAVI. Patients with SS > 22 receive less complete revascularization and have a higher risk of cardiovascular death, stroke, or MI than patients without CAD or low SS.


A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.
How the ER-associated degradation (ERAD) machinery accurately identifies terminally misfolded proteins is poorly understood. For luminal ERAD substrates, this recognition depends on their folding and glycosylation status as well as on the conserved ER lectin Yos9p. Here we show that Yos9p is part of a stable complex that organizes key components of ERAD machinery on both sides of the ER membrane, including the transmembrane ubiquitin ligase Hrd1p. We further demonstrate that Yos9p, together with Kar2p and Hrd3p, forms a luminal surveillance complex that both recruits nonnative proteins to the core ERAD machinery and assists a distinct sugar-dependent step necessary to commit substrates for degradation. When Hrd1p is uncoupled from the Yos9p surveillance complex, degradation can occur independently of the requirement for glycosylation. Thus, Yos9p/Kar2p/Hrd3p acts as a gatekeeper, ensuring correct identification of terminally misfolded proteins by recruiting misfolded forms to the ERAD machinery, contributing to the interrogation of substrate sugar status, and preventing glycosylation-independent degradation.


Feasibility and efficacy of a digital CBT intervention for symptoms of Generalized Anxiety Disorder: A randomized multiple-baseline study.
Cognitive behavioral therapy (CBT) is a first-line treatment for anxiety, but it is not widely available as clinical guidelines recommend. We examined the feasibility and efficacy of a novel smartphone-based fully automated digital CBT intervention, 'Daylight™', to improve symptoms of Generalized Anxiety Disorder (GAD). In this multiple-baseline design, 21 adults (20 F; mean age 43yrs. range 19-65yrs.) with moderate-to-severe symptoms of GAD were randomized to one of three baseline durations (2-, 4-, or 6-weeks) and then received access to digital CBT. Participants completed daily ratings of anxiety and worry, weekly measures of anxiety, depressive symptoms, and sleep, and measures of anxiety, worry, wellbeing, quality of life, CBT skill acquisition, and work performance at initial assessment prior to baseline randomization, post-intervention, and follow-up. Digital CBT was found to be feasible in terms of engagement, satisfaction, and safety. For preliminary efficacy, improvements were detected in daily and weekly outcomes of anxiety for most participants. Despite individual differences, significant improvements occurred with the introduction of digital CBT and not during baseline. Overall, 70% of participants no longer had clinically significant symptoms of GAD, 61% no longer had significant depressive symptoms, and 40% no longer had significant sleep difficulty at post-intervention. The study sample was recruited using the internet and was mostly female, limiting the generalizability of the findings. Findings support the feasibility and efficacy of Daylight. Further examination in randomized controlled trials is now warranted.


Multisite Study of Women Living With HIV's Perceived Barriers to, and Interest in, Long-Acting Injectable Antiretroviral Therapy.
Adherence to antiretroviral therapy (ART) is imperative for viral suppression and reducing HIV transmission, but many people living with HIV report difficultly sustaining long-term adherence. Long-acting injectable (LAI) ART has the potential to transform HIV treatment and prevention. However, little LAI ART-related behavioral research has occurred among women, particularly outside of clinical trials. Six Women's Interagency HIV Study sites: New York, Chicago, Washington DC, Atlanta, Chapel Hill, and San Francisco. We conducted 59 in-depth interviews with women living with HIV across 6 Women's Interagency HIV Study sites (10 per site; 9 at Washington DC). We interviewed women who were not included in LAI ART clinical trials but who receive care at university settings that will administer LAI ART once it is approved. Interviews were recorded, transcribed, and analyzed using thematic content analysis. Most women enthusiastically endorsed monthly LAI ART and would prefer it over pills. The following 3 reasons emerged for this preference: (1) convenience and confidentiality, (2) avoiding daily reminders about living with HIV, and (3) believing that shots are more effective than pills. Challenges remain, however, specifically around (1) medical mistrust, (2) concerns about safety and effectiveness, (3) pill burden for HIV and other conditions, and (4) barriers to additional medical visits. Most women preferred LAI ART over daily pills given its benefits, including convenience, privacy, and perceived effectiveness. Future research should incorporate more women into LAI ART trials to better understand and align development with user concerns and preferences to enhance uptake.


Alternative lengthening of telomeres is enriched in, and impacts survival of TP53 mutant pediatric malignant brain tumors.
Although telomeres are maintained in most cancers by telomerase activation, a subset of tumors utilize alternative lengthening of telomeres (ALT) to sustain self-renewal capacity. In order to study the prevalence and significance of ALT in childhood brain tumors we screened 517 pediatric brain tumors using the novel C-circle assay. We examined the association of ALT with alterations in genes found to segregate with specific histological phenotypes and with clinical outcome. ALT was detected almost exclusively in malignant tumors (p = 0.001). ALT was highly enriched in primitive neuroectodermal tumors (12 %), choroid plexus carcinomas (23 %) and high-grade gliomas (22 %). Furthermore, in contrast to adult gliomas, pediatric low grade gliomas which progressed to high-grade tumors did not exhibit the ALT phenotype. Somatic but not germline TP53 mutations were highly associated with ALT (p = 1.01 × 10(-8)). Of the other alterations examined, only ATRX point mutations and reduced expression were associated with the ALT phenotype (p = 0.0005). Interestingly, ALT attenuated the poor outcome conferred by TP53 mutations in specific pediatric brain tumors. Due to very poor prognosis, one year overall survival was quantified in malignant gliomas, while in children with choroid plexus carcinoma, five year overall survival was investigated. For children with TP53 mutant malignant gliomas, one year overall survival was 63 ± 12 and 23 ± 10 % for ALT positive and negative tumors, respectively (p = 0.03), while for children with TP53 mutant choroid plexus carcinomas, 5 years overall survival was 67 ± 19 and 27 ± 13 % for ALT positive and negative tumors, respectively (p = 0.07). These observations suggest that the presence of ALT is limited to a specific group of childhood brain cancers which harbor somatic TP53 mutations and may influence the outcome of these patients. Analysis of ALT may contribute to risk stratification and targeted therapies to improve outcome for these children.


Transferrin receptor targeting by de novo sheet extension.
The de novo design of polar protein-protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood-brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.


Reaching out to Ebola victims: Coercion, persuasion or an appeal for self-sacrifice?
The 2014-2015 Ebola crisis in West Africa has highlighted the practical limits of upholding human rights and common ethical principles when applying emergency public-health measures. The role of medical teams in the implementation of quarantine and isolation has been equivocal, particularly when such measures are opposed by communities who are coerced by the temporary suspension of civil liberties. In their encounters with Ebola victims, outreach teams face moral dilemmas, where the boundaries are unclear between coercion, persuasion and appeals for self-sacrifice. For those teams, we propose a set of practical recommendations aimed at respecting the autonomy of epidemic victims and easing tensions within communities. We recognize that some of these recommendations are progressively achievable, depending on the specific stage or setting of an outbreak. Yet with the increasing availability of experimental treatments and research interventions, weighing patients' autonomy against the common good will become an even more pressing ethical obligation.


A versatile synthetic route to the preparation of 15 N heterocycles.
A robust medium-scale (approximately 3 g) synthetic method for 15 N labeling of pyridine (15 N-Py) is reported based on the Zincke reaction. 15 N enrichment in excess of 81% was achieved with approximately 33% yield. 15 N-Py serves as a standard substrate in a wide range of studies employing a hyperpolarization technique for efficient polarization transfer from parahydrogen to heteronuclei; this technique, called SABRE (signal amplification by reversible exchange), employs a simultaneous chemical exchange of parahydrogen and a to-be-hyperpolarized substrate (e.g., pyridine) on metal centers. In studies aimed at the development of hyperpolarized contrast agents for in vivo molecular imaging, pyridine is often employed either as a model substrate (for hyperpolarization technique development, quality assurance, and phantom imaging studies) or as a co-substrate to facilitate more efficient hyperpolarization of a wide range of emerging contrast agents (e.g., nicotinamide). Here, the produced 15 N-Py was used for the feasibility study of spontaneous 15 N hyperpolarization at high magnetic (HF) fields (7 T and 9.4 T) of an NMR spectrometer and an MRI scanner. SABRE hyperpolarization enabled acquisition of 2D MRI imaging of catalyst-bound 15 N-pyridine with 75 × 75 mm2 field of view (FOV), 32 × 32 matrix size, demonstrating the feasibility of 15 N HF-SABRE molecular imaging with 2.4 × 2.4 mm2 spatial resolution.


Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast.
The enzyme 3-hydroxy-3-methylglutaryl-co-enzyme-A (HMG-CoA) reductase is known as the rate limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1-552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene.


Follow-up of tetralogy of Fallot patients: tertiary centre versus satellite clinic.
Canadian Cardiovascular Society consensus guidelines recommend that tetralogy of Fallot patients be seen by a congenital cardiologist every 2 years. In Atlantic Canada, tetralogy of Fallot patients are followed up at either tertiary or satellite clinics, which are held in the community and attended by paediatric cardiologists. The effectiveness of satellite clinics in congenital cardiac disease follow-up is unproven. Our objective was to compare patient-reported quality of life measures to determine whether these were impacted by the site of follow-up. We included patients with tetralogy of Fallot undergoing surgical repair at the Izaak Walton Killam Health Centre from 1 November, 1972 to 31 May, 2002. Quality of life surveys, SF-10 or SF-36v2, were administered to consenting patients. We analysed the subjective health status by patient age and site of follow-up. Of the 184 eligible patients, 72 were lost to follow-up. Of the locatable patients, 61% completed the questionnaires. In all, 90% (101 out of 112) were followed up at recommended intervals. Of the 112 (68%) patients, 76 were followed up at a tertiary clinic. These patients were older, with a mean age of 18.4 years versus 14.7 years, and scored higher on the SF-36 physical component summary (52.6 versus 45.7, p = 0.02) compared with satellite clinic patients. The SF-36 mental component summary scores were similar for patients regardless of the site of follow-up. SF-10 physical and psychosocial scores were similar regardless of the site of follow-up. Tetralogy of Fallot patients followed at either satellite or tertiary clinics have similar subjective health status.


Recombination of nonchromosomal mutations: a 3-point cross in the green alga Chlamydomonas reinhardi.
The heterothallic, unicellular green alga Chlamydomonas reinhardi possesses both chromosomal and nonchromosomal systems of inheritance. Mutations belonging to the latter system are capable of recombination. The experiments reported show that double and triple recombinants can be obtained from crosses employing three phenotypically distinct mutations and that certain pairs of mutants recombine more freely than others.


Extensive testing or focused testing of patients with elevated liver enzymes.
Many patients have elevated serum aminotransferases reflecting many underlying conditions, both common and rare. Clinicians generally apply one of two evaluative strategies: testing for all diseases at once (extensive) or just common diseases first (focused). We simulated the evaluation of 10,000 adult outpatients with elevated with alanine aminotransferase to compare both testing strategies. Model inputs employed population-based data from the US (National Health and Nutrition Examination Survey) and Britain (Birmingham and Lambeth Liver Evaluation Testing Strategies). Patients were followed until a diagnosis was provided or a diagnostic liver biopsy was considered. The primary outcome was US dollars per diagnosis. Secondary outcomes included doctor visits per diagnosis, false-positives per diagnosis and confirmatory liver biopsies ordered. The extensive testing strategy required the lowest monetary cost, yielding diagnoses for 54% of patients at $448/patient compared to 53% for $502 under the focused strategy. The extensive strategy also required fewer doctor visits (1.35 vs. 1.61 visits/patient). However, the focused strategy generated fewer false-positives (0.1 vs. 0.19/patient) and more biopsies (0.04 vs. 0.08/patient). Focused testing becomes the most cost-effective strategy when accounting for pre-test probabilities and prior evaluations performed. This includes when the respective prevalence of alcoholic, non-alcoholic and drug-induced liver disease exceeds 51.1%, 53.0% and 13.0%. Focused testing is also the most cost-effective strategy in the referral setting where assessments for viral hepatitis, alcoholic and non-alcoholic fatty liver disease have already been performed. Testing for elevated liver enzymes should be deliberate and focused to account for pre-test probabilities if possible. Many patients have elevated liver enzymes reflecting one of many possible liver diseases, some of which are very common and some of which are rare. Tests are widely available for most causes but it is unclear whether clinicians should order them all at once or direct testing based on how likely a given disease may be given the patient's history and physical exam. The tradeoffs of both approaches involve the money spent on testing, number of office visits needed, and false positive results generated. This study shows that if there are no clues available at the time of evaluation, testing all at once saves time and money while causing more false positives. However, if there are strong clues regarding the likelihood of a particular disease, limited testing saves time, money and prevents false positives.


A trial of two cognitive-behavioural methods of treating drug-resistant residual psychotic symptoms in schizophrenic patients: I. Outcome.
Despite neuroleptic medication, many schizophrenic patients continue to experience residual positive psychotic symptoms. These residual symptoms cause distress and disability. We report a controlled trial of two cognitive-behavioural treatments to alleviate residual hallucinations and delusions. Forty-nine patients were recruited into the trial, of whom 27 entered the trial and completed post-treatment assessment, and 23 were reassessed at six-month follow-up. Patients were randomly allocated to either coping strategy enhancement (CSE) or problem solving (PS). Half the patients were allocated to a high-expectancy positive demand condition and half to a counter-demand condition to evaluate expectation of improvement. Patients receiving either cognitive-behavioural treatment showed significant reductions in psychotic symptoms compared with those in the waiting period, who showed no improvement. There was some evidence, although equivocal, that patients receiving CSE improved more than those receiving PS. There was no evidence that improvements generalised to negative symptoms or social functioning, nor was there evidence that expectancy of treatment benefit contributed to the treatment effect.


Liver iron concentrations and urinary hepcidin in beta-thalassemia.
Patients with beta-thalassemia, like those with genetic hemochromatosis, develop iron overload due to increased iron absorption, and their iron burden is further exacerbated by transfusion therapy. Hepcidin, a hepatic hormone, regulates systemic iron homeostasis by inhibiting the absorption of iron from the diet and the recycling of iron by macrophages. In turn, hepcidin release is increased by iron loading and inhibited by erythropoietic activity. Hepcidin deficiency is the cause of iron overload in most forms of hereditary hemochromatosis. We sought to determine hepcidin's role in the pathogenesis of iron overload in b-thalassemia. We assessed the degree of iron overload in thalassemia intermedia and major patients by measuring hepatic iron concentration in liver biopsy samples and serum ferritin, estimated erythropoietic drive by assaying soluble transferrin receptor and serum erythropoietin levels and correlated these with urinary hepcidin measurements. Urinary hepcidin levels in beta-thalassemia demonstrate severe hepcidin deficiency in thalassemia intermedia. There was a strong inverse relationship between urinary hepcidin levels and both erythropoietin and soluble transferrin receptor, markers of erythropoietic activity. In contrast, hepcidin levels were elevated in thalassemia major, presumably due to transfusions that reduce erythropoietic drive and deliver a large iron load. Despite similar liver iron concentrations in the two conditions, serum ferritin was much lower in thalassemia intermedia. In thalassemia intermedia, high erythropoietic drive causes severe hepcidin deficiency. The lack of hepcidin results in hyperabsorption of dietary iron, but also in iron depletion of macrophages, lowering their secretion of ferritin and, consequently, serum ferritin levels. In contrast, in thalassemia major, transfusions decrease erythropoietic drive and increase the iron load, resulting in relatively higher hepcidin levels. In the presence of higher hepcidin levels, dietary iron absorption is moderated and macrophages retain iron, contributing to higher serum ferritin. In the future, hepcidin measurements may allow a more accurate assessment of the degree of iron overload and the maldistribution of iron in thalassemia.


Uncoupling cation effects on cardiac contractility and sarcolemmal Ca2+ binding.
Studies have been performed correlating the effects of cationic uncouplers on intact cardiac muscle and on Ca2+ bound to isolated cardiac plasma membranes. Sarcolemmal vesicles were isolated from neonatal rat hearts. Ca2+ binding experiments were performed and Scatchard plot analysis indicated two classes of Ca2+ binding sites. The ability of certain cations to displace Ca2+ bound to these vesicles was measured. The effects of these same cations on the tension developed by neonatal rat papillary muscles and on the Ca2+ content of tissue culture cells (from neonatal rat heart) were measured. The results show that a) the selectivity sequence (Y3+ greater than Nd3+ greater than La3+, Cd2+ greater than Co2+ greater than Mg2+) of sarcolemmal Ca2+ binding sites is the same as the effective uncoupling sequence; b) the amount of Ca2+ bound at these sites (approximately 700 micromol/kg wet wt) is more than adequate to support tension development; c) the dependence of sarcolemmal Ca2+ binding at low-affinity sites and tension development on [Ca2+]o is essentially the same. It is then reasonable to propose that the Ca2+ bound to these sarcolemmal sites plays an important role in controlling the amount of Ca2+ available to the myofilaments and thus myocardial contractility.


Plasma microRNA-122 as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases.
The liver is frequently subject to insult because of viral infection, alcohol abuse, or toxic chemical exposure. Extensive research has been conducted to identify blood markers that can better discern liver damage, but little progress has been achieved in clinical practice. Recently, circulating microRNAs (miRNAs) have been reported as potential biomarkers for the noninvasive diagnosis of cancer. In this study, we investigated whether plasma miRNAs have diagnostic utility in identifying liver disease. The study was divided into 2 phases: marker selection by real-time quantitative PCR analysis of a small set of plasma samples, and marker validation with a large set of plasma samples from 83 patients with chronic hepatitis B viral infections, 15 patients with skeletal muscle disease, and 40 healthy controls. Two mouse model systems, d-galactosamine- and alcohol-induced liver injury, were also developed to evaluate whether differences in miRNA concentration were associated with various liver diseases. Among the miRNA candidates identified, miR-122 presented a disease severity-dependent change in plasma concentration in the patients and animal models. Compared with an increase in aminotransferase activity in the blood, the change in miR-122 concentration appeared earlier. Furthermore, this change was more specific for liver injury than for other organ damage and was more reliable, because the change was correlated with liver histologic stage. Our findings suggest that circulating miR-122 has potential as a novel, predictive, and reliable blood marker for viral-, alcohol-, and chemical-induced liver injury.


Partial restoration of blink reflex function after spinal accessory-facial nerve anastomosis.
Functional motor control requires perfect matching of the central connections of motoneurons with their peripheral inputs. It is not known, however, to what extent these central circuits are influenced by target muscles, either during development or after a lesion. Surgical interventions aimed at restoring function after peripheral nerve lesions provide an opportunity for studying this interaction in the mature human nervous system. A patient was studied in whom the spinal accessory nerve was anastomosed into a lesioned facial nerve, allowing voluntary contractions of the previously paralysed muscles. This procedure, in addition to replacing the facial neurons at peripheral synapses, allowed a new short latency trigeminospinal accessory reflex of the R1 blink reflex type to be demonstrated, implying that trigeminal neurons had sprouted towards spinal accessory motoneurons over a distance of at least 1 cm. These results show an unexpected influence of the periphery in remodelling central connectivity in humans. The motoneuronal excitability for this R1 reflex response was therefore studied to compare the convergent properties of facial motoneurons (normal side) with those of the spinal accessory motoneurons (operated side) using a classic double shock technique with variable interstimulus intervals (conditioning test stimulus). On the normal side, conditioning stimuli (to the ipsilateral or contralateral infraliminar supraorbital nerve) produced a clearcut facilitation of the R1 blink reflex when the interstimulus interval was 30-80 ms. By contrast, a similar procedure had no effect on the R1 blink reflex mediated via the trigeminal-spinal accessory reflex arc. These data indicate that despite the heterotopic sprouting of some axons from neurons in the XIth nucleus, motoneurons involved in the newly formed reflex arc remain totally inexcitable by other trigeminal afferents and seem unable to ensure a physiological functioning of the normal blink reflex. Thus the functional relevance of the recovered R1 blink response remains unclear.


Role of anorectic N-acylethanolamines in intestinal physiology and satiety control with respect to dietary fat.
Anandamide is a well-known agonist for the cannabinoid receptors. Along with endogenous anandamide other non-endocannabinoid N-acylethanolamines are also formed, apparently in higher amounts. These include mainly oleoylethanolamide (OEA), palmitoyelethanolamide (PEA) and linoleoylethanolamide (LEA), and they have biological activity by themselves being anorectic and anti-inflammatory. It appears that the major effect of dietary fat on the level of these molecules is in the gastrointestinal system, where OEA, PEA and LEA in the enterocytes may function as homeostatic signals, which are decreased by prolonged consumption of a high-fat diet. These lipid amides appear to mediate their signaling activity via activation of PPARα in the enterocyte followed by activation of afferent vagal fibers leading to the brain. Through this mechanism OEA, PEA and LEA may both reduce the consumption of a meal as well as increase the reward value of the food. Thus, they may function as homeostatic intestinal signals involving hedonic aspects that contribute to the regulation of the amounts of dietary fat to be ingested.


MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme.
Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.


Transfection of the human IGFBP-3 gene into Balb/c fibroblasts: a model for the cellular functions of IGFBPs.
The insulin-like growth factors (IGFs) are potent mitogens with both endocrine and autocrine-paracrine effects on cell growth. The insulin-like growth factor binding proteins (IGFBPs) are found in many human biological fluids and in the conditioned media of many cell cultures. These molecules are ontogenically and hormonally regulated. Nevertheless, the biological role(s) of the IGFBPs remain controversial. Both inhibitory and stimulatory effects of IGFBPs on cell growth have been suggested. In order to evaluate the actions of endogenously produced IGFBPs on cell growth, we have constructed a mammalian expression vector containing the hIGFBP-3 cDNA, and transfected the murine Balb/c cell line. The IGFBP-3 transfected Balb/c cells (Tx-BP-3) expressed readily detectable amounts of hIGFBP-3 protein and its mRNA. Growth of Tx-BP-3 in serum containing media was significantly slower, compared with control plasmid-transfected cells (Tx-P) grown under identical conditions. Fully confluent Tx-BP-3 cells arrested their growth at a cell density that was lower than did Tx-P cells. When transfected cells were grown in insulin-containing media, growth rates of the IGFBP-3 transfected cells were not restored to those observed in control plasmid-transfected cells. These results suggest that in this model, the expression of endogenous IGFBP-3 has an inhibitory effect on cell growth.


Low unesterified:esterified eicosapentaenoic acid (EPA) plasma concentration ratio is associated with bipolar disorder episodes, and omega-3 plasma concentrations are altered by treatment.
Omega (n)-3 and n-6 polyunsaturated fatty acids (PUFAs) are molecular modulators of neurotransmission and inflammation. We hypothesized that plasma concentrations of n-3 PUFAs would be lower and those of n-6 PUFAs higher in subjects with bipolar disorder (BD) compared to healthy controls (HCs), and would correlate with symptom severity in subjects with BD, and that effective treatment would correlate with increased n-3 but lower n-6 PUFA levels. Additionally, we explored clinical correlations and group differences in plasma levels of saturated and monounsaturated fatty acids. This observational, parallel group study compared biomarkers between HCs (n = 31) and symptomatic subjects with BD (n = 27) when ill and after symptomatic recovery (follow-up). Plasma concentrations of five PUFAs [linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA)], two saturated fatty acids (palmitic acid and stearic acid) and two monounsaturated fatty acids (palmitoleic acid and oleic acid) were measured in esterified (E) and unesterified (UE) forms. Calculated ratios included UE:E for the five PUFAs, ratios of n-3 PUFAs (DHA:ALA, EPA:ALA and EPA:DHA), and the ratio of n-6:n-3 AA:EPA. Comparisons of plasma fatty acid levels and ratios between BD and HC groups were made with Student t-tests, and between the BD group at baseline and follow-up using paired t-tests. Comparison of categorical variables was performed using chi-square tests. Pearson's r was used for bivariate correlations with clinical variables, including depressive and manic symptoms, current panic attacks, and psychosis. UE EPA was lower in subjects with BD than in HCs, with a large effect size (Cohen's d = 0.86, p < 0.002); however, it was not statistically significant after correction for multiple comparisons. No statistically significant difference was seen in any plasma PUFA concentration between the BD and HC groups after Bonferroni correction for 40 comparisons, at p < 0.001. Neither depressive severity nor mania severity was correlated significantly with any PUFA concentration. Exploratory comparison showed lower UE:E EPA in the BD than the HC group (p < 0.0001). At follow-up in the BD group, UE, E DHA:ALA, and UE EPA:ALA were decreased (p < 0.002). Exploratory correlations of clinical variables revealed that mania severity and suicidality were positively correlated with UE:E EPA ratio, and that several plasma levels and ratios correlated with panic disorder and psychosis. Depressive severity was not correlated with any ratio. No plasma fatty acid level or ratio correlated with self-reported n-3 PUFA intake or use of medication by class. A large effect size of reduced UE EPA, and a lower plasma UE:E concentration ratio of EPA in the symptomatic BD state may be important factors in vulnerability to a mood state. Altered n-3 PUFA ratios could indicate changes in PUFA metabolism concurrent with symptom improvement. Our findings are consistent with preclinical and postmortem data and suggest testing interventions that increase n-3 and decrease n-6 dietary PUFA intake.


A Model of Compound Heterozygous, Loss-of-Function Alleles Is Broadly Consistent with Observations from Complex-Disease GWAS Datasets.
The genetic component of complex disease risk in humans remains largely unexplained. A corollary is that the allelic spectrum of genetic variants contributing to complex disease risk is unknown. Theoretical models that relate population genetic processes to the maintenance of genetic variation for quantitative traits may suggest profitable avenues for future experimental design. Here we use forward simulation to model a genomic region evolving under a balance between recurrent deleterious mutation and Gaussian stabilizing selection. We consider multiple genetic and demographic models, and several different methods for identifying genomic regions harboring variants associated with complex disease risk. We demonstrate that the model of gene action, relating genotype to phenotype, has a qualitative effect on several relevant aspects of the population genetic architecture of a complex trait. In particular, the genetic model impacts genetic variance component partitioning across the allele frequency spectrum and the power of statistical tests. Models with partial recessivity closely match the minor allele frequency distribution of significant hits from empirical genome-wide association studies without requiring homozygous effect sizes to be small. We highlight a particular gene-based model of incomplete recessivity that is appealing from first principles. Under that model, deleterious mutations in a genomic region partially fail to complement one another. This model of gene-based recessivity predicts the empirically observed inconsistency between twin and SNP based estimated of dominance heritability. Furthermore, this model predicts considerable levels of unexplained variance associated with intralocus epistasis. Our results suggest a need for improved statistical tools for region based genetic association and heritability estimation.