Radioactive characterization of leachates and efflorescences in the neighbouring areas of a phosphogypsum disposal site as a preliminary step before its restoration.
After the recent closure of certain phosphoric acid plants located in the South-West of Spain, it has been decided to restore a big extension (more than six hundred hectares) of salt-marshes, where some million tonnes of phosphogypsum (PG), the main by-product generated by these plants, had been disposed of. This PG is characterized by its content of high activity concentrations of several radionuclides from the uranium series, mainly (226)Ra, (210)Pb, and (210)Po and, to a lesser extent, U-isotopes. The PG disposal area can be considered as a potential source of radionuclides into their nearby environment, through the waters which percolate from them and through the efflorescences formed in their surroundings. For this reason, a detailed radioactive characterization of the mentioned waters and efflorescences has been considered essential for a proper planning of the restoration tasks to be applied in the near future in the zone. To this end, U-isotopes, (234)Th, (230)Th, (226)Ra, (210)Pb and (210)Po activity concentrations have been determined by applying both alpha-particle and gamma-ray spectrometric techniques to selected water and efflorescence aliquots collected in the area. The analysis of the obtained results has enabled to obtain information about the geochemical behaviour in the area of the different radionuclides analyzed; and the conclusion to be drawn that, in the restoration plan under preparation, both the prohibition of outflowing waters from the disposal area to the neighbouring salt-marshes, and the removal of all the efflorescences now disseminated in their surroundings are essential.


Epithelioid sarcoma expresses epidermal growth factor receptor but gene amplification and kinase domain mutations are rare.
Epithelioid sarcoma is a rare, malignant, soft tissue neoplasm that can be classified into proximal, distal and fibroma-like subtypes. Regardless of subtype, epithelioid sarcoma often shows morphologic and immunophenotypic evidence of epithelial differentiation. Current therapeutic strategies include surgical resection, amputation, radiation or chemotherapy, although the overall prognosis remains poor. The epidermal growth factor receptor (EGFR) is a novel therapeutic target in carcinomas. In some carcinomas, EGFR kinase domain mutations or gene amplification may correlate with response to specific inhibitors. EGFR expression has been reported in some sarcoma types, but expression, amplification and mutations have not been studied in epithelioid sarcoma. We evaluated 15 cases of epithelioid sarcoma from 14 patients for EGFR expression using immunohistochemistry, EGFR copy number aberration using fluorescence in situ hybridization and screened for mutations in the tyrosine kinase domain of the EGFR gene using direct sequencing. In all, 14 of the 15 epithelioid sarcomas (93%) showed expression of EGFR by immunohistochemistry. A majority of the cases (n=11, 73%) showed strong (2+ to 3+) and homogeneous (>75% of cells) membrane staining. No amplification or polysomy of the EGFR gene or mutations of the tyrosine kinase domain of EGFR (exons 18-21) were detected. These results imply that although EGFR is expressed in most epithelioid sarcomas regardless of subtype, gene amplification and activating mutations in the tyrosine kinase domain appear to be rare or absent. Thus, the benefit of targeted therapy against EGFR in patients with epithelioid sarcoma remains to be determined.


Differential effect of 3-dimensional color Doppler echocardiography for the quantification of mitral regurgitation according to the severity and characteristics.
The aim of this study is to explore the differential effect of 3-dimensional color Doppler echocardiography for the quantification of mitral regurgitation (MR). Two-dimensional color Doppler echocardiography-based MR quantification has well-documented limitations. We consecutively enrolled 221 patients with MR. Adequate image quality was obtained by 2D- and 3D-color Doppler echocardiography in 211 (95.5%) patients. The quantitative differences between the MR volumes obtained by 2D- and 3D-proximal isovelocity surface area (PISA) were analyzed in various MR subgroups. In the validation cohort (n=52), MR volume obtained by 3D-PISA showed a better agreement with phase-contrast cardiac MRI than 2D-PISA (r=0.97 versus 0.84). In all 211 patients, 2D-PISA underestimated the MR volume when compared with 3D-PISA (52.4±19.6 versus 59.5±25.6 mL; P=0.005). A total of 33.3% with severe MR based on 3D-PISA were incorrectly assessed by 2D-PISA as having nonsevere MR. In the subgroup analysis, the MR severity (odds ratio, 6.96; 95% confidence interval, 3.04-15.94; P<0.001) and having an asymmetrical orifice (odds ratio, 11.48; 95% confidence interval, 3.72-35.4; P<0.001), and an eccentric jet (odds ratio, 3.82; 95% confidence interval, 1.27-11.48; P=0.017) were predictors of significant difference in MR volume (>15 mL) between 2D- and 3D-PISA methods. Quantification of MR by 3D-PISA method is clinically feasible and more accurate than the current 2D-PISA method. MR quantification by 2D-PISA significantly underestimated MR volume with severe, eccentric MR with an asymmetrical orifice. This article demonstrates that 3D-color Doppler echocardiography could be used as a valuable tool to confirm treatment strategy in patients with significant MR.


The development of one- and two-cell mouse embryos in the absence of human serum.
One- and two-cell embryos were obtained from F1 hybrid female mice stimulated with human menopausal gonadotrophin and randomly distributed into two groups--group 1 (no serum) and group 2 (10% patient's serum). Fifty of 53 (94.3%) one-cell embryos in group 1 had cleaved to the blastocyst stage and 44 of 49 (89.79%) in group 2 after 96 hours (no significant difference--chi-square test). In the two-cell embryos in group 1, 78 of 89 (87.6%) reached the blastocyst stage after 72 hours and 80 of 86 (93.02%) in group 2 (no significant difference--chi-square test). According to microscopic evaluation cleavage to the blastocyst stage without serum supplementation is possible. The value of serum is discussed.


Quantification of Boron Compound Concentration for BNCT Using Positron Emission Tomography.
Background: Boron neutron capture therapy requires a 2 mM 10B concentration in the tumor. The well-known BNCT patient treatment method using boronophenylalanine (BPA) as a boron-carrying agent utilizes [18F]fluoroBPA ([18F]FBPA) as an agent to qualify for treatment. Precisely, [18F]FBPA must have at least a 3:1 tumor to background tissue ratio to qualify the patient for BNCT treatment. Normal, hyperplasia, and cancer thyroids capture iodine and several other large ions, including BF4-, through a sodium-iodine symporter (NIS) expressed on the cell surface in normal conditions. In cancer, NIS is also expressed within the thyroid cell and is not functional. Methods: To visualize the thyroids and NIS, we have used a [18F]NaBF4 positron emission tomography (PET) tracer. It was injected into the tail veins of rats. The [18F]NaBF4 PET tracer was produced from NaBF4 by the isotopic exchange of natural 19F with radioactive 18F. Rats were subject to hyperplasia and tumor-inducing treatment. The NIS in thyroids was visualized by immunofluorescence staining. The boron concentration was calculated from Standard Uptake Values (SUV) in the PET/CT images and from the production data. Results: 41 MBq, 0.64 pmol of [18F]NaBF4 PET tracer that contained 0.351 mM, 53 nmol of NaBF4 was injected into the tail vein. After 17 min, the peak activity in the thyroid reached 2.3 MBq/mL (9 SUVmax). The natB concentration in the thyroid with hyperplasia reached 381 nM. Conclusions: Such an incorporation would require an additional 110 mg/kg dose of [10B]NaBF4 to reach the necessary 2 mM 10B concentration in the tumor. For future BNCT treatments of thyroid cancer, contrary to the 131I used now, there is no post-treatment radioactive decay, the patient can be immediately discharged from hospital, and there is no six-month moratorium for pregnancy. This method can be used for BNCT treatment compounds of the type R-BFn, where 1 <= n <= 3, labeled with 18F relatively easily, as in our example. A patient may undergo injection of a mixture of nonradioactive R-BFn to reach the necessary 10B concentration for BNCT treatment in the tumor together, with [18F]R-BFn for boron mapping.


Parents' Campaigns to Reduce their Children's Conduct Problems: Interactions with Temperamental Resistance to Control.
Longitudinal studies have found associations between parenting and the development of conduct problems, and have found that resistant to control temperament moderates these associations. Intervention studies have found associations between intervention-induced changes in parenting and subsequent reductions in children's conduct problems. However, no study to date has evaluated whether parents' self-initiated efforts to change their parenting practices affect children's conduct problems and whether effects depend on children's temperament. The current study asked whether parents' concerted efforts, or campaigns, to increase their involvement and limit-setting were effective in reducing growth in conduct problems from late childhood to early adolescence. It also asked whether the effects of campaigns varied according to children's levels of temperamental resistance to control. Analyses statistically controlled for parenting practices and conduct problems before the campaigns, socioeconomic status, gender, and ethnicity. Results indicated that campaigns that included increased involvement and limit-setting were beneficial only for youths who were rated in early childhood as temperamentally resistant to control.


Medical morbidity and mortality conferences: past, present and future.
Morbidity and mortality conferences (MMCs) have three potential aims-to improve patient safety by reducing adverse events and preventable deaths, to improve overall quality of care as part of the hospital governance structure and as educational learning events. At present, medical MMCs vary widely in format and attendance from hospital to hospital. The evidence for MMCs actually reducing adverse events and preventing avoidable deaths is disappointing. There is better evidence for their educational role. The majority of medical deaths in hospitals are frail older people with poor life expectancy in whom inadequate care is more likely to be due to errors of omission rather than commission. Medical MMCs should be multidisciplinary and led by a senior clinician to encourage discussion and reflection in a 'blame-free' environment. They should be learning events for both clinicians and the organisation as a whole with a structure to support this.


omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data.
Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.


p53-dependent BRCA1 nuclear export controls cellular susceptibility to DNA damage.
Subcellular localization regulates BRCA1 function, and BRCA1 is exported to the cytoplasm following DNA damage in a p53-dependent manner. Because more than 50% of solid tumors harbor p53 mutations, it is possible that genetically wild-type (wt) BRCA1 is functionally abnormal through compromised nuclear-cytoplasmic shuttling in sporadic breast cancer patients with dysfunctional p53. In this study, we have investigated the mechanisms of p53-dependent BRCA1 subcellular distribution and DNA damage-induced nuclear export, as well as the impact on the resulting cytotoxic response to therapy in human breast cancer. We first show that p53 mediates BRCA1 nuclear export via protein-protein binding, rather than by modulation of its transcription. Furthermore, it is the C-terminal (BRCT) region of BRCA1 that is critical for its interaction with p53, and p53 may promote BRCA1 nuclear export by interrupting the association of BRCA1 with BARD1. In sporadic breast cancer specimens, dysfunctional p53 strongly correlates with nuclear retention of sequence-verified wt BRCA1. This p53-dependent BRCA1 shuttling determines cellular susceptibility to DNA damage as augmentation of cytosolic BRCA1 significantly enhances cancer cell susceptibility to ionizing radiation. Taken together, our data suggest that p53 dysfunction compromises nuclear export of wt BRCA1 as a mechanism to increase cellular resistance to DNA damage in sporadic breast cancer. We propose that targeting nuclear BRCA1 to the cytoplasm may offer a unique strategy to sensitize p53-deficient sporadic breast cancers to DNA damage-based therapy.


Telehealth in Home-Based Primary Care: Factors and Challenges Associated With Integration Into Veteran Care.
To describe the structural characteristics and challenges associated with home telehealth (HT) use in the US Department of Veterans Affairs (VA) Home-Based Primary Care (HBPC) program. We designed a national survey to collect information about HBPC program structural characteristics. The survey included eight organizational and service domains, one of which was HT. HBPC program directors were surveyed online using REDCap. We received 232 surveys from 394 HBPC sites (59% response rate). HBPC structural domains were compared between sites using and not using HT technology. Open-ended responses were analyzed using content analysis. A total of 127 sites (76%) used HT, which was more likely when HBPC sites were aligned organizationally with the VA's Geriatrics and Extended Care Services division, when there were more disciplines on the HBPC team, and when primary care providers made home visits. Program directors overwhelmingly viewed HT as contributing to managing veterans' complex chronic conditions (81%), yet HT data were not readily integrated into care planning (24%). Challenges to HT use included veterans' acceptance and adherence, device issues, and collaboration between HBPC teams and HT staff. Corresponding to HBPC's complexity, HT use is primarily a self-organizing process that shapes the patterns of integration at each site. Although HT technology is compatible with core structures of the HBPC model, usability varies, and overall is low. To optimize HT use in HBPC, there are opportunities to redesign systems to mitigate challenges to adoption. As the Centers for Medicare and Medicaid Services' strives to increase access to both HBPC and telehealth benefits, evidenced by the continuation of its successful Independence at Home demonstration and the final changes in the proposed rule in April 2019 incorporating additional telehealth benefits for beneficiaries, this information will be relevant to VA and non-VA alike. J Am Geriatr Soc 67:1928-1933, 2019.


Calcium channel blockers versus other classes of drugs for hypertension.
Calcium channel blockers (CCBs) are a relatively new antihypertensive class. The effect of first-line CCBs on the prevention of cardiovascular events, as compared with other antihypertensive drug classes, is unknown. To determine whether CCBs used as first-line therapy for hypertension are different from other first-line drug classes in reducing the incidence of major adverse cardiovascular events. Electronic searches of the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASEand the WHO-ISH Collaboration Register (up to May 2009) were performed. We also checked the references of published studies to identify additional trials. Randomized controlled trial (RCT) comparing first-line CCBs with other antihypertensive classes, with at least 100 randomized hypertensive participants and with a follow-up of at least two years. Two authors independently selected the included trials, evaluated the risk of bias and entered the data for analysis. Eighteen RCTs (14 dihydropyridines, 4 non-dihydropyridines) with a total of 141,807 participants were included. All-cause mortality was not different between first-line CCBs and any other first-line antihypertensive classes. CCBs reduced the following outcomes as compared to beta-blockers: total cardiovascular events (RR 0.84, 95% CI [0.77, 0.92]), stroke (RR 0.77, 95% CI [0.67, 0.88]) and cardiovascular mortality (RR 0.90, 95% CI [0.81, 0.99]). CCBs increased total cardiovascular events (RR 1.05 , 95% CI [1.00, 1.09], p = 0.03) and congestive heart failure events (RR 1.37, 95% CI [1.25, 1.51]) as compared to diuretics. CCBs reduced stroke (RR 0.89, 95% CI [0.80, 0.98]) as compared to ACE inhibitors and reduced stroke (RR 0.85, 95% CI [0.73, 0.99]) and MI (RR 0.83, 95% CI [0.72, 0.96]) as compared to ARBs. CCBs also increased congestive heart failure events as compared to ACE inhibitors (RR 1.16, 95% CI [1.06, 1.27]) and ARBs (RR 1.20, 95% CI [1.06, 1.36]). The other evaluated outcomes were not significantly different. Diuretics are preferred first-line over CCBs to optimize reduction of cardiovascular events. The review does not distinguish between CCBs, ACE inhibitors or ARBs, but does provide evidence supporting the use of CCBs over beta-blockers. Many of the differences found in the current review are not robust and further trials might change the conclusions. More well-designed RCTs studying the mortality and morbidity of patients taking CCBs as compared with other antihypertensive drug classes are needed for patients with different stages of hypertension, different ages, and with different co-morbidities such as diabetes.


Genome Stability during Cell Proliferation: A Systems Analysis of the Molecular Mechanisms Controlling Progression through the Eukaryotic Cell Cycle.
Well-nourished cells in a favorable environment (well supplied with growth factors, cytokines, and/or hormones and free from stresses, ionizing radiation, etc.) will grow, replicate their genome, and divide into two daughter cells, fully prepared to repeat the process. This cycle of DNA replication and division underlies all aspects of biological growth, reproduction, repair and development. As such, it is essential that the cell's genome be guarded against damage during the replication/division process, lest the error(s) be irrevocably passed down to all future generations of progeny. Hence, cell cycle progression is closely guarded against major sources of errors, in particular DNA damage and misalignment of replicated chromosomes on the mitotic spindle. In this review article we examine closely the molecular mechanisms that maintain genomic integrity during the cell division cycle, and we find an unexpected and intriguing arrangement of concatenated and nested bistable toggle switches. The topology of the network seems to play crucial roles in maintaining the stability of the genome during cell proliferation.


Access to pain treatment as a human right.
Almost five decades ago, governments around the world adopted the 1961 Single Convention on Narcotic Drugs which, in addition to addressing the control of illicit narcotics, obligated countries to work towards universal access to the narcotic drugs necessary to alleviate pain and suffering. Yet, despite the existence of inexpensive and effective pain relief medicines, tens of millions of people around the world continue to suffer from moderate to severe pain each year without treatment. Significant barriers to effective pain treatment include: the failure of many governments to put in place functioning drug supply systems; the failure to enact policies on pain treatment and palliative care; poor training of healthcare workers; the existence of unnecessarily restrictive drug control regulations and practices; fear among healthcare workers of legal sanctions for legitimate medical practice; and the inflated cost of pain treatment. These barriers can be understood not only as a failure to provide essential medicines and relieve suffering but also as human rights abuses. According to international human rights law, countries have to provide pain treatment medications as part of their core obligations under the right to health; failure to take reasonable steps to ensure that people who suffer pain have access to adequate pain treatment may result in the violation of the obligation to protect against cruel, inhuman and degrading treatment.


Dietary effects on cardiovascular disease risk factors: beyond saturated fatty acids and cholesterol.
Hypercholesterolemia represents a significant risk for cardiovascular disease (CVD). While diet intervention remains the initial choice for the prevention and treatment of CVD, the nature of the dietary modification remains controversial. For example, reducing calories from total fat, without decreasing saturated fat intake results in insignificant changes in low density lipoprotein cholesterol (LDL-C). Similarly, diet interventions that focus solely on lowering dietary cholesterol and saturated fat intake not only decrease LDL-C, but also high density lipoprotein cholesterol (HDL-C) and therefore may not improve the lipoprotein profile. This brief review summarizes dietary interventions that lower LDL-C without affecting HDL-C levels. These interventions include soy protein, soluble fiber, soy lecithin and plant sterols. This review also includes some of the reported dietary interventions, such as polyphenols, isoflavones, folic acid and vitamins B6 and B12, which reduce the risk of CVD without changes in lipoprotein cholesterol.


MiRNA Transcriptome Profiling of Spheroid-Enriched Cells with Cancer Stem Cell Properties in Human Breast MCF-7 Cell Line.
Breast cancer is the second leading cause of cancer-related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome in spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p<0.05) differentially expressed miRNAs were identified between spheroids and parental cells using miRNA-NGS. MiRNA-NGS analysis revealed 25 up-regulated and 109 down-regulated miRNAs which includes some miRNAs previously reported in the regulation of breast CSCs. A number of miRNAs (miR-4492, miR-4532, miR-381, miR-4508, miR-4448, miR-1296, and miR-365a) which have not been previously reported in breast cancer were found to show potential association with breast cancer chemoresistance and self-renewal capability. The gene ontology (GO) analysis showed that the predicted genes were enriched in the regulation of metabolic processes, gene expression, DNA binding, and hormone receptor binding. The corresponding pathway analyses inferred from the GO results were closely related to the function of signalling pathway, self-renewability, chemoresistance, tumorigenesis, cytoskeletal proteins, and metastasis in breast cancer. Based on these results, we proposed that certain miRNAs identified in this study could be used as new potential biomarkers for breast cancer stem cell diagnosis and targeted therapy.


Efficacy of a miniature centrifugal rotary pump (TinyPump) for transfusion-free cardiopulmonary bypass in neonatal piglets.
We have developed a miniaturized semiclosed cardiopulmonary bypass (CPB) circuit incorporating a centrifugal blood pump (TinyPump) with a volume of 5 ml. The current study was undertaken to evaluate the hemolytic performance of the TinyPump in comparison with the BioPump and to investigate the impact of different CPB circuit volumes on hemodilution, coagulation, and the inflammatory response. Twelve 1-week-old piglets (3.4 +/- 0.2 kg) were used. The circuit comprised a centrifugal pump, a membrane oxygenator, and a cardiotomy reservoir. Cardiopulmonary bypass was conducted with mild hypothermia at 150 ml/kg/min for 3 hours. Transfusion was not performed. Priming volume was 68 ml for the circuit with the TinyPump and 111 ml for the circuit with the BioPump. Although the TinyPump required higher speed, plasma free hemoglobin levels after CPB were not different between the groups. After CPB, the TinyPump group had a significantly higher hematocrit (27% +/- 3% vs. 23% +/- 3%) and lower platelet reduction rate, lower thrombin-antithrombin complex levels, and lower interleukin-6 levels. Better lung compliance with less water content was observed in the TinyPump group. The TinyPump maintained CPB with acceptable hemolysis and lower inflammatory responses. This miniaturized CPB circuit may make transfusion-free open heart surgery feasible in neonates and would help to prevent postoperative organ dysfunction.


Simulations reveal the role of composition into the atomic-level flexibility of bioactive glass cements.
Bioactive glass ionomer cements (GICs), the reaction product of a fluoro-alumino-silicate glass and polyacrylic acid, have been in effective use in dentistry for over 40 years and more recently in orthopaedics and medical implantation. Their desirable properties have affirmed GIC's place in the medical materials community, yet are limited to non-load bearing applications due to the brittle nature of the hardened composite cement, thought to arise from the glass component and the interfaces it forms. Towards helping resolve the fundamental bases of the mechanical shortcomings of GICs, we report the 1st ever computational models of a GIC-relevant component. Ab initio molecular dynamics simulations were employed to generate and characterise three fluoro-alumino-silicate glasses of differing compositions with focus on resolving the atomic scale structural and dynamic contributions of aluminium, phosphorous and fluorine. Analyses of the glasses revealed rising F-content leading to the expansion of the glass network, compression of Al-F bonding, angular constraint at Al-pivots, localisation of alumino-phosphates and increased fluorine diffusion. Together, these changes to the structure, speciation and dynamics with raised fluorine content impart an overall rigidifying effect on the glass network, and suggest a predisposition to atomic-level inflexibility, which could manifest in the ionomer cements they form.


Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR.
We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.


Mechanisms of endothelial dysfunction in rheumatoid arthritis: lessons from animal studies.
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by articular and extra-articular manifestations involving cardiovascular diseases (CVDs), which account for 30% to 50% of all deaths. In patients with RA, atherosclerosis lesions occur earlier and have a more rapid evolution than in the general population. Beyond mortality, the impact of CVD on quality of life, combined with the associated increase in health-care costs, renders CVD in RA a major public health problem. Recent studies showed that patients with RA are characterized by the presence of endothelial dysfunction (ED), which is recognized as a key event in the development of atherosclerosis. By definition, ED is a functional and reversible alteration of endothelial cells, leading to a shift of the actions of the endothelium toward reduced vasodilation, proinflammatory state and proliferative and prothrombotic properties. Although the improvement of endothelial function is becoming an important element of the global management of patients with RA, the mechanistic determinants of ED in RA are still poorly understood. Animal models of RA provide the unique opportunity to unravel the pathophysiological features of ED in RA. The present review summarizes the available data on mechanisms underlying ED in animal models of RA and proposes attractive prospects in order to discover novel therapeutic strategies of RA-associated ED.


Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.
Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.


Occurrence of Tomato yellow leaf curl virus in Tomato in Martinique, Lesser Antilles.
During April of 2002, symptoms of stunting and chlorotic curled leaves of reduced size, similar to those caused by Tomato yellow leaf curl virus (TYLCV), were observed for the first time in commercial tomato (Solanum lycopersicum) in the northwest region of Martinique. Six months later, many tomato fields had more than 80% of plants expressing these symptoms and yield was drastically reduced. Samples from two symptomatic plants were collected and analyzed by PCR. Primers PC1 (5'-TGACTATGTCGAAGCGACCAGG-3') and PC2 (5'-CGACATTACAGCCTCAGACTGG-3') were used to amplify a 950-bp fragment within the coat protein gene (CP) of TYLCV species (1). Primer pair MP16-MP82 (2) amplified a 550-bp fragment from the conserved nonanucleotide sequence (TAATATTAC) to the 5' end of the CP gene. Products of expected sizes were obtained with both pairs of primers from symptomatic samples but not from uninfected ones. The two overlapping PCR products were cloned into a pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. A BLAST analysis was conducted with begomovirus sequences available in the GenBank database at the NCBI, and DNAMAN software (Lynnon Corporation, Quebec, Canada) was used for further comparisons. The 1275-bp sequence (GenBank Accession No. EF490995) shared 99% nucleotide identity with the partial sequences of TYLCV from Antigua and Barbuda (GenBank Accession No. EF028240), Saint Kitts and Nevis (GenBank Accession No. EF028239), and the two overlapping sequences from Guadeloupe (GenBank Accessions No. AY319645 and AY319646). It was at least 98% identical to TYLCV isolates from Florida (GenBank Accession No AY530931), Dominican Republic (GenBank Accession No. AF024715), and Cuba (GenBank Accession No. AJ223505). These results confirm the introduction of TYLCV into Martinique, possibly from a nearby Caribbean country, and reveal its southward spread in the Lesser Antilles. The nearness of the islands in the Lesser Antilles (20 to 100 km distant) probably permitted the rapid spread of TYLCV through the movement of plant material or wind transport of viruliferous whiteflies from one island to the next. Monitoring the spread of TYLCV in this Caribbean archipelago is important for regional virus management and in forecasting the spread of TYLCV to nearby countries in South America. References: (1) Y. Martinez et al. Rev. Prot. Veg. 18:168, 2003. (2) P. Umaharan et al. Phytopathology 88:1262, 1998.


Balancing the decision of cell proliferation and cell fate.
The control of cellular proliferation is key in the proper development of a complex organism, the maintenance of tissue homeostasis and the ability to respond to various hormonal and other inducers. Key in the control of proliferation is the retinoblastoma (Rb) protein which regulates the activity of a family of transcription factors known as E2Fs. The E2F proteins are now recognized to regulate the expression of a large number of genes associated with cell proliferation including genes encoding DNA replication as well as mitotic activities. What has also become clear over the past several years is the intimate relationship between the control of cell proliferation and the control of cell fate, particularly the activation of apoptotic pathways. Central in this connection is the Rb/E2F pathway that not only provides the primary signals for proliferation but at the same time, connects with the p53-dependent apoptotic pathway. This review addresses this inter-connection and the molecular mechanisms that control the decision between proliferation and cell death.


Store-operated Ca2+ entry controls ameloblast cell function and enamel development.
Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.


Inhibition of atherosclerosis associated with reduction of arterial intramural stress in rabbits.
Atherosclerotic lesions commonly develop at arterial branch sites, which are also the sites of high arterial intramural stress produced by intraluminal pressure. We investigated the effect of reduced intramural stress on the development of atherosclerotic lesions. We exposed the origin of the left renal artery in five rabbits and the aortic bifurcation in another five, lowered the mean arterial pressure to 35 to 45 mm Hg, and poured a dental acrylic liquid around the branch to form a rigid cast. When the rabbits recovered and the arterial pressure increased to normal, the casts prevented the arteries from expanding, thereby maintaining a low intramural stress. These rabbits plus two unoperated, two sham-operated, two with silicone rubber casts placed at similar pressures, and four with casts placed at 95 mm Hg pressure were given a 2% cholesterol-enriched diet for 7 to 11 weeks, and then their arteries were examined. In all rabbits, atherosclerotic lesions developed at the origins of the intercostal, celiac, superior mesenteric, and both renal arteries, and at the aortic bifurcation, with these notable exceptions: no lesions developed at the origins of casted renal arteries or at the casted aortic bifurcations when the cast was placed at a low pressure. Measurements of the diameter and thickness of the aorta in the left renal branch and aortic bifurcation areas, with and without the casts, indicated that there was no significant narrowing of the aortic lumen or thinning of the aorta due to the cast. In conclusion, the inhibition of the development of atherosclerotic lesions appears to be associated with the reduction of arterial intramural stress.


Latin American immigrants have limited access to health insurance in Japan: a cross sectional study.
Japan provides universal health insurance to all legal residents. Prior research has suggested that immigrants to Japan disproportionately lack health insurance coverage, but no prior study has used rigorous methodology to examine this issue among Latin American immigrants in Japan. The aim of our study, therefore, was to assess the pattern of health insurance coverage and predictors of uninsurance among documented Latin American immigrants in Japan. We used a cross sectional, mixed method approach using a probability proportional to estimated size sampling procedure. Of 1052 eligible Latin American residents mapped through extensive fieldwork in selected clusters, 400 immigrant residents living in Nagahama City, Japan were randomly selected for our study. Data were collected through face-to-face interviews using a structured questionnaire developed from qualitative interviews. Our response rate was 70.5% (n = 282). Respondents were mainly from Brazil (69.9%), under 40 years of age (64.5%) and had lived in Japan for 9.45 years (SE 0.44; median, 8.00). We found a high prevalence of uninsurance (19.8%) among our sample compared with the estimated national average of 1.3% in the general population. Among the insured full time workers (n = 209), 55.5% were not covered by the Employee's Health Insurance. Many immigrants cited financial trade-offs as the main reasons for uninsurance. Lacking of knowledge that health insurance is mandatory in Japan, not having a chronic disease, and having one or no children were strong predictors of uninsurance. Lack of health insurance for immigrants in Japan is a serious concern for this population as well as for the Japanese health care system. Appropriate measures should be taken to facilitate access to health insurance for this vulnerable population.


Systemic therapy for hepatocellular carcinoma.
The prognosis is poor for patients with advanced hepatocellular carcinoma (HCC). Sorafenib is the only accepted standard of care for advanced disease. The benefits of this agent are modest and the precise mechanism of antitumor activity in HCC is unknown. Since the approval of sorafenib, there has been intense investigation into strategies that block angiogenic pathways. Unfortunately, the results of three randomized phase III trials that compared newer anti-angiogenic treatments to sorafenib failed to demonstrate their superiority or non-inferiority. Thus, there remains a critical need for both continued molecular characterization and aggressive drug development in hepatocellular carcinoma.


EMF radiation at 2450 MHz triggers changes in the morphology and expression of heat shock proteins and glucocorticoid receptors in rat thymus.
Electromagnetic fields (EMFs) can act as inducers or mediators of stress response through the production of heat shock proteins (HSPs) that modulate immune response and thymus functions. In this study, we analyzed cellular stress levels in rat thymus after exposure of the rats to a 2.45 GHz radio frequency (RF) using an experimental diathermic model in a Gigahertz Transverse Electromagnetic (GTEM) chamber. In this experiment, we used H&E staining, the ELISA test and immunohistochemistry to examine Hsp70 and Hsp90 expression in the thymus and glucocorticoid receptors (GR) of 64 female Sprague–Dawley rats exposed individually to 2.45 GHz (at 0, 1.5, 3.0 or 12.0 W power). The 1 g averaged peak and mean SAR values in the thymus and whole body of each rat to ensure that sub-thermal levels of radiation were being reached. The thymus tissue presented several morphological changes, including increased distribution of blood vessels along with the appearance of red blood cells and hemorrhagic reticuloepithelial cells. Levels of Hsp90 decreased in the thymus when animals were exposed to the highest power level (12 W), but only one group did not show recovery after 24 h. Hsp70 presented no significant modifications in any of the groups. The glucocorticoid receptors presented greater immunomarking on the thymic cortex in exposed animals. Our results indicate that non-ionizing sub-thermal radiation causes changes in the endothelial permeability and vascularization of the thymus, and is a tissue-modulating agent for Hsp90 and GR.


Socio-economic status, family disruption and residential stability in childhood: relation to onset, recurrence and remission of major depression.
Childhood adversity significantly increases the risk of depression, but it is unclear whether this risk is most pronounced for depression occurring early in life. In the present study, we examine whether three aspects of childhood adversity--low socio-economic status (SES), family disruption, and residential instability--are related to increased risk of depression during specific stages of the life course. We also examine whether these aspects of childhood adversity are related to the severity of depression. A sample of 1089 of the 4140 births enrolled in the Providence, Rhode Island cohort of the National Collaborative Perinatal Project was interviewed between the ages of 18 and 39. Measures of parental SES, childhood family disruption and residential instability were obtained upon mother's enrolment and at age 7. Age at onset of major depressive episode, lifetime number of depressive episodes, and age at last episode were ascertained via structured diagnostic interviews. Survival analysis was used to identify risk factors for depression onset and remission and Poisson regression was used to model the recurrence rate of depressive episodes. Low parental SES, family disruption and a high level of residential instability, defined as three or more family moves, were related to elevated lifetime risks of depression; the effects of family disruption and residential instability were most pronounced on depression onset by age 14. Childhood adversity was also related to increased risk of recurrence and reduced likelihood of remission. Childhood social disadvantage significantly influences risk of depression onset both in childhood and in adulthood. Early childhood adversity is also related to poor prognosis.


Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.


Immune Profile in Blood Following Non-convulsive Epileptic Seizures in Rats.
Non-convulsive status epilepticus (NCSE) is a prolonged epileptic seizure with subtle symptoms that may delay clinical diagnosis. Emerging experimental evidence shows brain pathology and epilepsy development following NCSE. New diagnostic/prognostic tools are therefore needed for earlier and better stratification of treatment. Here we examined whether NCSE initiates a peripheral immune response in blood serum from rats that experienced electrically-induced NCSE. ELISA analysis showed an acute transient increase in serum protein levels including interleukin-6 6 h post-NCSE, similar to the immune reaction in the brain. At 4 weeks post-NCSE, when 75% of rats subjected to NCSE had also developed spontaneous seizures, several immune proteins were altered. In particular, markers associated with microglia, macrophages and antigen presenting cells, such as CD68, MHCII, and galectin-3, were increased and the T-cell marker CD4 was decreased in serum compared to both non-stimulated controls and NCSE rats without spontaneous seizures, without correlation to interictal epileptiform activity. Analyses of serum following intracerebral injection of lipopolysaccharide (LPS) showed an acute increase in interleukin-6, but at 4 weeks unaltered levels of MHCII and galectin-3, an increase in CD8 and CD11b and a decrease in CD68. None of the increased serum protein levels after NCSE or LPS could be confirmed in spleen tissue. Our data identifies the possibility to detect peripheral changes in serum protein levels following NCSE, which may be related to the development of subsequent spontaneous seizures.


Role of Dynamics in the Autoinhibition and Activation of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) Ion Channels.
The hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating.


The MomConnect helpdesk: how an interactive mobile messaging programme is used by mothers in South Africa.
South Africa's MomConnect mobile messaging programme, which aims to promote safe motherhood and improve pregnancy outcomes for South African women, includes a helpdesk feature which allows women registered on the system to ask maternal and child health (MCH)-related questions and to provide feedback on health services received at public health clinics. Messages sent to the helpdesk are answered by staff located at the National Department of Health. We examined event data from the MomConnect helpdesk database to identify any patterns in messages received, such as correlation of frequency or types of messages with location. We also explored what these data could tell us about the helpdesk's effectiveness in improving health service delivery at public health clinics. We found that approximately 8% of registered MomConnect users used the helpdesk, and that usage was generally proportional to the use of antenatal care (ANC) services in provinces (as indicated by number of ANC first visits and number of MomConnect registrations), except in two provinces. Language, category and key topics of helpdesk messages were correlated with provinces. Most users accessed the helpdesk to seek maternal information, and where feedback about health services was provided, there were significantly more compliments than complaints. The MomConnect helpdesk is an important resource providing expectant mothers and mothers of infants with an interactive option for accessing MCH-related information-above that included in the standard MomConnect messages-and advances achievement of the health goals of the MomConnect programme.


Genome Wide Approaches to Identify Protein-DNA Interactions.
Transcription factors are DNA-binding proteins that play key roles in many fundamental biological processes. Unraveling their interactions with DNA is essential to identify their target genes and understand the regulatory network. Genome-wide identification of their binding sites became feasible thanks to recent progress in experimental and computational approaches. ChIP-chip, ChIP-seq, and ChIP-exo are three widely used techniques to demarcate genome-wide transcription factor binding sites. This review aims to provide an overview of these three techniques including their experiment procedures, computational approaches, and popular analytic tools. ChIP-chip, ChIP-seq, and ChIP-exo have been the major techniques to study genome- wide in vivo protein-DNA interaction. Due to the rapid development of next-generation sequencing technology, array-based ChIP-chip is deprecated and ChIP-seq has become the most widely used technique to identify transcription factor binding sites in genome-wide. The newly developed ChIP-exo further improves the spatial resolution to single nucleotide. Numerous tools have been developed to analyze ChIP-chip, ChIP-seq and ChIP-exo data. However, different programs may employ different mechanisms or underlying algorithms thus each will inherently include its own set of statistical assumption and bias. So choosing the most appropriate analytic program for a given experiment needs careful considerations. Moreover, most programs only have command line interface so their installation and usage will require basic computation expertise in Unix/Linux.


BMP-2/β-TCP Local Delivery for Bone Regeneration in MRONJ-Like Mouse Model.
Medication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (β-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/β-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/β-TCP transplanted group. For treatment studies, BMP-2/β-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/β-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/β-TCP could become a suitable therapy for the management of MRONJ.


Cooperation and assortativity with dynamic partner updating.
The natural tendency for humans to make and break relationships is thought to facilitate the emergence of cooperation. In particular, allowing conditional cooperators to choose with whom they interact is believed to reinforce the rewards accruing to mutual cooperation while simultaneously excluding defectors. Here we report on a series of human subjects experiments in which groups of 24 participants played an iterated prisoner's dilemma game where, critically, they were also allowed to propose and delete links to players of their own choosing at some variable rate. Over a wide variety of parameter settings and initial conditions, we found that dynamic partner updating significantly increased the level of cooperation, the average payoffs to players, and the assortativity between cooperators. Even relatively slow update rates were sufficient to produce large effects, while subsequent increases to the update rate had progressively smaller, but still positive, effects. For standard prisoner's dilemma payoffs, we also found that assortativity resulted predominantly from cooperators avoiding defectors, not by severing ties with defecting partners, and that cooperation correspondingly suffered. Finally, by modifying the payoffs to satisfy two novel conditions, we found that cooperators did punish defectors by severing ties, leading to higher levels of cooperation that persisted for longer.


Evolution of polyketide synthesis in a Dothideomycete forest pathogen.
Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.


Altered neuroendocrine regulation of gonadotropin secretion in women distance runners.
We tested the hypothesis that the neuroendocrine control of gonadotropin secretion is altered in certain women distance runners with secondary amenorrhea. To this end, we quantitated the frequency and amplitude of spontaneous pulsatile LH secretion during a 24-h interval in nine such women. The ability of the pituitary gland to release LH normally was assessed by administration of graded bolus doses of GnRH during the subsequent 8 h. Compared to normally menstruating women, six of nine amenorrheic distance runners had a distinct reduction in spontaneous LH pulse frequency, with one, three, six, five, four, or two pulses per 24 h (normal, 8-15 pulses/24 h). This reduction in LH pulse frequency occurred without any significant alterations in plasma concentrations of estradiol and free testosterone or 24-h integrated serum concentrations of LH, FSH, or PRL. Moreover, in long-distance runners, the capacity of the pituitary gland to release LH was normal or accentuated in response to exogenous pulses of GnRH. In the six women athletes with diminished spontaneous LH pulsatility, acute ovarian responsiveness also was normal, since serum estradiol concentrations increased normally in response to the GnRH-induced LH pulses. Although long-distance runners had significantly lower estimated percent body fat compared to control women, specific changes in pulsatile gonadotropin release did not correlate with degree of body leanness. In summary, certain long-distance runners with secondary amenorrhea or severe oligomenorrhea have unambiguously decreased spontaneous LH pulse frequency with intact pituitary responsiveness to GnRH. This neuroendocrine disturbance may be relevant to exercise-associated amenorrhea, since pulsatile LH release is a prerequisite for cyclic ovarian function. We speculate that such alterations in pulsatile LH release in exercising women reflect an adaptive response of the hypothalamic pulse generator controlling the intermittent GnRH signal to the pituitary gland. The basis for amenorrhea in the remaining runners who have normal pulsatile properties of LH release is not known.


Modelling the impact of a combined varicella and zoster vaccination programme on the epidemiology of varicella zoster virus in England.
This study updates previous work on modelling the incidence of varicella and Herpes Zoster (HZ) following the introduction of childhood vaccination. The updated model includes new data on age-specific contact patterns, as well as data on the efficacy of zoster vaccination in the elderly and allows for HZ among vaccinees. The current study also looks at two-dose varicella childhood programmes, and assesses the combined impact of varicella vaccination in childhood and zoster vaccination of the elderly. The results suggest that a two-dose schedule is likely to reduce the incidence of varicella to very low levels, provided first dose coverage is around 90% and second dose coverage is in excess of 70%. Single dose varicella vaccination programmes are expected to result in large numbers of breakthrough cases. Childhood vaccination is expected to increase the incidence of zoster for more than 40 years after introduction of the programme, the magnitude of this increase being influenced primarily by the duration of boosting following exposure to the varicella zoster virus. Though this increase in zoster incidence can be partly offset by vaccination of the elderly, the effectiveness of this combined strategy is limited, as much of the increase occurs in those adults too young to be vaccinated. Childhood vaccination at intermediate levels of coverage (70% and 60% for first and second dose coverage respectively) is expected to lead to an increase in adult varicella. At high coverage (90% and 80% coverage) this is unlikely to be the case. These results will be used to inform a cost-effectiveness analysis of combined varicella and zoster vaccination programmes.


Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress.
Chronic psychological stress, including water avoidance stress (WAS), induces intestinal mucosal barrier dysfunction and impairs mucosal defences against luminal bacteria. The aim of this study was to determine the ability of a defined probiotic regimen to prevent WAS induced intestinal pathophysiology. Male rats were subjected to either WAS or sham stress for one hour per day for 10 consecutive days. Additional animals received seven days of Lactobacillus helveticus and L rhamnosus in the drinking water prior to stress and remained on these probiotics for the duration of the study. Rats were then sacrificed, intestinal segments assessed in Ussing chambers, and mesenteric lymph nodes cultured to determine bacterial translocation. All animals remained healthy for the duration of the study. Chronic WAS induced excess ion secretion (elevated baseline short circuit current) and barrier dysfunction (increased conductance) in both the ileum and colon, associated with increased bacterial adhesion and penetration into surface epithelial cells. Approximately 70% of rats subjected to WAS had bacterial translocation to mesenteric lymph nodes while there was no bacterial translocation in controls. Probiotic pretreatment alone had no effect on intestinal barrier function. However, WAS induced increased ileal short circuit current was reduced with probiotics whereas there was no impact on altered conductance. Pretreatment of animals with probiotics also completely abrogated WAS induced bacterial adhesion and prevented translocation of bacteria to mesenteric lymph nodes. These findings indicate that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract.


Monitoring and optimization of the microcirculation during CPB.
In this review the impact of cardiopulmonary bypass (CPB) on the microcirculation is discussed. The impact of conventional non-pulsatile perfusion is contrasted with the microcirculatory impact of pulsatile CPB which is associated with better preservation of microcirculatory function. Evidence that alterations in microcirculatory function that persist following cardiac surgery are associated with adverse clinical outcomes is reviewed. Recent studies using novel techniques employing near infrared reflectance spectroscopy (NIRS) and sublingual microscopy to explore alterations in microcirculatory function during CPB are reviewed and the implications of these observations for studies investigating minimally invasive extracorporeal circulation (MiECC) are discussed.


Dermatologic emergencies.
Life-threatening dermatologic conditions include Rocky Mountain spotted fever; necrotizing fasciitis; toxic epidermal necrolysis; and Stevens-Johnson syndrome. Rocky Mountain spotted fever is the most common rickettsial disease in the United States, with an overall mortality rate of 5 to 10 percent. Classic symptoms include fever, headache, and rash in a patient with a history of tick bite or exposure. Doxycycline is the first-line treatment. Necrotizing fasciitis is a rapidly progressive infection of the deep fascia, with necrosis of the subcutaneous tissues. It usually occurs after surgery or trauma. Patients have erythema and pain out of proportion to the physical findings. Immediate surgical debridement and antibiotic therapy should be initiated. Stevens-Johnson syndrome and toxic epidermal necrolysis are acute hypersensitivity cutaneous reactions. Stevens-Johnson syndrome is characterized by target lesions with central dusky purpura or a central bulla. Toxic epidermal necrolysis is a more severe reaction with full-thickness epidermal necrosis and exfoliation. Most cases of Stevens-Johnson syndrome and toxic epidermal necrolysis are drug induced. The causative drug should be discontinued immediately, and the patient should be hospitalized for supportive care.


Challenges in realizing a self-contained hydraulically-driven contractile fiber actuator.
The field of soft robots would benefit from electrically controlled contractile actuators in the form of fibers that achieve a strain of 20% in less than a second while exerting high force. This work explores possible designs for achieving this goal using self-contained electroosmotic fluid pumping within a tube-shaped structure. The most promising configuration is a combination of a bellows and a McKibben-type muscle, since pumping fluid from the former to the latter results in contraction of both portions. Realizing such a device entails challenges in fabrication and electrokinetic fluid pumping in closed systems. Further studies of electroosmotic flow in salt-free organic solvents are needed.


Early hospital discharge of children with cancer treated for fever and neutropenia: identification and management of the low-risk patient.
Children with leukemia and solid tumors are often hospitalized for empiric broad-spectrum antibiotic therapy because of fever during periods of chemotherapy-induced neutropenia. Conventional practice dictates that parenteral antibiotics be continued until the patient is afebrile and has recovered from neutropenia, ie, until the absolute neutrophil count (ANC) exceeds 500 cells per cubic millimeter. However, the practice in our center has been to discontinue parenteral antibiotic therapy and discharge many such patients before resolution of neutropenia. Since the feasibility and safety of this approach has not been studied, we reviewed the records of 114 consecutive hospitalizations for fever and neutropenia in 61 patients during a 13-month period. Seventy-seven children (68%) were discharged to their homes while still neutropenic after they had been afebrile for 1 to 2 days on parenteral antibiotics, had negative blood cultures, appeared well, and usually had some evidence of bone marrow recovery. Five patients (4.4%) developed recurrent fever and required rehospitalization within 7 days of discharge. Only three of the 77 patients (3.9%) who were sent home with neutropenia had recurrent fever. Each had a brief and uneventful second hospitalization. Two of the 37 children discharged with an ANC over 500 cells per cubic millimeter required rehospitalization. A declining ANC and advanced malignancy were risk factors in predicting recurrence of fever following discharge. A rising monocyte count was a predictor of imminent recovery from neutropenia. These results suggest that "early" discharge of an afebrile yet still neutropenic patient is safe when the patient is in remission, has no evidence of serious infection, appears clinically stable, and has indications of bone marrow recovery. The conventional approach of routinely continuing the hospitalization until resolution of neutropenia may be unnecessary in such low-risk patients.


Science, technology, engineering, and mathematics (STEM) participation among college students with an autism spectrum disorder.
Little research has examined the popular belief that individuals with an autism spectrum disorder (ASD) are more likely than the general population to gravitate toward science, technology, engineering, and mathematics (STEM) fields. This study analyzed data from the National Longitudinal Transition Study-2, a nationally representative sample of students with an ASD in special education. Findings suggest that students with an ASD had the highest STEM participation rates although their college enrollment rate was the third lowest among 11 disability categories and students in the general population. Disproportionate postsecondary enrollment and STEM participation by gender, family income, and mental functioning skills were found for young adults with an ASD. Educational policy implications are discussed.


Red Blood Cell Transfusion and Postoperative Infection in Patients Having Coronary Artery Bypass Grafting Surgery: An Analysis of the Society of Thoracic Surgeons Adult Cardiac Surgery Database.
Coronary artery bypass grafting (CABG) is the most common cardiac surgical procedure in the world and up to one-third of patients are transfused red blood cells (RBCs). RBC transfusion may increase the risk for health care-associated infection (HAI) after CABG, but previous studies have shown conflicting results and many did not establish exposure temporality. Our objective was to explore whether intraoperative RBC transfusion is associated with increased odds of postoperative HAI. We hypothesized that intraoperative RBC transfusion would be associated with increased odds of postoperative HAI. We performed an observational cohort study of isolated CABG patients in the Society of Thoracic Surgeons adult cardiac surgery database from July 1, 2017, to June 30, 2019. The exposure was intraoperative RBC transfusion modeled as 0, 1, 2, 3, or 4+ units. The authors focused on intraoperative RBC transfusion as a risk factor, because it has a definite temporal relationship before postoperative HAI. The study's primary outcome was a composite HAI variable that included sepsis, pneumonia, and surgical site infection (both deep and superficial). Mixed-effects modeling, which controlled for hospital as a clustering variable, was used to explore the relationship between intraoperative RBC transfusion and postoperative HAI. Among 362,954 CABG patients from 1076 hospitals included in our analysis, 59,578 patients (16.4%) received intraoperative RBCs and 116,186 (32.0%) received either intraoperative or postoperative RBCs. Risk-adjusted odds ratios for HAI in patients who received 1, 2, 3, and 4+ intraoperative RBCs were 1.11 (95% confidence interval [CI], 1.03-1.20; P = .005), 1.13 (95% CI, 1.05-1.21; P = .001), 1.15 (95% CI, 1.04-1.27; P = .008), and 1.14 (95% CI, 1.02-1.27; P = .02) compared to patients who received no RBCs. Intraoperative RBC transfusion is associated with a small increase in odds of HAI in CABG patients. Future studies should explore whether reductions in RBC transfusion can also reduce HAIs.


Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.
The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.


Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain.
The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.


Selective or predominant triceps muscle weakness in African-American patients with myasthenia gravis.
Myasthenia gravis (MG) can lead to weakness in different patterns of muscle groups. Limb muscle weakness is most typically seen in a limb girdle pattern, although variants exist. In the current study, we aimed to describe a unique MG phenotype consisting of selective or predominant triceps muscle weakness. We performed a retrospective review of MG patients who developed focal or predominant triceps muscle weakness between 2006 and 2016. The clinical, electrophysiological and serological characteristics of these patients were examined. 8 MG patients were identified, including 7 males, all of whom were African-American. Two patients underwent muscle biopsy, and one patient underwent cervical spine decompression surgery. All showed significant improvement following immunosuppressive treatment, although one patient experienced a relapse of muscle weakness. This case series highlights a relatively uncommon MG clinical phenotype of selective triceps muscle weakness, mainly in African-American males, in line with previous literature. Familiarity with this phenotype is important in order to facilitate diagnosis and appropriate treatment for this group, and avoid unnecessary investigations or treatments.


Analysis of heterologous and homologous promoters and enhancers in vitro and in vivo by gene transfer into Japanese medaka (Oryzias latipes) Xiphophorus.
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a number of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish cell lines and in embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoter-enhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. Fro the data obtained, a set of expression vectors for basic research as well as for aquaculture purposes were established.


Effect of a behaviour-change intervention on handwashing with soap in India (SuperAmma): a cluster-randomised trial.
Diarrhoea and respiratory infections are the two biggest causes of child death globally. Handwashing with soap could substantially reduce diarrhoea and respiratory infections, but prevalence of adequate handwashing is low. We tested whether a scalable village-level intervention based on emotional drivers of behaviour, rather than knowledge, could improve handwashing behaviour in rural India. The study was done in Chittoor district in southern Andhra Pradesh, India, between May 24, 2011, and Sept 10, 2012. Eligible villages had a population of 700-2000 people, a state-run primary school for children aged 8-13 years, and a preschool for children younger than 5 years. 14 villages (clusters) were selected, stratified by population size (<1200 vs >1200), and randomly assigned in a 1:1 ratio to intervention or control (no intervention). Clusters were enrolled by the study manager. Random allocation was done by the study statistician using a random number generator. The intervention included community and school-based events incorporating an animated film, skits, and public pledging ceremonies. Outcomes were measured by direct observation in 20-25 households per village at baseline and at three follow-up visits (6 weeks, 6 months, and 12 months after the intervention). Observers had no connection with the intervention and observers and participant households were told that the study was about domestic water use to reduce the risk of bias. No other masking was possible. The primary outcome was the proportion of handwashing with soap at key events (after defecation, after cleaning a child's bottom, before food preparation, and before eating) at all follow-up visits. The control villages received a shortened version of the intervention before the final follow-up round. Outcome data are presented as village-level means. Handwashing with soap at key events was rare at baseline in both the intervention and control groups (1% [SD 1] vs 2% [1]). At 6 weeks' follow-up, handwashing with soap at key events was more common in the intervention group than in the control group (19% [SD 21] vs 4% [2]; difference 15%, p=0·005). At the 6-month follow-up visit, the proportion handwashing with soap was 37% (SD 7) in the intervention group versus 6% (3) in the control group (difference 31%; p=0·02). At the 12-month follow-up visit, after the control villages had received the shortened intervention, the proportion handwashing with soap was 29% (SD 9) in the intervention group and 29% (13) in the control group. This study shows that substantial increases in handwashing with soap can be achieved using a scalable intervention based on emotional drivers. Wellcome Trust, SHARE.


Characterization of pathogenic SORL1 genetic variants for association with Alzheimer's disease: a clinical interpretation strategy.
Accumulating evidence suggests that genetic variants in the SORL1 gene are associated with Alzheimer disease (AD), but a strategy to identify which variants are pathogenic is lacking. In a discovery sample of 115 SORL1 variants detected in 1908 Dutch AD cases and controls, we identified the variant characteristics associated with SORL1 variant pathogenicity. Findings were replicated in an independent sample of 103 SORL1 variants detected in 3193 AD cases and controls. In a combined sample of the discovery and replication samples, comprising 181 unique SORL1 variants, we developed a strategy to classify SORL1 variants into five subtypes ranging from pathogenic to benign. We tested this pathogenicity screen in SORL1 variants reported in two independent published studies. SORL1 variant pathogenicity is defined by the Combined Annotation Dependent Depletion (CADD) score and the minor allele frequency (MAF) reported by the Exome Aggregation Consortium (ExAC) database. Variants predicted strongly damaging (CADD score >30), which are extremely rare (ExAC-MAF <1 × 10-5) increased AD risk by 12-fold (95% CI 4.2-34.3; P=5 × 10-9). Protein-truncating SORL1 mutations were all unknown to ExAC and occurred exclusively in AD cases. More common SORL1 variants (ExAC-MAF≥1 × 10-5) were not associated with increased AD risk, even when predicted strongly damaging. Findings were independent of gender and the APOE-ɛ4 allele. High-risk SORL1 variants were observed in a substantial proportion of the AD cases analyzed (2%). Based on their effect size, we propose to consider high-risk SORL1 variants next to variants in APOE, PSEN1, PSEN2 and APP for personalized risk assessments in clinical practice.


CoLoC-seq probes the global topology of organelle transcriptomes.
Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.


Direct antiproliferative effects of melatonin on two metastatic cell sublines of mouse melanoma (B16BL6 and PG19).
The effects of melatonin on the growth of two highly tumorigenic rodent melanoma cells were studied in vitro. PG19, an amelanotic mouse melanoma cell line, and B16BL6, a melanotic melanoma cell line selected for its invasive potential in vitro, were cultured in the presence of different concentrations of melatonin (10 microM to 0.1 pM). Five days later, viable cells were determined in a haemocytometer by the trypan blue exclusion test. Melatonin at concentrations of 1 nM and 10 pM (within the range of concentrations that correspond to physiological night-time and daytime levels in human blood) significantly inhibited proliferation in both melanoma cell lines. Subphysiological (0.1 pM) or supraphysiological (10 microM to 100 nM) concentrations of melatonin lacked this effect. These results support the hypothesis that, at physiological concentrations, melatonin exerts a direct inhibitory effect on PG19 and B16BL6 cells proliferation.


Fold recognition using predicted secondary structure sequences and hidden Markov models of protein folds.
We present an analysis of the blind predictions submitted to the fold recognition category for the second meeting on the Critical Assessment of techniques for protein Structure Prediction. Our method achieves fold recognition from predicted secondary structure sequences using hidden Markov models (HMMs) of protein folds. HMMs are trained only with experimentally derived secondary structure sequences of proteins having similar fold, therefore protein structures are described by the models at a remarkably simplified level. We submitted predictions for five target sequences, of which four were later found to be suitable for threading. Our approach correctly predicted the fold for three of them. For a fourth sequence the fold could have been correctly predicted if a better model for its structure was available. We conclude that we have additional evidence that secondary structure information represents an important factor for achieving fold recognition.


Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations.
Accurate diagnosis of monocytic neoplasms often is dependent on quantitating monoblasts, promonocytes, and monocytes. However, distinguishing these populations by morphologic assessment alone can be difficult and subject to significant intraobserver variability. We evaluated a 4-color flow cytometry technique using different anti-CD14 antibodies that recognize the MO2 and MY4 epitopes to identify monoblasts, promonocytes, and monocytes. Normal control specimens and 18 cases of monocytic neoplasia were evaluated and results correlated with morphologic findings. We found the MY4 epitope first appearing at the early promonocyte stage and the MO2 epitope appearing only on mature monocytes after the promonocyte stage. Expression of the MY4 and MO2 epitopes by neoplastic monocyte populations was found to usually parallel the normal monocyte differentiation patterns, although exceptions were noted. Our study suggests that 4-color flow cytometry may be an especially useful adjunct for morphologically difficult cases by providing an immunophenotypic measure of neoplastic monoblasts and promonocytes.


The impact of amplification on differential expression analyses by RNA-seq.
Currently, quantitative RNA-seq methods are pushed to work with increasingly small starting amounts of RNA that require amplification. However, it is unclear how much noise or bias amplification introduces and how this affects precision and accuracy of RNA quantification. To assess the effects of amplification, reads that originated from the same RNA molecule (PCR-duplicates) need to be identified. Computationally, read duplicates are defined by their mapping position, which does not distinguish PCR- from natural duplicates and hence it is unclear how to treat duplicated reads. Here, we generate and analyse RNA-seq data sets prepared using three different protocols (Smart-Seq, TruSeq and UMI-seq). We find that a large fraction of computationally identified read duplicates are not PCR duplicates and can be explained by sampling and fragmentation bias. Consequently, the computational removal of duplicates does improve neither accuracy nor precision and can actually worsen the power and the False Discovery Rate (FDR) for differential gene expression. Even when duplicates are experimentally identified by unique molecular identifiers (UMIs), power and FDR are only mildly improved. However, the pooling of samples as made possible by the early barcoding of the UMI-protocol leads to an appreciable increase in the power to detect differentially expressed genes.


The effect of a patient centred care bundle intervention on pressure ulcer incidence (INTACT): A cluster randomised trial.
Hospital-acquired pressure ulcers are a serious patient safety concern, associated with poor patient outcomes and high healthcare costs. They are also viewed as an indicator of nursing care quality. To evaluate the effectiveness of a pressure ulcer prevention care bundle in preventing hospital-acquired pressure ulcers among at risk patients. Pragmatic cluster randomised trial. Eight tertiary referral hospitals with >200 beds each in three Australian states. 1600 patients (200/hospital) were recruited. Patients were eligible if they were: ≥18 years old; at risk of pressure ulcer because of limited mobility; expected to stay in hospital ≥48h and able to read English. Hospitals (clusters) were stratified in two groups by recent pressure ulcer rates and randomised within strata to either a pressure ulcer prevention care bundle or standard care. The care bundle was theoretically and empirically based on patient participation and clinical practice guidelines. It was multi-component, with three messages for patients' participation in pressure ulcer prevention care: keep moving; look after your skin; and eat a healthy diet. Training aids for patients included a DVD, brochure and poster. Nurses in intervention hospitals were trained in partnering with patients in their pressure ulcer prevention care. The statistician, recruiters, and outcome assessors were blinded to group allocation and interventionists blinded to the study hypotheses, tested at both the cluster and patient level. The primary outcome, incidence of hospital-acquired pressure ulcers, which applied to both the cluster and individual participant level, was measured by daily skin inspection. Four clusters were randomised to each group and 799 patients per group analysed. The intraclass correlation coefficient was 0.035. After adjusting for clustering and pre-specified covariates (age, pressure ulcer present at baseline, body mass index, reason for admission, residence and number of comorbidities on admission), the hazard ratio for new pressure ulcers developed (pressure ulcer prevention care bundle relative to standard care) was 0.58 (95% CI: 0.25, 1.33; p=0.198). No adverse events or harms were reported. Although the pressure ulcer prevention care bundle was associated with a large reduction in the hazard of ulceration, there was a high degree of uncertainty around this estimate and the difference was not statistically significant. Possible explanations for this non-significant finding include that the pressure ulcer prevention care bundle was effective but the sample size too small to detect this.


Oncologist factors that influence referrals to subspecialty palliative care clinics.
Recent research and professional guidelines support expanded use of outpatient subspecialty palliative care in oncology, but provider referral practices vary widely. We sought to explore oncologist factors that influence referrals to outpatient palliative care. Multisite, qualitative interview study at three academic cancer centers in the United States with well-established palliative care clinics. Seventy-four medical oncologists participated in semistructured interviews between February and October 2012. The interview guide asked about experiences and decision making regarding outpatient palliative care use. A multidisciplinary team analyzed interview transcripts using constant comparative methods to inductively develop and refine themes related to palliative care referral decisions. We identified three main oncologist barriers to subspecialty palliative care referrals at sites with comprehensive palliative care clinics: persistent conceptions of palliative care as an alternative philosophy of care incompatible with cancer therapy, a predominant belief that providing palliative care is an integral part of the oncologist's role, and a lack of knowledge about locally available services. Participants described their views of subspecialty palliative care as evolving in response to increasing availability of services and positive referral experiences, but emphasized that views of palliative care as valuable in addition to standard oncology care were not universally shared by oncologists. Improving provision of palliative care in oncology will likely require efforts beyond increasing service availability. Raising awareness of ways in which subspecialty palliative care complements standard oncology care and developing ways for oncologists and palliative care physicians to collaborate and integrate their respective skills may help.


Enabling integrative genomic analysis of high-impact human diseases through text mining.
Our limited ability to perform large-scale translational discovery and analysis of disease characterizations from public genomic data repositories remains a major bottleneck in efforts to translate genomics experiments to medicine. Through comprehensive, integrative genomic analysis of all available human disease characterizations we gain crucial insight into the molecular phenomena underlying pathogenesis as well as intra- and inter-disease differentiation. Such knowledge is crucial in the development of improved clinical diagnostics and the identification of molecular targets for novel therapeutics. In this study we build on our previous work to realize the next important step in large-scale translational discovery and analysis, which is to automatically identify those genomic experiments in which a disease state is compared to a normal control state. We present an automated text mining method that employs Natural Language Processing (NLP) techniques to automatically identify disease-related experiments in the NCBI Gene Expression Omnibus (GEO) that include measurements for both disease and normal control states. In this manner, we find that 62% of disease-related experiments contain sample subsets that can be automatically identified as normal controls. Furthermore, we calculate that the identified experiments characterize diseases that contribute to 30% of all human disease-related mortality in the United States. This work demonstrates that we now have the necessary tools and methods to initiate large-scale translational bioinformatics inquiry across the broad spectrum of high-impact human disease.


Detection of circulating parasite antigen in murine fascioliasis by two-site enzyme-linked immunosorbent assays.
A 2-site enzyme immunoassay was developed for the detection of Fasciola hepatica antigen in the serum of fascioliasis infected mice. The assay utilizes high titer rabbit immunoglobulins to parasite excretory/secretory antigens (FhES) as capture antibody, and also as detection antibody when linked to horseradish peroxidase (HRP) or to biotin for reaction with avidin-peroxidase. The assays were compared with a conventional (antibody detection) ELISA to determine diagnostic utility. Using mean rates of detection of fascioliasis, the HRP-based antigen capture assay diagnosed the infection at 1 week postinfection and showed that circulating antigen levels are maximal 3 weeks after infection. The earliest mean diagnosis for the antibody detection and the biotin-based antigen capture ELISAs were 2 and 3 weeks postinfection, respectively. The addition of known quantities of FhES antigens to normal mouse serum gave estimates of lower limits of detectability for the HRP- and biotin-based assays of 25 ng and 0.25 ng, respectively. Routine use of the biotin-avidin system in the antigen capture test resulted in high background activity making this method insensitive.


Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome: insights from the LUNG SAFE study.
Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073.