Citrus Viroids: Symptom Expression and Effect on Vegetative Growth and Yield of Clementine Trees Grafted on Trifoliate Orange.
Citrus are natural hosts of five viroid species: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV). CEVd and specific sequence variants of HSVd are the causal agents of the wellknown diseases of citrus, exocortis and cachexia. Other viroids have been found to induce different degrees of stunting. Since commercial citrus trees are commonly infected with mixtures of these viroids, only limited information is available on their effect in species other than Etrog citron. A field assay was conducted to establish the effect of each viroid on Commune clementine trees grafted on Pomeroy trifoliate orange. Infected trees were periodically monitored over a 12-year period (1990 to 2002) for symptom expression, growth, and fruit yield. Only CEVd caused bark scaling on the trifoliate orange rootstock and marked dwarfing, both characteristic of exocortis disease as initially described. In addition, very conspicuous bumps were observed in the wood of the rootstock after removing the bark. Only those HSVd variants, previously characterized as pathogenic in several cachexia-sensitive species, induced pits and gum deposits characteristic of this disease in the clementine scion. Bark cracking symptoms on the trifoliate orange rootstock were also observed. They were associated with CVd-IV, HSVd, or CEVd infection, but in the latter, they were only clearly observed in trees that showed mild scaling. Other abnormalities (deep pits, crests, and gummy pits) were not associated with viroid infection. No specific symptoms resulted from infection with CBLVd and CVd-III. HSVd, CVd-IV, and CBLVd had little or no effect in growth and yield, whereas CEVd and CVd-III caused a significant reduction of growth and yield, which became more pronounced over time with CEVd infection. Yield reduction was associated mainly with loss of production of large fruits. In general, there was a good correlation between reduction in vegetative growth and yield.


Determination of the magnetic properties and orientation of the heme axial ligands of PpcA from Geobacter metallireducens by paramagnetic NMR.
The rising interest in the use of Geobacter bacteria for biotechnological applications demands a deep understanding of how these bacteria are able to thrive in a variety of environments and perform extracellular electron transfer. The Geobacter metallireducens bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular acceptors, including heavy metals, toxic organic compounds or electrode surfaces. The periplasmic c-type cytochrome PpcA from this bacterium is a member of a family composed of five periplasmic triheme cytochromes, which are important to bridge the electron transfer between the cytoplasm and the extracellular environment. To better understand the functional mechanism of PpcA it is essential to obtain structural data for this cytochrome. In this work, the geometry of the heme axial ligands, as well as the magnetic properties of the hemes were determined for the oxidized form of the cytochrome, using the 13C NMR chemical shifts of the heme α-substituents. The results were further compared with those previously obtained for the homologous cytochrome from Geobacter sulfurreducens. The orientations of the axial histidine planes and the magnetic properties of the hemes are conserved in both proteins. Overall, the results obtained allowed the definition of the orientation of the magnetic axes of PpcA from G. metallireducens, which will be used as constraints to assist the solution structure determination of the cytochrome in the oxidized form.


Parcellating cognitive heterogeneity in early psychosis-spectrum illnesses: A cluster analysis.
Cognitive impairment is argued to represent a core feature of psychosis-spectrum illnesses. However, within-diagnosis heterogeneity is common, and risk factors for poor cognition remain to be examined after statistically accounting for heterogeneity. Accordingly, we used a data-driven technique (cluster analysis) to empirically-derive cognitive clusters across diagnoses and examined whether concurrent substance use or a history of a neurodevelopmental/behavioral disorder differed between clusters. Data from 135 young help-seekers (aged 12-30 years) with a psychosis-spectrum illness were retrospectively analyzed. Ward's hierarchical cluster analysis classified three cognitive clusters characterized by: (1) normal-range; (2) mixed; and (3) grossly-impaired performance. Despite mostly comparable clinical and demographic measures, cluster 1 had superior socio-occupational functioning and the highest estimated premorbid IQ, followed sequentially by clusters 2 and 3. Proportions of cannabis and amphetamine users did not differ significantly across clusters, nor did rates of patients with a neurodevelopmental/behavioral disorder history. Cluster 3 was however comprised of fewer 'risky' drinkers, possibly reflecting reduced opportunity for social drinking associated with cognitive impairment. Estimated premorbid IQ predicted cluster membership (2 vs. 1 & 3 vs. 1), as did clinician-rated socio-occupational functioning and 'not being enrolled in school or tertiary education' (3 vs. 1). Our results suggest that concurrent substance use and history of a neurodevelopmental/behavioral disorder do not adequately explain cluster-level cognitive variance in this sample. Future work should integrate neurobiological measures associated with cognition (e.g. white matter integrity) to discern whether clusters reflect neurobiological subtypes better representative of pathophysiology than present symptom-based classifications.


Atrial myxomas and thrombi: comparison of imaging features on CT.
The purpose of our study was to compare the imaging features of atrial myxomas and thrombi using CT and to assess the accuracy of CT for determining the origin of myxomas in comparison with surgical findings. From July 2006 until June 2008, 23 patients (15 women, eight men; mean age, 63 +/- 14 years) with atrial myxomas (n = 13) and thrombi (n = 11) who underwent dual-source CT coronary angiography were included in this retrospective study. Two independent and blinded readers evaluated quantitative (CT attenuation and size) and qualitative (location, origin, shape, mobility, prolapse, and calcifications) parameters at CT. The shape and origin of myxomas were compared with the findings at surgery. No significant differences regarding the CT attenuation of myxomas in comparison with thrombi were found (43 +/- 14 HU vs 57 +/- 30 HU; p = 0.23). Myxomas were significantly larger than thrombi (33 +/- 16 mm vs 21 +/- 7 mm; p < 0.05). The lesions were found equally in the left and right atria (p = 0.11). The origin (p < 0.001), shape (p < 0.05), mobility (p < 0.01), and occurrence of prolapse (p < 0.01) differed significantly between the lesions. Calcifications did not differ between the lesions (p = 0.2). In comparison with surgery, the origin of myxomas was correctly evaluated by CT in 11 of 13 patients (fossa ovalis, n = 5; interatrial septum, n = 4; and lateral atrial wall, n = 2), whereas CT misclassified the origin of two myxomas (posterior and lateral wall left atria at CT vs fossa ovalis at surgery). Atrial myxomas and thrombi can be differentiated by their distinguishing features of size, origin, shape, mobility, and prolapse. CT is accurate in determining the origin of myxomas but may fail in some cases.


Genome Sequence and Analysis of Escherichia coli MRE600, a Colicinogenic, Nonmotile Strain that Lacks RNase I and the Type I Methyltransferase, EcoKI.
Escherichia coli strain MRE600 was originally identified for its low RNase I activity and has therefore been widely adopted by the biomedical research community as a preferred source for the expression and purification of transfer RNAs and ribosomes. Despite its widespread use, surprisingly little information about its genome or genetic content exists. Here, we present the first de novo assembly and description of the MRE600 genome and epigenome. To provide context to these studies of MRE600, we include comparative analyses with E. coli K-12 MG1655 (K12). Pacific Biosciences Single Molecule, Real-Time sequencing reads were assembled into one large chromosome (4.83 Mb) and three smaller plasmids (89.1, 56.9, and 7.1 kb). Interestingly, the 7.1-kb plasmid possesses genes encoding a colicin E1 protein and its associated immunity protein. The MRE600 genome has a G + C content of 50.8% and contains a total of 5,181 genes, including 4,913 protein-encoding genes and 268 RNA genes. We identified 41,469 modified DNA bases (0.83% of total) and found that MRE600 lacks the gene for type I methyltransferase, EcoKI. Phylogenetic, taxonomic, and genetic analyses demonstrate that MRE600 is a divergent E. coli strain that displays features of the closely related genus, Shigella. Nevertheless, comparative analyses between MRE600 and E. coli K12 show that these two strains exhibit nearly identical ribosomal proteins, ribosomal RNAs, and highly homologous tRNA species. Substantiating prior suggestions that MRE600 lacks RNase I activity, the RNase I-encoding gene, rna, contains a single premature stop codon early in its open-reading frame.


Why Hantavirus Prevalence Does Not Always Increase With Host Density: Modeling the Role of Host Spatial Behavior and Maternal Antibodies.
For wildlife diseases, one often relies on host density to predict host infection prevalence and the subsequent force of infection to humans in the case of zoonoses. Indeed, if transmission is mainly indirect, i.e., by way of the environment, the force of infection is expected to increase with host density, yet the laborious field data supporting this theoretical claim are often absent. Hantaviruses are among those zoonoses that have been studied extensively over the past decades, as they pose a significant threat to humans. In Europe, the most widespread hantavirus is the Puumala virus (PUUV), which is carried by the bank vole and causes nephropathia epidemica (NE) in humans. Extensive field campaigns have been carried out in Central Finland to shed light on this supposed relationship between bank vole density and PUUV prevalence and to identify other drivers for the infection dynamics. This resulted in the surprising observation that the relationship between bank vole density and PUUV prevalence is not purely monotonic on an annual basis, contrary to what previous models predicted: a higher vole density does not necessary result in a higher infection prevalence, nor in an increased number of humans reported having NE. Here, we advance a novel individual-based spatially-explicit model which takes into account the immunity provided by maternal antibodies and which simulates the spatial behavior of the host, both possible causes for this discrepancy that were not accounted for in previous models. We show that the reduced prevalence in peak years can be attributed to transient immunity, and that the density-dependent spatial vole behavior, i.e., the fact that home ranges are smaller in high density years, plays only a minor role. The applicability of the model is not limited to the study and prediction of PUUV (and NE) occurrence in Europe, as it could be easily adapted to model other rodent-borne diseases, either with indirect or direct transmission.


Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
Genotyping for 10 mutations in the CYP21 gene was performed in 88 families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Southern blot analysis was used to detect CYP21 deletions or large gene conversions, and allele-specific hybridizations were performed with DNA amplified by the polymerase chain reaction to detect smaller mutations. Mutations were detected on 95% of chromosomes examined. The most common mutations were an A----G change in the second intron affecting pre-mRNA splicing (26%), large deletions (21%), Ile-172----Asn (16%), and Val-281----Leu (11%). Patients were classified into three mutation groups based on degree of predicted enzymatic compromise. Mutation groups were correlated with clinical diagnosis and specific measures of in vivo 21-hydroxylase activity, such as 17-hydroxyprogesterone, aldosterone, and sodium balance. Mutation group A (no enzymatic activity) consisted principally of salt-wasting (severely affected) patients, group B (2% activity) of simple virilizing patients, and group C (10-20% activity) of nonclassic (mildly affected) patients, but each group contained patients with phenotypes either more or less severe than predicted. These data suggest that most but not all of the phenotypic variability in 21-hydroxylase deficiency results from allelic variation in CYP21. Accurate prenatal diagnosis should be possible in most cases using the described strategy.


Effect of Serotype on Pneumococcal Competition in a Mouse Colonization Model.
Competitive interactions between Streptococcus pneumoniae strains during host colonization could influence the serotype distribution in nasopharyngeal carriage and pneumococcal disease. We evaluated the competitive fitness of strains of serotypes 6B, 14, 19A, 19F, 23F, and 35B in a mouse model of multiserotype carriage. Isogenic variants were constructed using clinical strains as the capsule gene donors. Animals were intranasally inoculated with a mixture of up to six pneumococcal strains of different serotypes, with separate experiments involving either clinical isolates or isogenic capsule-switch variants of clinical strain TIGR4. Upper-respiratory-tract samples were repeatedly collected from animals in order to monitor changes in the serotype ratios using quantitative PCR. A reproducible hierarchy of capsular types developed in the airways of mice inoculated with multiple strains. Serotype ranks in this hierarchy were similar among pneumococcal strains of different genetic backgrounds in different strains of mice and were not altered when tested under a range of host conditions. This rank correlated with the measure of the metabolic cost of capsule synthesis and in vitro measure of pneumococcal cell surface charge, both parameters considered to be predictors of serotype-specific fitness in carriage. This study demonstrates the presence of a robust competitive hierarchy of pneumococcal serotypes in vivo that is driven mainly, but not exclusively, by the capsule itself. Streptococcus pneumoniae (pneumococcus) is the leading cause of death due to respiratory bacterial infections but also a commensal frequently carried in upper airways. Available vaccines induce immune responses against polysaccharides coating pneumococcal cells, but with over 90 different capsular types (serotypes) identified, they can only target strains of the selected few serotypes most prevalent in disease. Vaccines not only protect vaccinated individuals against disease but also protect by reducing carriage of vaccine-targeted strains to induce herd effects across whole populations. Unfortunately, reduction in the circulation of vaccine-type strains is offset by increase in carriage and disease from nonvaccine strains, indicating the importance of competitive interactions between pneumococci in shaping the population structure of this pathogen. Here, we showed that the competitive ability of pneumococcal strains to colonize the host strongly depends on the type of capsular polysaccharide expressed by pneumococci and only to a lesser degree on strain or host genetic backgrounds or on variation in host immune responses.


Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex.
The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.


[Topical ozone application: An innovative therapy for infantile atopic dermatitis].
To evaluate the clinical efficacy and safety of the innovative topical ozone therapy for infantile atopic dermatitis. Methods: Sixty children with atopic dermatitis were divided into a treatment group and a control group. The treatment group was showered with ozonated water (3-5 times a week) and smeared with ozonated oil (twice a day), while the control group was washed with warm running water and smeared with base oil, adding moisturizer if necessary. The treatment course was 2 weeks. Efficacy and side effect were evaluated. Results: The skin exudation was reduced and erosion was healing after 3-5 days topical ozone therapy for infantile atopic dermatitis. The effective rates were 80.0% and 20.0% in the treatment group and control group for 1 week, and 89.6% and 30.7% for 2 weeks, respectively, with significant difference between the 2 groups (P<0. 001). Conclusion: Innovative treatment of infantile atopic dermatitis with topical ozone application is safe and effective, which is worth popularizing in clinic.


Scaling up impact on nutrition: what will it take?
Despite consensus on actions to improve nutrition globally, less is known about how to operationalize the right mix of actions-nutrition-specific and nutrition-sensitive-equitably, at scale, in different contexts. This review draws on a large scaling-up literature search and 4 case studies of large-scale nutrition programs with proven impact to synthesize critical elements for impact at scale. Nine elements emerged as central: 1) having a clear vision or goal for impact; 2) intervention characteristics; 3) an enabling organizational context for scaling up; 4) establishing drivers such as catalysts, champions, systemwide ownership, and incentives; 5) choosing contextually relevant strategies and pathways for scaling up, 6) building operational and strategic capacities; 7) ensuring adequacy, stability, and flexibility of financing; 8) ensuring adequate governance structures and systems; and 9) embedding mechanisms for monitoring, learning, and accountability. Translating current political commitment to large-scale impact on nutrition will require robust attention to these elements.


A simplified risk-ranking system for prioritizing toxic pollution sites in low- and middle-income countries.
In low- and middle-income countries (LMICs), chemical exposures in the environment due to hazardous waste sites and toxic pollutants are typically poorly documented and their health impacts insufficiently quantified. Furthermore, there often is only limited understanding of the health and environmental consequences of point source pollution problems, and little consensus on how to assess and rank them. The contributions of toxic environmental exposures to the global burden of disease are not well characterized. The aim of this study was to describe the simple but effective approach taken by Blacksmith Institute's Toxic Sites Identification Program to quantify and rank toxic exposures in LMICs. This system is already in use at more than 3000 sites in 48 countries such as India, Indonesia, China, Ghana, Kenya, Tanzania, Peru, Bolivia, Argentina, Uruguay, Armenia, Azerbaijan, and Ukraine. A hazard ranking system formula, the Blacksmith Index (BI), takes into account important factors such as the scale of the pollution source, the size of the population possibly affected, and the exposure pathways, and is designed for use reliably in low-resource settings by local personnel provided with limited training. Four representative case studies are presented, with varying locations, populations, pollutants, and exposure pathways. The BI was successfully applied to assess the extent and severity of environmental pollution problems at these sites. The BI is a risk-ranking tool that provides direct and straightforward characterization, quantification, and prioritization of toxic pollution sites in settings where time, money, or resources are limited. It will be an important and useful tool for addressing toxic pollution problems in LMICs. Although the BI does not have the sophistication of the US Environmental Protection Agency's Hazard Ranking System, the case studies presented here document the effectiveness of the BI in the field, especially in low-resource settings. Understanding of the risks posed by toxic pollution sites helps assure better use of resources to manage sites and mitigate risks to public health. Quantification of these hazards is an important input to assessments of the global burden of disease.


HOMINID BIPEDALISM: INDEPENDENT EVIDENCE FOR THE FOOD-CARRYING THEORY.
Habitual food carrying has been suggested as a possible major factor in making bipedal locomotion biologically advantageous so that it was selected for in early hominid evolution. This speculation, supported by slight observations of captive macaques, has now acquired greater plausibility from four recent independent reports of wild and semi-feral bipedal, food-carrying apes and monkeys from the Congo, Tanganyika, Japan, and a Puerto Rican monkey colony. The most striking evidence of the relationship between food-transport and bipedal walking comes from a troop of Japanese monkeys where the locomotor habit emerged as part of a chain of new behaviors initiated with a changed food supply.


Long-term lamivudine treatment of children with chronic hepatitis B: durability of therapeutic responses and safety.
Lamivudine has been demonstrated safe and efficacious in the short term in a large cohort of children with chronic hepatitis B (CHB), but optimal duration of treatment has not been elucidated and limited data on the safety of long-term lamivudine administration have been reported. In addition, the durability of favourable therapeutic outcomes after lamivudine therapy in children has not been well characterized. The aim of this study was to examine the safety of lamivudine and the durability of clinical responses in a group of children who received up to 3 years of treatment for CHB. One hundred and fifty-one children from centres in nine countries who had previously received lamivudine in a large prospective trial were enrolled. During the first year, children had been randomized to either lamivudine or placebo treatment. Subsequently, in a separate extension study, those who remained hepatitis B e antigen (HBeAg) positive were given lamivudine for up to 2 years and those who were HBeAg negative were observed for additional 2 years. Results of these studies have been previously reported. In this study, these children were followed for 2 additional years. Data gathered from medical record review included weight, height, signs and symptoms of hepatitis, alanine aminotransferase (ALT) levels, serologic markers, hepatitis B virus (HBV) DNA levels and serious adverse events (SAEs). Other pharmacological treatments for CHB were allowed according to the practices of individual investigators and were documented. Subjects were divided into two groups for analysis, those who had achieved virological response (VR), defined as HBeAg negative and undetectable HBV DNA by the bDNA assay by the end of the extension study at 3 years, and those who had not. In those who had achieved VR by the end of the extension study, long-term durability of HBeAg seroconversion was 82% and >90% in those who had received lamivudine for 52 weeks and at least 2 years respectively. This compares to 75% for those who had achieved seroconversion after placebo. In those who had not achieved VR by the end of the extension study, an additional 11% did so by the end of the study; they had all received lamivudine in the previous trial, and none had received further treatment during the study. Eight children lost hepatitis B surface antigen during the study and all had received lamivudine at some point during the previous trials. Evaluation of safety data revealed no SAEs related to lamivudine. There was no effect of treatment on weight or height z scores. Clinically benign ALT flares (>10 times normal) were seen in 2% of children. Favourable outcomes from lamivudine treatment of CHB in children are maintained for at least several years after completion of treatment. Up to 3 years of lamivudine treatment is safe in children.


A Risk-Scoring System Combined With a Fecal Immunochemical Test Is Effective in Screening High-Risk Subjects for Early Colonoscopy to Detect Advanced Colorectal Neoplasms.
Age, sex, smoking, and family history are risk factors for colorectal cancer in Asia. The Asia-Pacific Colorectal Screening (APCS) scoring system was developed to identify subjects with a high risk for advanced neoplasm (AN). We tested an algorithm that combined APCS scores with fecal immunochemical test (FIT) in colorectal cancer screening. We performed a multicenter prospective study, enrolling asymptomatic individuals older than 40 years old in 12 Asia-Pacific regions from December 2011 to December 2013. APCS scores were calculated for each individual (0-1 = low risk [LR], 2-3 = medium risk [MR], and 4-7 = high risk [HR] for AN). LR and MR subjects were offered FIT and referred for early colonoscopies if FIT results were positive. HR subjects were offered colonoscopies. The proportions of subjects with ANs were determined for each group based on colonoscopy findings; odd ratios for LR and MR subjects were calculated compared to LR individuals. We calculated the sensitivity of the APCS-FIT algorithm in identifying subjects with AN. A total of 5657 subjects were recruited: 646 subjects (11.4%) were considered LR, 3243 subjects (57.3%) were considered MR, and 1768 subjects (31.3%) were considered HR for AN. The proportions of individuals with an AN in these groups were 1.5%, 5.1%, and 10.9%, respectively. Compared with LR group, MR and HR subjects had a 3.4-fold increase and a 7.8-fold increase in risk for AN, respectively. A total of 70.6% subjects with AN (95% confidence interval: 65.6%-75.1%) and 95.1% subjects with invasive cancers (95% confidence interval: 82.2%-99.2%) were correctly instructed to undergo early colonoscopy examination. The APCS scoring system, which is based on age, sex, family history, and smoking, is a useful tool for determining risk for colorectal cancer and advanced adenoma in asymptomatic subjects. Use of the APCS score-based algorithm in triaging subjects for FIT or colonoscopy can substantially reduce colonoscopy workload.


Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation.
To investigate the role of endoplasmic reticulum (ER) stress in cancer radiotherapy and its molecular mechanism. Tunicamycin (TM) was applied to induce ER stress in human esophageal cancer cell line EC109, and the radiosensitization effects were detected by acute cell death and clonogenic survival assay. Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide. Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase. Autophagic response was determined by acridine orange (AO) staining and Western blotting of microtubule-associated protein-1 light chain-3 (LC3) and autophagy related gene 5 (ATG5). In order to test the biological function of autophagy, specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy, and its effect on cell apoptosis was thus detected. Additionally, involvement of the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway was also detected by Western blotting. Finally, male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations. Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation (IR), which suggested an obvious radiosensitization effect of TM. Moreover, TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. We also observed an increase of AO positive cells, and the protein level of LC3-II and ATG5 was induced by TM treatment, which suggested an autophagic response in EC109 cells. However, inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability, which suggested a cytoprotective role of autophagy in stressed EC109 cells. Furthermore, TM treatment also activated mTORC1, and in turn reduced Akt phosphorylation, which suggested the PI3K/Akt/mTOR signal pathway was involved in the TM-induced autophagic response in EC109 cells. Tumor xenograft results also showed synergistic retarded tumor growth by TM treatment and IR, as well as the involvement of the PI3K/Akt/mTOR pathway. Our data showed that TM treatment sensitized human esophageal cancer cells to radiation via apoptosis and autophagy both in vitro and in vivo.


Color tuning and white light emission via in situ doping of luminescent lanthanide metal-organic frameworks.
Isostructural lanthanide metal-organic frameworks (MOFs) are synthesized through the spontaneous self-assembly of H3BTPCA (1,1',1″-(benzene-1,3,5-triyl)tripiperidine-4-carboxylic acid) ligands and lanthanide ions (we term these MOFs Ln-BTPCA, Ln = La(3+), Tb(3+), Sm(3+), etc.). Prompted by the observation that the different lanthanide ions have identical coordination environment in these MOFs, we explored and succeeded in the preparation of mixed-lanthanide analogues of the single-lanthanide MOFs by way of in situ doping using a mixture of lanthanide salts. With careful adjustment of the relative concentration of the lanthanide ions, the color of the luminescence can be modulated, and white light-emission can indeed be achieved. The mechanisms possibly responsible for the observed photophysical properties of these mixed-lanthanide MOFs are also discussed.


Efficacy and Biological Activity of Imatinib in Metastatic Dermatofibrosarcoma Protuberans (DFSP).
To report on imatinib mesylate (IM) in patients with metastatic dermatofibrosarcoma protuberans (DFSP)/fibrosarcomatous (FS)-DFSP and on the impact of the treatment on tumor biology. Ten consecutive patients treated with IM from 2007 to 2015 for a metastatic relapse from DFSP/FS-DFSP were identified. FISH analysis for COL1A1-PDGFB was performed. Two IM-treated and 4 naïve FS-DFSP were transcriptionally profiled by RNAseq on HiScanSQ platform. Differential gene expression was analyzed with edgeR (Bioconductor), followed by hierarchical clustering and Principal Component Analysis. All cases featured fibrosarcomatous in the metastasis and retained the COL1A1-PDGFB. Best RECIST response was: 8 partial response, 1 stable disease, and 1 progressive disease. Median progression-free survival was 11 months. Five patients received surgery after IM and all relapsed. IM was restored in 4 patients with a new response. After IM, the most upregulated genes included those encoding for immunoglobulins and those affecting functions and differentiation of endothelial cells. Pathway enrichment analysis revealed upregulation in genes involved in antigen processing and presentation, natural killer-mediated cytotoxicity, and drug and xenobiotics metabolism. Conversely, a significant down-regulation of kinase signaling pathways was detected. All metastatic cases were fibrosarcomatous. Most patients responded to IM, but PFS was shorter than reported in published series which included both DFSP and FS-DFSP. All patients operated after IM had a relapse, suggesting that IM cannot eradicate metastatic cases and that the role of surgery is limited. Transcriptional profile of naïve and posttreatment samples pointed the contribution of immune infiltrates in sustaining the response to IM.


Ambulance clinicians' experiences of relationships with patients and significant others.
Ambulance clinicians (ACs) have to provide advanced care and treatment to patients in a challenging and emotionally demanding environment, therefore they establish interpersonal relationships embracing both patients and significant others. Relationships in emergency care were earlier found to be short-lived and lacking a holistic understanding of the patient. In their relationship with the ambulance clinicians, it is for patients to surrender and become dependent, which may be interpreted as both a negative and a positive experience. The aim of this study was to elucidate ambulance clinicians' experiences of relationships with patients and significant others. Data were collected from four focus group conversations, with a total of 18 participating ambulance clinicians. An inductive qualitative content analysis method was chosen. The analysis resulted in one main category: 'To be personal in a professional role' and three generic categories: 'To be there for the affected person', 'To be personally involved' and 'To have a professional mission'. There were subsequently nine sub-categories. The main category was described as intertwining the experience of being both personal and professional. The ambulance clinicians adapt to a situation while having the affected person in focus. They involve themselves as persons but at the same time use the power of their professional role. The relationship with patients and significant others from the ambulance clinicians' perspective can be understood as embracing both personal and professional aspects. This study provides an understanding of the ambulance clinicians' professional role as embracing a personal perspective, which is important when developing an emergency ambulance service focusing on care that involves more than just emergency medical treatment.


Clusters of red blood cells in microcapillary flow: hydrodynamic versus macromolecule induced interaction.
We present experiments on RBCs that flow through micro-capillaries under physiological conditions. The strong flow-shape coupling of these deformable objects leads to a rich variety of cluster formation. We show that the RBC clusters form as a subtle imbrication between hydrodynamic interactions and adhesion forces because of plasma proteins, mimicked by the polymer dextran. Clusters form along the capillaries and macromolecule-induced adhesion contributes to their stability. However, at high yet physiological flow velocities, shear stresses overcome part of the adhesion forces, and cluster stabilization due to hydrodynamics becomes stronger. For the case of pure hydrodynamic interaction, cell-to-cell distances have a pronounced bimodal distribution. Our 2D-numerical simulations on vesicles capture the transition between adhesive and non-adhesive clusters at different flow velocities.


Changes in MRI signal intensity during hypercapnic challenge under conscious and anesthetized conditions.
Most functional magnetic resonance imaging (fMRI) studies in animals are conducted under anesthesia to minimize motion artifacts. However, methods and techniques have been developed recently for imaging fully conscious rats. Functional MRI studies on conscious animals report enhanced BOLD signal changes as compared to the anesthetized condition. In this study, rats were exposed to different concentrations of carbon dioxide (CO(2)) while conscious and anesthetized to test whether cerebrovascular reactivity may be contributing to these enhanced BOLD signal changes. Hypercapnia produced significantly greater increases in MRI signal intensity in fully conscious animals (6.7-13.3% changes) as when anesthetized with 1% isoflurane (3.2-4.9% changes). In addition, the response to hypercapnia was more immediate in the conscious condition (< 30s) with signal risetimes twice as fast as in the anesthetized state (60s). Both cortical and subcortical brain regions showed a robust, dose- dependent increase in MRI signal intensity with hypercapnic challenge while the animals were conscious but little or no change when anesthetized. Baseline variations in MRI signal were higher while animals were conscious but this was off set by greater signal intensity changes leading to a greater contrast-to-noise ratio, 13.1 in conscious animals, as compared to 8.0 in the anesthetized condition. In summary, cerebral vasculature appears to be more sensitive to hypercapnic challenge in the conscious condition resulting in enhanced T2* MRI signal intensity and the potential for better BOLD signal changes during functional imaging.


Cognitive decline preceding the onset of psychosis in patients with 22q11.2 deletion syndrome.
Patients with 22q11.2 deletion syndrome (22q11DS) have an elevated (25%) risk of developing schizophrenia. Recent reports have suggested that a subgroup of children with 22q11DS display a substantial decline in cognitive abilities starting at a young age. To determine whether early cognitive decline is associated with risk of psychotic disorder in 22q11DS. Prospective longitudinal cohort study. As part of an international research consortium initiative, we used the largest data set of intelligence (IQ) measurements in patients with 22q11DS reported to date to investigate longitudinal IQ trajectories and the risk of subsequent psychotic illness. A total of 829 patients with a confirmed hemizygous 22q11.2 deletion, recruited through 12 international clinical research sites, were included. Both psychiatric assessments and longitudinal IQ measurements were available for a subset of 411 patients (388 with ≥1 assessment at age 8-24 years). Diagnosis of a psychotic disorder, initial IQ, longitudinal IQ trajectory, and timing of the last psychiatric assessment with respect to the last IQ test. Among 411 patients with 22q11DS, 55 (13.4%) were diagnosed as having a psychotic disorder. The mean (SD) age at the most recent psychiatric assessment was 16.1 (6.2) years. The mean (SD) full-scale IQ at first cognitive assessment was lower in patients who developed a psychotic disorder (65.5 [12.0]) compared with those without a psychotic disorder (74.0 [14.0]). On average, children with 22q11DS showed a mild decline in IQ (full-scale IQ, 7.04 points) with increasing age, particularly in the domain of verbal IQ (9.02 points). In those who developed psychotic illness, this decline was significantly steeper (P < .001). Those with a negative deviation from the average cognitive trajectory observed in 22q11DS were at significantly increased risk for the development of a psychotic disorder (odds ratio = 2.49; 95% CI, 1.24-5.00; P = .01). The divergence of verbal IQ trajectories between those who subsequently developed a psychotic disorder and those who did not was distinguishable from age 11 years onward. In 22q11DS, early cognitive decline is a robust indicator of the risk of developing a psychotic illness. These findings mirror those observed in idiopathic schizophrenia. The results provide further support for investigations of 22q11DS as a genetic model for elucidating neurobiological mechanisms underlying the development of psychosis.


RIG-I detects viral genomic RNA during negative-strand RNA virus infection.
RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.


Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer.
Infection-driven inflammation has been implicated in the pathogenesis of ~15-20% of human tumors. Expression of microRNA-155 (miR-155) is elevated during innate immune response and autoimmune disorders as well as in various malignancies. However, the molecular mechanisms providing miR-155 with its oncogenic properties remain unclear. We examined the effects of miR-155 overexpression and proinflammatory environment on the frequency of spontaneous hypoxanthine phosphoribosyltransferase (HPRT) mutations that can be detected based on the resistance to 6-thioguanine. Both miR-155 overexpression and inflammatory environment increased the frequency of HPRT mutations and down-regulated WEE1 (WEE1 homolog-S. pombe), a kinase that blocks cell-cycle progression. The increased frequency of HPRT mutation was only modestly attributable to defects in mismatch repair machinery. This result suggests that miR-155 enhances the mutation rate by simultaneously targeting different genes that suppress mutations and decreasing the efficiency of DNA safeguard mechanisms by targeting of cell-cycle regulators such as WEE1. By simultaneously targeting tumor suppressor genes and inducing a mutator phenotype, miR-155 may allow the selection of gene alterations required for tumor development and progression. Hence, we anticipate that the development of drugs reducing endogenous miR-155 levels might be key in the treatment of inflammation-related cancers.


Measuring the diaspora for virus-specific CD8+ T cells.
The CD8(+) T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of D(b)NP(366)- and D(b)PA(224)-specific CD8(+) T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8(+) tetramer(+) populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the "whole mouse" virus-specific CD8(+) T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8(+)D(b)NP(366)+ and CD8(+)D(b)PA(224)+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 "activation marker" were detected consistently on virus-specific CD8(+) T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69(hi) T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of "resting" CD8(+) memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.


A survey of current and anticipated use of standard and specialist equipment by UK optometrists.
To investigate current and anticipated use of equipment and information technology (IT) in community optometric practice in the UK, and to elicit optometrists' views on adoption of specialist equipment and IT. An anonymous online questionnaire was developed, covering use of standard and specialist diagnostic equipment, and IT. The survey was distributed to a random sample of 1300 UK College of Optometrists members. Four hundred and thirty-two responses were received (response rate = 35%). Enhanced (locally commissioned) or additional/separately contracted services were provided by 73% of respondents. Services included glaucoma repeat measures (30% of respondents), glaucoma referral refinement (22%), fast-track referral for wet age-related macular degeneration (48%), and direct cataract referral (40%). Most respondents (88%) reported using non-contact/pneumo tonometry for intra-ocular pressure measurement, with 81% using Goldmann or Perkins tonometry. The most widely used item of specialist equipment was the fundus camera (74% of respondents). Optical Coherence Tomography (OCT) was used by 15% of respondents, up from 2% in 2007. Notably, 43% of those anticipating purchasing specialist equipment in the next 12 months planned to buy an OCT. 'Paperless' records were used by 39% of respondents, and almost 80% of practices used an electronic patient record/practice management system. Variations in responses between parts of the UK reflect differences in the provision of the General Ophthalmic Services contract or community enhanced services. There was general agreement that specialised equipment enhances clinical care, permits increased involvement in enhanced services, promotes the practice and can be used as a defence in clinico-legal cases, but initial costs and ongoing maintenance can be a financial burden. Respondents generally agreed that IT facilitates administrative flow and secure exchange of health information, and promotes a state-of-the-art practice image. However, use of IT may not save examination time; its dynamic nature necessitates frequent updates and technical support; the need for adequate training is an issue; and security of data is also a concern. UK optometrists increasingly employ modern equipment and IT services to enhance patient care and for practice management. While the clinical benefits of specialist equipment and IT are appreciated, questions remain as to whether the investment is cost-effective, and how specialist equipment and IT may be used to best advantage in community optometric practice.


Using stated preference and revealed preference modeling to evaluate prescribing decisions.
The use of stated preference analyses to evaluate choice of health care products has been growing in recent years. This paper shows how revealed preference data can be enriched with stated preference data and highlights the relative advantages of revealed and stated preference data. The techniques were applied to a study of determinants of physicians' prescriptions of alcoholism medications. Analyses were conducted on the relationship between physicians' perceptions of existing alcoholism medication attributes and their prescribing rates of those medications. Analyses were also conducted on physicians' decisions to prescribe hypothetical alcoholism medications with varying attributes such as efficacy, side effects, compliance, mode of action, and price. Finally, analyses were conducted on the combined stated and revealed preference data. Joint estimation suggests that parameters from the revealed and stated preference data are equal, up to scale. Joint analyses highlight how stated preference data can be used to estimate parameters for attributes that are not observed in the marketplace, that do not vary in the marketplace, or that are highly collinear with other attributes in actual markets.


Halogenated building blocks for 2D crystal engineering on solid surfaces: lessons from hydrogen bonding.
Halogen bonding has emerged as a promising tool in two-dimensional (2D) crystal engineering. Since halogen bonds are similar to hydrogen bonds in a number of aspects, the existing knowledge of hydrogen bonded systems can be applied to halogenated systems. Here we evaluate the applicability of a retrosynthetic approach based on topological similarity between hydrogen and halogen bonds to obtain predictable halogen bonded networks. The self-assembly of 1,3-dibromo-5-alkoxybenzene derivatives was studied in analogy with well-explored alkoxy isophthalic acids using a combination of experimental and theoretical tools. Scanning tunneling microscopy (STM) characterization of the networks formed at the liquid-graphite interface revealed that while the retrosynthetic approach works at the level of small clusters of molecules within the 2D network, the overall structure of the network deviates from the anticipated structure. The monolayers consist of fractured rows of halogen-bonded modules instead of the expected continuous lamellar structure. Each module consists of a discrete number of halogen-bonded molecules. The interactions responsible for the stabilization of halogen bonded dimers are delineated through detailed density functional theory (DFT) calculations coupled with natural bonding orbitals (NBO) and perturbation analysis. A modified force field that includes an extra charged site to imitate the σ hole on the halogen atom was developed and applied to extract total potential energies of the anticipated and observed networks. Plausible reasons for the deviation from the anticipated structure are discussed. Finally, a modified molecular design that allows successful application of the hydrogen bond-halogen bond analogy was tested experimentally.


Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways.
Living cells possess a panel of specialized DNA polymerases that deal with the large diversity of DNA lesions that occur in their genomes. How specialized DNA polymerases gain access to the replication intermediate in the vicinity of the lesion is unknown. Using a model system in which a single replication blocking lesion can be bypassed concurrently by two pathways that leave distinct molecular signatures, we analyzed the complex interplay among replicative and specialized DNA polymerases. The system involves a single N-2-acetylaminofluorene guanine adduct within the NarI frameshift hot spot that can be bypassed concurrently by Pol II or Pol V, yielding a -2 frameshift or an error-free bypass product, respectively. Reconstitution of the two pathways using purified DNA polymerases Pol III, Pol II and Pol V and a set of essential accessory factors was achieved under conditions that recapitulate the known in vivo requirements. With this approach, we have identified the key replication intermediates that are used preferentially by Pol II and Pol V, respectively. Using single-hit conditions, we show that the beta-clamp is critical by increasing the processivity of Pol II during elongation of the slipped -2 frameshift intermediate by one nucleotide which, surprisingly, is enough to support subsequent elongation by Pol III rather than degradation. Finally, the proofreading activity of the replicative polymerase prevents the formation of a Pol II-mediated -1 frameshift product. In conclusion, failure or success of TLS pathways appears to be the net result of a complex interplay among DNA polymerases and accessory factors.


A Survey of American Thyroid Association Members Regarding the 2015 Adult Thyroid Nodule and Differentiated Thyroid Cancer Clinical Practice Guidelines.
Background: The 2015 American Thyroid Association (ATA) clinical practice guidelines (CPGs) on management of thyroid nodules (TNs) and differentiated thyroid cancer (DTC) in adults were developed to inform clinicians, patients, researchers, and health policy makers about the best available evidence, and its limitations, relating to management of these conditions. Methods: We conducted a cross-sectional electronic survey of ATA members' perspectives of these CPGs, using a standardized survey (Clinician Guidelines Determinant Questionnaire) developed by the Guidelines International Network. A survey link was electronically mailed to members in February of 2019, with reminders sent to nonrespondents 2 and 5 weeks later. Data were descriptively summarized, after excluding missing responses. Results: The overall response rate was 19.8% (348/1761). The effective response rate was 20.2% (348/1720), after excluding a recently deceased member and individuals who had either invalid e-mail addresses or whose e-mails were returned. Of the respondents, 37.9% (132/348) were female, 60.4% (209/346) were endocrinologists, 27.5% (95/346) were surgeons, and 3.5% (12/346) were nuclear medicine specialists. The majority of respondents (71.9%; 250/348) were at a mid- or advanced-career level, and more than half were in academia (57.5%; 195/339). The majority (69.8%; 243/348) practiced in North America. The vast majority of respondents indicated that the CPGs explained the underlying evidence (92.3%; 298/323) and 92.9% (300/323) agreed or strongly agreed with the content. Most respondents stated that they regularly used the CPGs in their practice (83.0%; 268/323). Most respondents (83.0%; 268/323) also agreed or strongly agreed that the recommendations were easy to incorporate in their practice. The most popular CPG format was an electronic desktop file (78.8%; 252/320). Shorter more frequent CPGs were favored by 55.0% (176/320) of respondents, and longer traditional CPGs were favored by 39.7% (127/320). Conclusions: The clinical content and evidence explanations in the adult TN and DTC CPGs are widely accepted and applied among ATA survey respondents. Future ATA CPG updates need to be optimized to best meet users' preferences regarding format, frequency, and length.


Hyperlipidemia and atherosclerosis.
These studies provide new insight into the complex mechanisms wherby hyperlipidemia causes progressive atherosclerosis. It has been shown that physical injury to the endothelial lining of arteries sets off a process which probably is an attempt at healing the injury but which can lead to atherosclerosis. It has also been found that chemical agents such as homocystine can produce a similar series of events leading to atherosclerosis. These events include focal loss of endothelium, exposure of subendothelial connective tissue, and adherence of platelets followed by release of factors that stimulate intimal smooth muscle proliferation. The present studies indicate that the effects of chronic hyperlipidemia are complex in that the condition results not only in the deposition of lipids in the atheromatous lesions but that it may produce the primary endothelial injury that initiates the process of atherosclerosis as well.


Food Inhibits the Oral Bioavailability of the Major Green Tea Antioxidant Epigallocatechin Gallate in Humans.
The bioavailability of the most abundant and most active green tea antioxidant, epigallocatechin gallate (EGCG) remains uncertain. Therefore, the systemic absorption of EGCG was tested in healthy fasted humans. It was administered as capsules with water or with a light breakfast, or when incorporated within a strawberry sorbet. The results for plasma EGCG clearly revealed that taking EGCG capsules without food was better; the AUC was 2.7 and 3.9 times higher than when EGCG capsules were taken with a light breakfast (p = 0.044) or with EGCG imbedded in the strawberry sorbet (p = 0.019), respectively. This pattern was also observed for Cmax and Cav. Therefore, ingesting food at the same time as EGCG, whether it was imbedded or not in food, substantially inhibited the absorption of the catechin. As with some types of medications that are affected by food, it appears that EGCG should be taken without food in order to maximise its systemic absorption. Therefore, based on these findings, ingesting EGCG with water on an empty stomach is the most appropriate method for the oral delivery of EGCG in clinical trials where EGCG is to be investigated as a potential bioactive nutraceutical in humans.


Quality of care in patients with chronic hepatitis C virus infection: a cohort study.
Medicare has proposed quality-of-care indicators for chronic hepatitis C virus (HCV) infection. The extent to which these standards are met in practice is largely unknown. To evaluate the quality of health care that patients with HCV receive and the factors associated with receipt of quality care. Retrospective cohort study. Nationwide U.S. health insurance company research database. 10 385 patients with HCV enrolled in the database between 2003 and 2006. Patients were included if they were eligible for at least 1 quality indicator. Quality of HCV care received by patients, as measured by 7 explicit quality indicators included in Medicare's 2009 Physician Quality Reporting Initiative. Proportions of patients meeting quality indicators varied, ranging from 21.5% for vaccination to 79% for the HCV genotype testing indicator. Overall, 18.5% of patients (95% CI, 18% to 19%) received all recommended care. Older age and presence of comorbid conditions were associated with lower quality, whereas elevated liver enzyme levels, cirrhosis, and HIV infection were associated with higher quality. Patients who saw both generalists and specialists received the best care (odds ratio of receiving care for which a patient is eligible: specialists alone, 0.79 [CI, 0.66 to 0.95]; primary care physician alone, 0.44 [CI, 0.40 to 0.48]). The study had an observational retrospective design, used a convenience sample, and had no information on patient ethnicity. It may be that the indicators or the reporting of the indicators of HCV care--and not the care itself--is suboptimum. Health care quality, based on Medicare criteria, is suboptimum for HCV. Care that included both specialists and generalists is associated with the best quality. Our results support the development of specialist and primary care collaboration to improve the quality of HCV care. Saint Louis University Liver Center.


Uremic solutes and risk of end-stage renal disease in type 2 diabetes: metabolomic study.
Here we studied plasma metabolomic profiles as determinants of progression to end-stage renal disease (ESRD) in patients with type 2 diabetes (T2D). This nested case-control study evaluated 40 cases who progressed to ESRD during 8-12 years of follow-up and 40 controls who remained alive without ESRD from the Joslin Kidney Study cohort. Controls were matched with cases for baseline clinical characteristics, although controls had slightly higher eGFR and lower levels of urinary albumin excretion than cases. Plasma metabolites at baseline were measured by mass spectrometry-based global metabolomic profiling. Of the named metabolites in the library, 262 were detected in at least 80% of the study patients. The metabolomic platform recognized 78 metabolites previously reported to be elevated in ESRD (uremic solutes). Sixteen were already elevated in the baseline plasma of our cases years before ESRD developed. Other uremic solutes were either not different or not commonly detectable. Essential amino acids and their derivatives were significantly depleted in the cases, whereas certain amino acid-derived acylcarnitines were increased. All findings remained statistically significant after adjustment for differences between study groups in albumin excretion rate, eGFR, or HbA1c. Uremic solute differences were confirmed by quantitative measurements. Thus, abnormal plasma concentrations of putative uremic solutes and essential amino acids either contribute to progression to ESRD or are a manifestation of an early stage(s) of the disease process that leads to ESRD in T2D.


Age and founder effect of SOD1 A4V mutation causing ALS.
The alanine to valine mutation at codon 4 (A4V) of SOD1 causes a rapidly progressive dominant form of amyotrophic lateral sclerosis (ALS) with exclusively lower motor neuron disease and is responsible for 50% of SOD1 mutations associated with familial ALS in North America. This mutation is rare in Europe. The authors investigated the origin (geographic and time) of the A4V mutation. Several cohorts were genotyped: North American patients with confirmed A4V mutation (n = 54), Swedish (n = 3) and Italian (n = 6) A4V patients, patients with ALS with SOD1 non-A4V mutations (n = 66) and patients with sporadic ALS (n = 96), healthy white (n = 96), African American (n = 17), Chinese (n = 53), Amerindian (n = 11), and Hispanic (n = 12) subjects. High-throughput SNP genotyping was performed using Taqman assay in 384-well format. A novel biallelic CA repeat in exon 5 of SOD1, tightly linked to A4V, was genotyped on sequencing gels. Association statistics were estimated using Haploview. p Values less than 0.05 were considered significant. Age of A4V was estimated using a novel method based on r(2) degeneration with genetic distance and a Bayesian method incorporated in DMLE+. A single haplotype of 10 polymorphisms across a 5.86-cM region was associated with A4V (p = 3.0e-11) when white controls were used, suggesting a founder effect. The strength of association of this haplotype progressively decreased when African American, Chinese, Hispanic, and Amerindian subjects were used as controls. The associated European haplotype was different from the North American haplotype, indicating two founder effects for A4V (Amerindian and European). The estimated age of A4V with the r(2) degeneration method was 458 +/- 59 years (range 398-569) and in agreement with the Bayesian method (554-734 years with 80-90% posterior probability). North American SOD1 alanine to valine mutation at codon 4 descended from two founders (Amerindian and European) 400-500 years ago.


Breast cancer: views of general practitioners on its detection and treatment.
A considerable proportion of women with breast cancer in this country present for treatment in late stages of the disease; Britain's primary health-care system means that in most cases the possible diagnosis of breast cancer is first made in general practice. This offers ample opportunity for early diagnosis and a better understanding about the way the general practitioners perceive the problems associated with the disease. With that specific aim, the present article reports on a survey conducted simultaneously in Oxford and in Edinburgh. Results show that general practitioners in the survey are deeply concerned about breast cancer and see a definite role for themselves in its early detection and long-term management. They also see a need for more health education that includes the encourgement of breast self-examination programmes. Most are in favour of open-access breast clinics for women and, in general, they feel positive about self-education programmes.


Scintillation light detection in the 6-m drift-length ProtoDUNE Dual Phase liquid argon TPC.
DUNE is a dual-site experiment for long-baseline neutrino oscillation studies, neutrino astrophysics and nucleon decay searches. ProtoDUNE Dual Phase (DP) is a 6 × 6 × 6 m 3 liquid argon time-projection-chamber (LArTPC) that recorded cosmic-muon data at the CERN Neutrino Platform in 2019-2020 as a prototype of the DUNE Far Detector. Charged particles propagating through the LArTPC produce ionization and scintillation light. The scintillation light signal in these detectors can provide the trigger for non-beam events. In addition, it adds precise timing capabilities and improves the calorimetry measurements. In ProtoDUNE-DP, scintillation and electroluminescence light produced by cosmic muons in the LArTPC is collected by photomultiplier tubes placed up to 7 m away from the ionizing track. In this paper, the ProtoDUNE-DP photon detection system performance is evaluated with a particular focus on the different wavelength shifters, such as PEN and TPB, and the use of Xe-doped LAr, considering its future use in giant LArTPCs. The scintillation light production and propagation processes are analyzed and a comparison of simulation to data is performed, improving understanding of the liquid argon properties.


Influence of hospital characteristics on quality of care in patients with community-acquired pneumonia.
In-hospital care of patients with community-acquired pneumonia (CAP) varies across hospitals. Understanding of the underlying factors is the basis for tailored quality improvements. Using data from a randomised controlled Swiss-wide multicentre trial, we compared length of stay (LOS) and other patient outcomes according to (A) the use of a procalcitonin (PCT)-based antibiotic stewardship protocol, (B) institution type (university vs non-university), and (C) historical time period in relation to the introduction of Diagnosis Related Group (DRG) reimbursement (2012). We included 784 patients hospitalised with CAP from six institutions into this secondary analysis. We used multivariable regression models adjusted for age, comorbidities and disease severity to determine the influence of institution characteristics on LOS and patient outcomes. LOS was significantly shorter in the institution using a PCT-based antibiotic stewardship protocol (9.2 vs 5.3 days; adjusted mean difference 3.92 days; 95% confidence interval [CI] 5.16-2.68) with shorter antibiotic treatment. There was no difference in LOS in university vs non-university hospitals, but antibiotic courses in university-type hospitals were longer (11.0 vs 8.3 days; adjusted mean difference 2.59 days; 95% CI, 1.69-3.49). No significant difference in LOS was found when comparing the time period before and after the introduction of the DRG system in Switzerland. We found differences in LOS associated with theuse of a PCT-based antibiotic stewardship protocol, which remained robust after multivariable adjustment. Importantly, the type of institution and model of reimbursement did not influence LOS in our CAP cohort. More health services research studies are needed to establish causal effects.


Recommendations for the management of irritable bowel syndrome in family practice. IBS Consensus Conference Participants.
To help family physicians manage patients with irritable bowel syndrome (IBS), a consensus conference was convened in June 1997 at which 5 internationally recognized experts in IBS presented position papers on selected topics previously circulated to the conference participants. Five working groups comprising family physicians, gastroenterologists and allied health care professionals from across Canada were then charged with developing recommendations for the diagnosis, patient education, psychosocial management, dietary advice and pharmacotherapy, respectively. An evidence-based approach was used where possible; otherwise, recommendations were made by consensus. The participants concluded that family physicians can make a positive diagnosis of IBS using symptom criteria. The pathophysiology is poorly understood, but motility and sensory disturbances appear to play a role. Neither psychological nor specific dietary factors cause IBS, but both can trigger symptoms. Drug therapy is not recommended for the routine treatment of IBS, but short-term trials of drug therapy may be targeted to predominant symptoms in selected patients. A step-wise, patient-centred approach to management is outlined.


Probing bacterial-fungal interactions at the single cell level.
Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into the dynamic interactions between the basidiomycete Coprinopsis cinerea and the bacterium Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of B. subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.


NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.
1. The ability of bombesin or platelet-derived growth factor (PDGF) to stimulate Ca2+ inflow (assessed by measuring changes in the intracellular free Ca2+ concentration in cells loaded with fura-2) in NIH-3T3 cells transformed with the EJ/T24-Ha-ras-1 oncogene is inhibited when compared with the action of the agonists on wild-type cells. 2. The effects of transformation with the ras oncogene are associated with complete inhibition of the ability of bombesin to release Ca2+ from intracellular stores, a substantial decrease in the number of bombesin receptors, no change in the ability of foetal calf serum or ionomycin to release Ca2+ from intracellular stores and the activation of protein kinase C. 3. The effects of transformation with the H-ras oncogene on the ability of bombesin or PDGF to stimulate Ca2+ inflow were mimicked by a 30 min exposure of wild-type cells to phorbol dibutyrate. This action of phorbol dibutyrate was completely blocked by prior treatment of wild-type cells for 24 h with the phorbol ester. 4. It is concluded that one of the actions of the H-ras oncogene in fibroblasts is to inhibit agonist-stimulated Ca2+ inflow by a mechanism which involves the activation of protein kinase C.


Effect of L-arginine infusion on airway NO in cystic fibrosis and primary ciliary dyskinesia syndrome.
Airway nitric oxide concentrations in patients with cystic fibrosis or primary ciliary dyskinesia syndrome have been shown to be lower than in healthy subjects. Decreased NO concentrations may contribute to impaired ciliary clearance, respiratory tract infections, or obstructive lung disease in these conditions. Nasal and exhaled NO concentrations were compared before and after infusion of 500 mg x kg(-1) L-arginine, the substrate of NO synthases, in 11 cystic fibrosis (CF) patients, seven primary ciliary dyskinesia (PCD) syndrome patients, and 11 control subjects. Baseline nasal and exhaled NO concentrations were significantly lower in both CF and PCD syndrome patients than in controls (p<0.01). In controls, the maximum increase of NO was seen immediately after L-arginine infusion in the upper airways (1.8-fold) and 3 h after the infusion in the lower airways (1.4-fold). Although NO concentrations also increased significantly in both CF (1.9-fold and 1.6-fold, respectively) and PCD syndrome patients (1.4-fold and 1.8-fold, respectively), concentrations remained subnormal compared with baseline values of controls. Pulmonary function remained unchanged in both patient groups. In conclusion, the low airway nitric oxide formation in both cystic fibrosis and primary ciliary dyskinesia syndrome patients can be augmented by L-arginine administration. The finding that pulmonary function remained unchanged in both conditions may be due to the fact that normalization of airway nitric oxide concentrations could not be achieved.


Attitudes to genetic testing in families with multiple cases of bipolar disorder.
This study assesses interest in genetic testing for gene variations associated with bipolar disorder and associated information needs. Two hundred individuals (95 unaffected and 105 affected with either bipolar disorder, schizoaffective disorder--manic type, or recurrent major depression) from families with multiple cases of bipolar disorder were assessed, using mailed, self-administered questionnaires. The percentage of participants reporting interest in genetic testing was associated with the degree of certainty with which any test would indicate the development of bipolar disorder. Interest in genetic testing, given a 25% lifetime risk scenario, was lowest (with 77% of participants indicating interest), and highest for the 100% lifetime risk scenario (92%). Eighty percent of participants indicated interest in genetic testing of their own children; of these 30% reported wanting their children tested at birth, and 33% in early childhood. Forty-one percent of participants reported that they would be interested in preimplantation genetic diagnosis, and 54% in prenatal testing. The possibility of ascertainment bias cannot be ruled out. Interest in hypothetical genetic testing for bipolar disorder may not necessarily translate into actual utilization. These results indicate that uptake of genetic testing for genotyping for low-risk alleles related to bipolar disorder is likely to be lower than for testing for high-penetrance gene mutations that follow Mendelian inheritance. The discrepancy between the desired age of testing children and the accepted current practice may be a source of distress and conflict for parents and health professionals alike.


Internalization of macromolecules from the medium in Suctoria.
Takophrya infusionum like all other Suctoria lacks an oral cavity. Its feeding apparatus consists of tentacles, long narrow tubes through which the contents of the living prey are ingested. For normal growth, reproduction, and longevity of clones, Tokophrya needs supplements deriving from the medium in addition to living prey. Since Tokophrya lacks a mouth, these supplements can reach the cytoplasm only through the complex structure of the cortex, which is composed of a three-membraned pellicle and a dense epiplasm. In addition, external to the cortex, an extraneous coat covers the whole organism. Only the outer pellicular plasma membrane is continuous; the other two and the epiplasm are interrupted by the outer plasma membrane which invaginates at intervals forming the so-called pits. The invaginated plasma membrane dips down into the cytoplasm where it extends to form a saccule. Experiments with cationized ferritin and Thorotrast provide evidence that internalization of these macromolecules takes place through the pits by pinocytosis. The membrane of the saccules of the pits forms invaginations which pinch off giving rise to small, flattened vesicles containing the tracers. The tracers were never found free in the cytoplasm but exclusively in the flat vesicles. These vesicles are thus the vehicles transporting macromolecules from the medium to the cytoplasm. The saccules of the pits are the natural loci of pinocytosis and together with the flattened vesicles perform an important function in Suctoria, supplying the organisms with macromolecules from the medium.


Helicate, macrocycle, or catenate: Dynamic topological control over subcomponent self-assembly.
The aqueous reaction between equimolar amounts of 2-(2-(2-aminoethoxy)ethoxy)ethanamine, 1,10-phenanthroline-2,9-dialdehyde and copper(I) produced a dimeric helical macrocycle in quantitative yield. This ring could also be generated by the addition of two equivalents of the diamine to an acyclic helicate containing four mono-imine residues: A transimination occurred, the chelate effect being implicated as a driving force. In the case of a helicate containing mono-imines derived from anilines, the substitution of diamine for monoamine was reversible upon lowering the pH. The aliphatic diamine was protonated at a higher pH than the arylamine, which left the arylamine free for incorporation instead of the alkyl diamine. This reaction thus opened the possibility of switching between closed macrocyclic and open helicate topologies by changing the pH. An additional closed topology became accessible through the use of a diamine that incorporates two rigid phenylene spacer groups between a flexible chain and the imine-forming nitrogen atoms. The resulting catenate consists of a pair of topologically interlinked macrocycles. The presence of the phenylene groups appeared to dictate the topology of the final product, making the formation of a single macrocycle energetically disfavoured.


Histone 3 s10 phosphorylation: "caught in the R loop!".
In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.


Effect of Lactobacillus acidophilus strain NP51 on Escherichia coil O157:H7 fecal shedding and finishing performance in beef feedlot cattle.
A 2-year study was conducted during the summer months (May to September) to test the effectiveness of feeding Lactobacillus acidophilus strain NP51 on the proportion of cattle shedding Escherichia coli O157:H7 in the feces and evaluate the effect of the treatment on finishing performance. Steers (n = 448) were assigned randomly to pens, and pens of cattle were assigned randomly to NP51 supplementation or no supplementation (control). NP51 products were mixed with water and applied as the feed was mixed daily in treatment-designated trucks at the rate of 10(9) CFU per steer. Fecal samples were collected (n = 3,360) from the rectum from each animal every 3 weeks, and E. coli O157:H7 was isolated by standard procedures, using selective enrichment, immunomagnetic separation, and PCR confirmation. The outcome variable was the recovery of E. coli O157:H7 from feces, and was modeled using logistic regression accounting for year, repeated measures of pens of cattle, and block. No significant differences were detected for gain, intakes, or feed efficiency of control or NP51-fed steers. The probability for cattle to shed E. coli O157:H7 varied significantly between 2002 and 2003 (P = 0.004). In 2002 and 2003, the probability for NP51-treated steers to shed E. coli O157:H7 over the test periods was 13 and 21%, respectively, compared with 21 and 28% among controls. Over the 2 years, NP51-treated steers were 35% less likely to shed E. coli O157: H7 than were steers in untreated pens (odds ratio = 0.58, P = 0.008). This study is consistent with previous reports that feeding NP51 is effective in reducing E. coli O157:H7 fecal shedding in feedlot cattle.


The effects of urease immobilization on the transport characteristics and protein adsorption capacity of cellulose acetate based hemodialysis membranes.
In this study, cellulose acetate (CA) based hemodialysis membranes were prepared by a dry phase inversion method and the influences of urease immobilization on the clearing performance and protein adsorption capacity of the membranes were investigated. Permeation experiments have shown that modification of CA membranes with urease immobilization not only enhanced the transport rate of urea but also increased the permeation coefficients of uric acid and creatinine by changing the structure of the membrane. Furthermore, the protein adsorption capacity of the CA membranes decreased. On the other hand, the mechanical strength of the modified CA membrane did not change significantly compared with that of the unmodified one. A mathematical model was derived to determine the rate of mass transfer of urea through modified CA membranes. Model predictions along with the experimental data suggest that urease immobilization can be used as an alternative method in preparing CA based hemodialysis membranes with improved transport characteristics and biocompatibility through reduced protein adsorption capacities.


Distinct serum metabolomics profiles associated with malignant progression in the KrasG12D mouse model of pancreatic ductal adenocarcinoma.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths worldwide with less than a 6% 5-year survival rate. PDAC is associated with poor prognosis based on the late stage diagnosis of the disease. Current diagnostic tests lack the sensitivity and specificity to identify markers of early staging. Metabolomics has provided biomarkers for various diseases, stressors, and environmental exposures. In this study we utilized the p48-Cre/LSL-KrasG12D mouse model with age-matched wild type mice. This model shows malignant progression to PDAC analogous to the human disease stages via early and late pancreatic intra-epithelial neoplasia (PanIN) lesions. Serum was collected from mice with early PanIN lesions (at 3-5 months) and with late PanIN or invasive PDAC lesions (13-16 months), as determined by histopathology. Metabolomics analysis of the serum samples was conducted through UPLC-TOFMS (Ultra Performance Liquid Chromatography coupled to Time-of-flight Mass Spectrometry). Multivariate data analysis revealed distinct metabolic patterns in serum samples collected during malignant progression towards invasive PDAC. Animals with early or late stage lesions were distinguished from their respective controls with 82.1% and 81.5% accuracy, respectively. This also held up for randomly selected subgroups in the late stage lesion group that showed less variability between animals. One of the metabolites, citrate, was validated through tandem mass spectrometry and showed increased levels in serum with disease progression. Furthermore, serum metabolite signatures from animals with early stage lesions identified controls and animals with late stage lesions with 81.5% accuracy (p<0.01) and vice-versa with 73.2% accuracy (p<0.01). We conclude that metabolomics analysis of serum samples can identify the presence of early and late stage pancreatic cancer.


Reticular Erythematous Mucinosis in an African Woman with HIV Infection: Case Report and Literature Review.
Reticular erythematous mucinosis is a rare and persistent form of primary idiopathic mucinosis, often referred to as plaque-like cutaneous mucinosis or midline mucinosis. It presents with reticulate patches or erythematous plaques with predilection for the anterior and posterior trunk. Affected patients are frequently asymptomatic. Pruritus or burning sensations were reported after exposure to the sun. The aetiology remains obscure; its pathogenesis is poorly understood, particularly in immunocompromised patients such as HIV-infected patients. The disease associations are not uniformly documented. Antimalarial agents significantly improve and shorten the course of the disease. We report a case of a 31-year-old African woman with underlying HIV infection who displayed the classical clinical and histological features of reticular erythematous mucinosis. This condition is rare among the HIV-infected patients, particularly in those of African descent, in whom lichen myxoedematosus/scleromyxoedema variants and acral persistent papular mucinoses were most frequently reported. The higher incidence of photosensitivity in HIV-infected individuals including the patients with skin of colour may play a potential role in reticular erythematous mucinosis. Its relationship with lupus erythematosus and photosensitivity in the context of HIV infection is discussed. To the best of our knowledge, this is the first reported case of reticular erythematous mucinosis in an African HIV-infected patient. This case highlights the need for diagnostic awareness in cases presenting with erythematous plaques and patches in a net-like pattern developing on the midline and sun-exposed areas of the trunk.


The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response.
The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE(+) cells in memory responses is particularly unclear. IgE(+) B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE(+) GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE(+) GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE(+) GC cells, whereas sequential switching gives rise to IgE(+) PCs. We propose a comprehensive model for the generation and memory of IgE responses.


A novel approach to characterization of a relatively unstable intercalation compound under ambient conditions: revisiting a kaolinite-acetone intercalation compound.
Characteristics of a kaolinite-acetone intercalation compound prepared using a kaolinite N-methylformamide intercalation compound (Kaol-NMF) as an intermediate were obtained by a set of techniques with attention to suppressing evaporation and deintercalation of acetone. X-ray diffraction (XRD) with spectroscopic analyses, Fourier-transform infrared spectroscopy (FTIR) accompanied by solid-state 13C and 29Si nuclear magnetic resonance (NMR) spectroscopy with cross polarization (CP) and magic angle spinning (MAS) enable us to demonstrate full replacement of a pre-intercalated NMF monolayer with an acetone monolayer between the layers of kaolinite with an increase in the basal spacing from 1.08 nm (Kaol-NMF) to 1.12 nm. In addition, the appearance of an additional OH stretching band at 3630 cm-1 and the shift of the C[double bond, length as m-dash]O stretching band to a lower wavenumber, from 1714 to 1701 cm-1, in the FTIR spectrum, along with a downfield shift of the signal due to C[double bond, length as m-dash]O groups from 209 ppm, where a singlet was observed in the liquid-state 13C NMR spectrum of acetone in CDCl3, to 219 ppm in the 13C CP/MAS NMR spectrum, indicate hydrogen bond formation between interlayer hydroxyl groups of kaolinite and C[double bond, length as m-dash]O groups of the intercalated acetone molecules. These careful characterization studies provide information on an interaction between kaolinite and acetone under ambient conditions.


Carcinoma of the gallbladder--a possible regional predisposition in the western Cape and northern Cape.
An analysis of 19 patients with confirmed carcinoma of the gallbladder who were treated over a period of 68 months emphasised known clinical features of the disease, i.e. nonspecific symptomatology, late presentation and poor prognosis. Comparison with 883 patients undergoing surgery for benign calculous biliary tract disease during the same period identified a more advanced mean age (67.0 v. 55.3 years), larger mean maximum stone diameter (20.3 v. 11.9 mm) and a higher proportion of patients resident in the Cape interior and west coast region (57.8% v. 11.8%) as features associated with carcinoma of the gallbladder. Further investigation of the regional predisposition suggested by this study may identify new factors linked to the development of gallbladder cancer.


[The effect of videophone communication (with skype and webcam)for elderly patients with dementia and their caregivers].
We conducted an intervention study to clarify how effectively videophone (Skype) was used in the communication for elderly patients with dementia cared at home and their caregivers. For a period of 12 weeks, a patient-caregiver pair(n = 6) communicated with a nurse via computer for 30 minutes once a week. The patient and the caregiver worked as a pair. Before and after the 12-week study period started, the intervention and control group (n = 7) patients were assessed with a cognitive scale (HDS-R), VAS, and a depression scale (SDS) for caregivers. The result on the 12th week showed signs of improvement on the intervention group in HDS-R and SDS. The intervention group kept no change in VAS, an expression of subjective feelings of happiness. Meanwhile, the control group significantly decreased in VAS. By the questionnaire for caregivers, many said that the videophone communication was a pleasure of the family and resulted in more family exchanges. Therefore, we consider that a videophone communication is useful for cognitive rehabilitation and the feelings of the patient, and it is also good for a satisfaction of the family.


Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder.
Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.


The crystal structure of the human titin:obscurin complex reveals a conserved yet specific muscle M-band zipper module.
M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig domain able to interact with two alternative ligands-obscurin and obscurin-like-1 (Obsl1)-in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes. O1, although structurally related to OL1 and M10, both members of the intermediate set (I-set) Ig family, presents an intriguing switch of its βA' strand. This leads to structural differences between the complexes, particularly for the "open side" of the chevron-shaped assembly. A bioinformatics analysis reveals that the βA'-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in neonatal rat cardiomyocytes are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher-order interactions.


Critical role of R-loops in processing replication blocks.
Blocks in replication result from impediments to the advancing replication machinery and are lethal if not resolved. The replication fork must be reassembled for DNA synthesis to proceed. Fork assembly outside the chromosomal origin of replication (oriC) is mediated by recombination or via a helicase-dependent pathway. ColE1 plasmid origins of replication and oriK sites initiate primosome assembly by an RNA-DNA hybrid structure known as R-loop. We review evidence suggesting that R-loops are frequent during normal cell growth and that R-loops are critical for the maintenance of genome integrity. We propose that downstream of a replication block, RNA at R-loops is extended by DNA polymerase I, opening up the DNA duplex and leading to the recruitment of the replisome. This would allow replication to proceed while the original block is repaired or bypassed. Unlike recombination and helicase-dependent fork restoration, this mechanism would operate preferentially in transcribed areas of the genome, which are known to be particularly susceptible to DNA damage. Our model emphasizes the intimate relationship between transcription and repair, offers a unifying interpretation of phenotypes attributed to bacterial strains deficient in R-loop fork assembly, and calls for a renewed focus on R-loop formation and regulation.


Anti-inflammatory properties of Salograviolide A purified from Lebanese plant Centaurea ainetensis.
Anti-inflammatory activities of medicinal plants have largely been attributed to their content of sesquiterpene lactones (SLs). SLs are predominantly found in the sunflower family Asteraceae and have been isolated from many plants of this family, particularly Centaurea. The anti-inflammatory activities of extract of Centaurea ainetensis, a Lebanese endemic plant, and the isolated active molecule were assessed for their potential ant-inflammatory activities. Plant extract from Centaurea ainetensis, and the isolated active ingredient Salograviolide A (SA), a sesquiterpene lactones guaianolide, were used for the study. Western blotting and electrophoretic mobility shift assays were used to test the effects of the plant extract and SA on interleukin-1 (IL-1) induced increase in cyclooxygenase-2 (COX-2) levels and in nuclear factor-kappaB (NF-kappaB) translocation in an intestinal epithelial cell (IEC) of inflammation. Their effects on inflammation score and cytokine levels were also studied in an iodoacetoamide-induced rat model of inflammation. Plant extract and SA were shown to reverse the effects observed by IL-1 on COX-2 levels and NF-kappaB translocation in IEC. SA decreased the level of inflammatory cytokines and the level of inflammation in the animal model. These findings suggest that SA may be useful in the development of natural therapies for inflammatory diseases.


Epidemiology of human leptospirosis in Malaysia, 2004-2012.
Leptospirosis is an emerging disease, especially in countries with a tropical climate such as Malaysia. A dramatic increase in the number of cases has been reported over the last decade; however, information on the epidemiological trends of this disease is lacking. The objective of this study is to provide an epidemiological description of human leptospirosis cases over a 9-year period (2004-2012) and disease relationship with meteorological, geographical, and demographical information. A retrospective study was undertaken to describe the patterns of human leptospirosis cases and their association with intrinsic (sex, age, and ethnicity) and extrinsic (location, rainfall, and temperature) factors. Data was grouped according to age, sex, ethnicity, seasonality and geographical distribution, and analyzed using statistical tools to understand the influence of all the different factors on disease incidence. A total of 12,325 cases of leptospirosis were reported between 2004 and 2012 with an upward trend in disease incidence, with the highest in 2012. Three hundred thirty-eight deaths were reported with an overall case fatality rate of 2.74%, with higher incidence in males (9696; 78.7%) compared with female patients (2629; 21.3%), and overall male to female ratio of 3.69:1. Patients aged cohorts between 30-39 years old (16.22 per 100,000 population) had the highest disease incidence while the lowest incidence occurred between <1 to 9 years old (3.44 per 100,000 population). The average incidence was highest amongst Malays (10.97 per 100,000 population), followed by Indians (7.95 per 100,000 population). Stratification according to geographical distribution showed that the state of Malacca had the highest average disease incidence (11.12 per 100,000 population) followed by Pahang (10.08 per 100,000 population). The states of Terengganu, Kelantan, and Perak recorded similar rates of incidence (≈8.00 per 100,000 population), while Johor with the least number of reported cases (1.80 per 100,000 population). Positive relationships were recorded between the number of reported cases with the number of raining days per month and monthly average temperature (p-value<0.05). However, no significant association was noted between rainfall volume and number of reported Leptospirosis cases. This collaborative efforts between medical, academic and governmental institutions has enabled the construction of this comprehensive database that is essential to understand the disease trends in Malaysia and add insights into the prevention and control of this disease.