Habitat fragmentation caused by human activities alters metapopulation dynamics and decreases biological connectivity through reduced migration and gene flow, leading to lowered levels of population genetic diversity and to local extinctions. The threatened Yarra pygmy perch, Nannoperca obscura, is a poor disperser found in small, isolated populations in wetlands and streams of southeastern Australia. Modifications to natural flow regimes in anthropogenically-impacted river systems have recently reduced the amount of habitat for this species and likely further limited its opportunity to disperse. We employed highly resolving microsatellite DNA markers to assess genetic variation, population structure and the spatial scale that dispersal takes place across the distribution of this freshwater fish and used this information to identify conservation units for management. The levels of genetic variation found for N. obscura are amongst the lowest reported for a fish species (mean heterozygosity of 0.318 and mean allelic richness of 1.92). We identified very strong population genetic structure, nil to little evidence of recent migration among demes and a minimum of 11 units for conservation management, hierarchically nested within four major genetic lineages. A combination of spatial analytical methods revealed hierarchical genetic structure corresponding with catchment boundaries and also demonstrated significant isolation by riverine distance. Our findings have implications for the national recovery plan of this species by demonstrating that N. obscura populations should be managed at a catchment level and highlighting the need to restore habitat and avoid further alteration of the natural hydrology.
Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.
Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.
Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.
Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.
Cocaine dependence is a substantial public health problem, yet there are no clearly effective medication treatments. Amphetamine and topiramate have both shown promise for the treatment of cocaine dependence in preclinical and early-stage clinical studies.
Eighty-one cocaine-dependent adults were randomized to receive a combination of mixed amphetamine salts extended-release (MAS-ER) and topiramate or placebo for twelve weeks under double-blind conditions. MAS-ER doses were titrated over two weeks to a maximum dose of 60 mg daily and topiramate doses were titrated over six weeks to a maximum dose of 150 mg twice daily. All participants received a supportive behavioral intervention. The primary outcome was the proportion of individuals who achieved three consecutive weeks of abstinence as measured by urine toxicology confirmed self-report.
The overall proportion of participants who achieved three consecutive weeks of abstinence was larger in the extended-release mixed amphetamine salts and topiramate group (33.3%) than in placebo group (16.7%). There was a significant moderating effect of baseline total number of cocaine use days (Wald χ2=3.75, df =1, P=.05) on outcome suggesting that the combination treatment was most effective for participants with a high baseline frequency of cocaine use.
The results of this study supported our hypothesis that the combination of MASER and topiramate would be superior to placebo in achieving three weeks of consecutive abstinence. These findings provide evidence that the combination of MAS-ER and topiramate is efficacious in promoting abstinence in cocaine-dependent individuals.
cocaine dependence; amphetamines; topiramate; treatment; clinical trial; substance dependence
Maternal-fetal Escherichia coli infections, such as neonatal bacteremia and meningitis, are important causes of morbidity and mortality. From 2006 to 2010, we studied newborns and their mothers who were colonized with E. coli in a French hospital in order to document (i) the epidemiology and genetic characteristics of extended-spectrum-beta-lactamase (ESBL)-producing E. coli strains, (ii) the prevalence of associated virulence genes, (iii) the prevalence of clone sequence type 131 (ST131), and (iv) the genetic relationship among ESBL-producing strains. Among the 2,755 E. coli cultures recovered from vaginal or neonatal samples, 68 were ESBL producers (2.46%). We found a wide diversity of ESBL genes, with the majority being blaCTX-M-14, blaCTX-M-1, and blaCTX-M-15, distributed among the 4 main phylogenetic groups. Genes encoding virulence factors were found in 90.7% of the isolates, with ≥2 virulence genes present in 76% of cases. The prevalence of ST131 among ESBL-producing E. coli isolates was 9.4% (6/64). Five of these 6 ST131 isolates possessed blaCTX-M-15 enzymes (and also were resistant to quinolones), and one possessed blaCTX-M-2 enzymes. Two possessed virulence genes, suggesting the presence of pathogenicity island IIJ96 (PAI IIJ96)-like domains. Pulsed-field gel electrophoresis (PFGE) revealed a high level of genomic diversity overall, except for 3 closely related isolates belonging to clonal group ST131. Repetitive PCR showed that the six ST131 isolates were closely related to ST131 control strains (>95% similarity). This study shows a high prevalence of ESBL-producing E. coli strains and clonal group ST131 in the French maternal-fetal population. These results suggest a widespread distribution of ESBL enzymes in the community and highlight the early transmission between mothers and neonates. These findings are worrisome, especially for this particularly vulnerable population.
Promyelocytic leukemia zinc finger protein (PLZF) is a protein involved in various signaling, growth regulatory, and differentiation pathways, including development/function of some T cells. Here, we aimed at the detection of PLZF during colorectal carcinogenesis, using immunofluorescence, and at the evaluation of the colocalization of PLZF with CD2 and CD56 positive cells (T, γδ, NK, and NKT cells), using confocal-microscopy, along colorectal carcinogenesis, since its earliest stages, that is, dysplastic aberrant crypt foci (ACF). Furthermore, we analyzed PLZF in the normal colonic mucosa (NM) according to anthropometric parameters of the subject. NM exhibited strong CD56 fluorescent staining. This infiltration was lost in both ACF and colorectal carcinoma (CRC), while PLZF presence increased from NM to ACF and CRC. Strong association was found between CD56+ colonic mucosa cell infiltration and body mass index. Interestingly, an increased stromal PLZF-reactivity was present in NM of obese subjects. This study shows that overexpression of PLZF and exclusion of NK cells in dysplastic microenvironment are very early events in the stepwise sequence leading to CRC and that lower levels of CD56+ cells in NM, together with increased levels of PLZF+ cells, can be a reflection of colon cancer risk due to obesity.
Coordinated migration and placement of interneurons and projection neurons lead to functional connectivity in the cerebral cortex; defective neuronal migration and the resultant connectivity changes underlie the cognitive defects in a spectrum of neurological disorders. Here we show that primary cilia play a guiding role in the migration and placement of postmitotic interneurons in the developing cerebral cortex, and that this process requires the ciliary protein, Arl13b. Through live imaging of interneuronal cilia we show migrating interneurons display highly dynamic primary cilia and we correlate cilia dynamics with the interneuron’s migratory state. We demonstrate that the guidance cue receptors essential for interneuronal migration localize to interneuronal primary cilia, but their concentration and dynamics are altered in the absence of Arl13b. Expression of Arl13b variants known to cause Joubert syndrome induce defective interneuronal migration, suggesting that defects in cilia-dependent interneuron migration may underlie the neurological defects in Joubert syndrome patients.
Ciliopathies; microfluidics; Arl13b; Joubert Syndrome
[…] commonly used equations for eGFR should not be applied in AKI patients, neither at the debut of AKI nor during its recovery. The common equations for eGFR will markedly overestimate GFR and may potentially result in overdosing of drugs with renal excretion and risk of side effects. […]
Existing systems for grading severity of acute kidney injury (AKI) rely on a change of serum creatinine concentration over a defined time interval. The rate of change in serum creatinine increases by degree of reduction in glomerular filtration rate, but is mitigated by low creatinine generation rate (CGR). Failure to appreciate variation in CGR may lead to erroneous conclusions regarding severity of AKI and distorted predictions regarding patient outcomes based on AKI severity.
Cohort study of 103 patients who received continuous venovenous hemodialysis (CVVHD) over a 2-year period in a tertiary care hospital setting. Study participants entered the cohort when they were anuric, receiving a stable and uninterrupted dose of CVVHD with serum creatinine in steady state. They were followed until hospital discharge. CGR was measured based on dialyzate effluent volume and effluent creatinine concentration (prospective cohort) and via effluent volume and serum creatinine concentration (retrospective cohort).
CGR (mean 10.5, range 1.7–22.4 mg/kg/day) was substantially lower in this patient population than what would be predicted from existing equations. Correlates of CGR in multivariable analysis included the length of hospitalization prior to measurement and presence of an oncologic diagnosis. Lower CGR was independently associated with in-hospital mortality in unadjusted analysis and after multivariable adjustment for measures of severity of illness.
Grading systems for severity of AKI fail to account for variation in CGR, limiting their ability to predict relevant outcomes. Calculation of CGR is superior to other risk metrics in predicting hospital mortality in this population.
acute kidney injury; continuous renal replacement therapy; creatinine generation; critical care nephrology; mortality
Several screening tools for attention-deficit/hyperactivity disorder (ADHD) have been validated in non–substance-abusing populations, but limited data are available regarding their utility in adults with current substance use disorders. The aim of this study was to determine the sensitivity, specificity, and positive and negative predictive values of 3 commonly used ADHD screening instruments in cocaine-dependent individuals.
Adults seeking treatment for cocaine dependence (N = 102) were administered 3 self-report instruments between May 2009 and April 2011: the Conners Adult ADHD Rating Scale (CAARS), the Wender Utah Rating Scale (WURS), and the Adult ADHD Self-Report Scale-Version 1.1 (ASRS-V1.1). They then met with master’s-level clinicians who administered the Conners Adult ADHD Diagnostic Interview for DSM-IV (CAADID). With the CAADID serving as the gold standard, the validity of the screening instruments was determined, both singly and in combination.
Twenty-five (25%) of the 102 patients met DSM-IV criteria for ADHD or ADHD not otherwise specified (NOS) based on the CAADID. Kappa scores determining agreement between the screening tools and the CAADID (with ADHD NOS labeled as ADHD or labeled as not ADHD) ranged from 0.37 to 0.69. Sensitivity scores for the broadest range of ADHD cases were 80.0%, 87.5%, and 60.9% for the CAARS, WURS, and ASRS-V1.1, respectively. Positive predictive value was highest for the CAARS, at 74.1%, and negative predictive value was highest for the WURS, at 95.1%. The highest sensitivity (96.0%) was found with coadministration of the WURS and CAARS.
While all of the screening instruments were found to have adequate sensitivity and specificity, the CAARS outperformed the other instruments in regard to agreement with the CAADID and positive predictive values. However, the WURS, with the highest sensitivity in regard to the broadest range of ADHD cases, may be the single best instrument for preliminary screening purposes. Further, because the ASRS-V1.1 is the simplest and shortest instrument to administer, it may have advantages when a large number of patients need to be screened.
To identify risk factors for endometrial cancer after benign results of endometrial biopsy or dilatation and curettage (D&C).
Nested case-control study from Rochester Epidemiology Project data. Among 370 Olmsted County, Minnesota, residents who received an endometrial cancer diagnosis between 1970 and 2008, we identified 90 patients (24.5%) who had previous benign endometrial biopsy or D&C results (no atypical hyperplasia). We compared them with 172 matched controls who had benign endometrial biopsy or D&C results without subsequent endometrial cancer.
Using a multivariable, conditional logistic regression model, we found that oral contraceptive use was protective (odds ratio [OR], 0.18; 95% confidence interval [CI], 0.08-0.45; P<.001), and personal history of colorectal cancer (OR, 4.44; 95% CI, 1.02-19.31; P<.05), endometrial polyp (OR, 4.12; 95% CI, 1.40-12.17; P=.01), and morbid obesity (OR, 3.40; 95% CI, 1.18-9.78; P<.03) were independently associated with subsequent endometrial cancer. Compared with the presence of no risk factor, presence of one and two or more risk factors increased the risk of endometrial cancer by 8.12 (95% CI, 3.08-21.44) and 17.87 (95% CI, 5.57-57.39) times, respectively. Assuming a 2.6% lifetime risk of EC, OR’s of 8.12 and 17.87 for 1 and 2 or more of the 4 afore-mentioned risk factors confer a lifetime risk of approximately 18% and 32%, respectively.
One-fourth of patients with endometrial cancer had previous benign endometrial biopsy or D&C results. Personal history of colorectal cancer, presence of endometrial polyps, and morbid obesity are the strongest risk factors for having endometrial cancer after a benign endometrial biopsy or D&C result, and oral contraceptive use is the strongest protective factor.
The value of a positron emission tomography and X-ray computed tomography (PET/CT) combined service in terms of diagnostic accuracy, cost-effectiveness and impact on clinical decision-making is well-documented in the literature. Its role in the management of patients presenting with cancer is shifting from early staging and restaging to the early assessment of the treatment response. Currently, the application of PET/CT has extended to non-oncological specialties—mainly neurology, cardiology and rheumatology. A further emerging application for PET/CT is the imaging of infection/inflammation. This article illustrates some of the PET/CT applications in both oncological and non-oncological disorders. In view of the absence of this modality in Oman, this article aims to increase the awareness of the importance of these imaging modalities and their significant impact on diagnosis and management in both oncological and non-oncological specialties for patients of all age groups as well as the decision-makers.
Positron Emission Tomography; X-Ray Computed Tomography; Medical Oncology; Oman
Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.
Landscape genetics provides a framework for pinpointing environmental features that determine the important exchange of migrants among populations. These studies usually test the significance of environmental variables on gene flow, yet ignore one fundamental driver of genetic variation in small populations, effective population size, Ne. We combined both approaches in evaluating genetic connectivity of a threatened ungulate, woodland caribou. We used least-cost paths to calculate matrices of resistance distance for landscape variables (preferred habitat, anthropogenic features and predation risk) and population-pairwise harmonic means of Ne, and correlated them with genetic distances, FST and Dc. Results showed that spatial configuration of preferred habitat and Ne were the two best predictors of genetic relationships. Additionally, controlling for the effect of Ne increased the strength of correlations of environmental variables with genetic distance, highlighting the significant underlying effect of Ne in modulating genetic drift and perceived spatial connectivity. We therefore have provided empirical support to emphasize preventing increased habitat loss and promoting population growth to ensure metapopulation viability.
Canadian Rockies; genetic drift; habitat fragmentation; landscape genetics; least-cost paths; Rangifer tarandus caribou
The accurate prognosis definition to tailor treatment for early luminal invasive breast carcinoma patients remains challenging.
Materials and Methods
Two hundred fourteen early luminal breast carcinomas were genotyped with single nucleotide polymorphisms (SNPs) array to determine the number of chromosomal breakpoints as a marker of genomic instability. Proliferation was assessed by KI67 (immunohistochemistry) and genomic grade index (transcriptomic analysis). IHC3 (IHC4 score for HER2 negative tumors) was also determined.
In the training set (109 cases), the optimal cut-off was 34 breakpoints with a specificity of 0.94 and a sensitivity of 0.57 (Area under the curve (AUC): 0.81[0.71; 0.91]). In the validation set (105 cases), the outcome of patients with > 34 breakpoints (11 events / 22 patients) was poorer (logrank test p < 0.001; Relative Risk (RR): 3.7 [1.73; 7.92]), than that of patients with < 34 breakpoints (19 events / 83 patients).Whereas genomic grade and KI67 had a significant prognostic value in univariate analysis in contrast to IHC3 that failed to have a statistical significant prognostic value in this series, the number of breakpoints remained the only significant parameter predictive of outcome (RR: 3.47, Confidence Interval (CI [1.29; 9.31], p = 0.014)) in multivariate analysis .
Genomic instability, defined herein as a high number of chromosomal breakpoints, in early stage luminal breast carcinoma is a stronger prognostic marker than proliferation.
A greater understanding of the regulatory processes contributing to lung development could be helpful to identify strategies to ameliorate morbidity and mortality in premature infants and to identify individuals at risk for congenital and/or chronic lung diseases. Over the past decade, genomics technologies have enabled the production of rich gene expression databases providing information for all genes across developmental time or in diseased tissue. These data sets facilitate systems biology approaches for identifying underlying biological modules and programs contributing to the complex processes of normal development, and those that may be associated with disease states. The next decade will undoubtedly see rapid and significant advances in redefining both lung development and disease at the systems level.
A study of acute lung injury reveals the involvement of transcription factor HIF1A in lung protection, where normoxic HIF1A stabilization functions to control alveolar epithelial glucose metabolism.
While acute lung injury (ALI) contributes significantly to critical illness, it resolves spontaneously in many instances. The majority of patients experiencing ALI require mechanical ventilation. Therefore, we hypothesized that mechanical ventilation and concomitant stretch-exposure of pulmonary epithelia could activate endogenous pathways important in lung protection.
Methods and Findings
To examine transcriptional responses during ALI, we exposed pulmonary epithelia to cyclic mechanical stretch conditions—an in vitro model resembling mechanical ventilation. A genome-wide screen revealed a transcriptional response similar to hypoxia signaling. Surprisingly, we found that stabilization of hypoxia-inducible factor 1A (HIF1A) during stretch conditions in vitro or during ventilator-induced ALI in vivo occurs under normoxic conditions. Extension of these findings identified a functional role for stretch-induced inhibition of succinate dehydrogenase (SDH) in mediating normoxic HIF1A stabilization, concomitant increases in glycolytic capacity, and improved tricarboxylic acid (TCA) cycle function. Pharmacologic studies with HIF activator or inhibitor treatment implicated HIF1A-stabilization in attenuating pulmonary edema and lung inflammation during ALI in vivo. Systematic deletion of HIF1A in the lungs, endothelia, myeloid cells, or pulmonary epithelia linked these findings to alveolar-epithelial HIF1A. In vivo analysis of 13C-glucose metabolites utilizing liquid-chromatography tandem mass-spectrometry demonstrated that increases in glycolytic capacity, improvement of mitochondrial respiration, and concomitant attenuation of lung inflammation during ALI were specific for alveolar-epithelial expressed HIF1A.
These studies reveal a surprising role for HIF1A in lung protection during ALI, where normoxic HIF1A stabilization and HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation.
Acute lung injury is a devastating lung disease caused by injuries or acute infections to the lung. In patients it manifests itself as acute respiratory distress syndrome. Severe pulmonary edema and uncontrolled lung inflammation are typical symptoms of acute lung injury, which make it hard for patients to breath efficiently. In the clinical course of the disease, patients require mechanical ventilation to support their lung function and to provide sufficient oxygen levels to vital organs such as the brain, the heart, or the kidneys. We hypothesized that stretch conditions—such as those that occur during mechanical ventilation—result in transcriptional adaptation of alveolar epithelial cells—the innermost lining of the lungs. In this study we identified an unexpected involvement of the transcription factor hypoxia-inducible factor HIF1A in lung protection. We observed that during acute lung injury, stabilization of HIF1A is mediated by increased levels of succinate, an intermediate product of the citrate cycle. Interestingly, we show that HIF1A in alveolar epithelia functions to induce a transcriptional program that improves the efficiency of carbohydrate metabolism by injured lungs, thereby helping to adapt to the injurious conditions of mechanical stretch and to reduce lung inflammation. These preclinical findings highlight the potential for pharmacological HIF1A stabilization in the treatment of acute lung injury.
To test the hypothesis that the use of closed suction pelvic drains placed at time of large bowel resection (LBR) for ovarian cancer (OC) decrease morbidity following anastomotic leak (AL).
Consecutive cases of LBR for OC between 01/01/1994 and 06/20/2011 were retrospectively identified. Drains were routinely used until bowel movement. AL was defined as: 1) feculent fluid from drains/wound/vagina, 2) radiographic evidence of AL, or 3) AL found at reoperation. Descriptive statistics, Wilcoxon rank-sum, Pearson's chi-square and Fisher's exact test were used.
43 cases met inclusion criteria. AL was characterized by method of diagnosis as follows: change in drain output only (DO, n=8); change in drain output associated with ambiguous clinical signs/symptoms (D-SSX, n=11); or clinical signs/symptoms only (SSX, n=24). The sensitivity of drains in diagnosing AL was 50%. Time to diagnosis was earlier in DO/D-SSX (median 7 vs. 11 days, P=0.003), however, no significant differences were observed in rates of reoperation, length of stay, time to chemotherapy (TTC), and 30- and 90-day mortality between DO/D-SSX and SSX. Comparing cases where no drains were placed (n=5) vs. those with drain (n=38), we observed no differences in outcomes. TTC though statistically significant (47 vs. 59 days, P=0.023) was not clinically significant.
Though a change in drain output correlated with earlier diagnosis, this did not appear to impact overall outcomes. We did not find strong evidence supporting routine prolonged drainage after LBR for OC. Additionally, absence of change in drain output does not rule out presence of AL.
large bowel resection; pelvic drain; anastomotic leak; ovarian cancer
A number of incurable retinal diseases causing vision impairments derive from alterations in visual phototransduction. Unraveling the structural determinants of even monogenic retinal diseases would require network-centered approaches combined with atomistic simulations.
The transducin G38D mutant associated with the Nougaret Congenital Night Blindness (NCNB) was thoroughly investigated by both mathematical modeling of visual phototransduction and atomistic simulations on the major targets of the mutational effect.
Mathematical modeling, in line with electrophysiological recordings, indicates reduction of phosphodiesterase 6 (PDE) recognition and activation as the main determinants of the pathological phenotype. Sub-microsecond molecular dynamics (MD) simulations coupled with Functional Mode Analysis improve the resolution of information, showing that such impairment is likely due to disruption of the PDEγ binding cavity in transducin. Protein Structure Network analyses additionally suggest that the observed slight reduction of theRGS9-catalyzed GTPase activity of transducin depends on perturbed communication between RGS9 and GTP binding site. These findings provide insights into the structural fundamentals of abnormal functioning of visual phototransduction caused by a missense mutation in one component of the signaling network. This combination of network-centered modeling with atomistic simulations represents a paradigm for future studies aimed at thoroughly deciphering the structural determinants of genetic retinal diseases. Analogous approaches are suitable to unveil the mechanism of information transfer in any signaling network either in physiological or pathological conditions.
Incurable retinal diseases causing vision impairments may be due to spontaneous mutations in one component of the visual phototransduction signaling network. Such alterations include the transducin single point mutation G38D associated with the Nougaret Congenital Night Blindness (NCNB). We combined a systems biology approach with atomistic simulations to gain insights into the structural fundamentals of the NCNB disease. Consistent with in vitro evidence, mathematical modeling suggests reduced effector recognition and activation as the main determinants of the pathological phenotype. Sub-microsecond molecular dynamics simulations improve the resolution of information, suggesting that such impairment is likely due to disruption of the effector binding cavity. Atomistic simulations also suggest that the observed slight reduction of the RGS9-catalyzed GTPase activity of transducin depends on perturbed inter-protein communication involving the nucleotide. The study highlighted manifold effects of a single point pathogenic mutation, thus paving the way for analogous studies towards a thorough understanding of the structural determinants of genetic retinal diseases.
Motivation: The assessment of protein structure prediction techniques requires objective criteria to measure the similarity between a computational model and the experimentally determined reference structure. Conventional similarity measures based on a global superposition of carbon α atoms are strongly influenced by domain motions and do not assess the accuracy of local atomic details in the model.
Results: The Local Distance Difference Test (lDDT) is a superposition-free score that evaluates local distance differences of all atoms in a model, including validation of stereochemical plausibility. The reference can be a single structure, or an ensemble of equivalent structures. We demonstrate that lDDT is well suited to assess local model quality, even in the presence of domain movements, while maintaining good correlation with global measures. These properties make lDDT a robust tool for the automated assessment of structure prediction servers without manual intervention.
Availability and implementation: Source code, binaries for Linux and MacOSX, and an interactive web server are available at http://swissmodel.expasy.org/lddt
Supplementary information: Supplementary data are available at Bioinformatics online.
Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The “NOTCH signaling pathway” was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1165-y) contains supplementary material, which is available to authorized users.
Paraganglioma; Head and neck; NOTCH signaling; CNV; MicroRNA; Paraganglioma cell culture
Rationale: Bronchopulmonary dysplasia (BPD) is a major complication of premature birth. Risk factors for BPD are complex and include prenatal infection and O2 toxicity. BPD pathology is equally complex and characterized by inflammation and dysmorphic airspaces and vasculature. Due to the limited availability of clinical samples, an understanding of the molecular pathogenesis of this disease and its causal mechanisms and associated biomarkers is limited.
Objectives: Apply genome-wide expression profiling to define pathways affected in BPD lungs.
Methods: Lung tissue was obtained at autopsy from 11 BPD cases and 17 age-matched control subjects without BPD. RNA isolated from these tissue samples was interrogated using microarrays. Standard gene selection and pathway analysis methods were applied to the data set. Abnormal expression patterns were validated by quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry.
Measurements and Main Results: We identified 159 genes differentially expressed in BPD tissues. Pathway analysis indicated previously appreciated (e.g., DNA damage regulation of cell cycle) as well as novel (e.g., B-cell development) biological functions were affected. Three of the five most highly induced genes were mast cell (MC)-specific markers. We confirmed an increased accumulation of connective tissue MCTC (chymase expressing) mast cells in BPD tissues. Increased expression of MCTC markers was also demonstrated in an animal model of BPD-like pathology.
Conclusions: We present a unique genome-wide expression data set from human BPD lung tissue. Our data provide information on gene expression patterns associated with BPD and facilitated the discovery that MCTC accumulation is a prominent feature of this disease. These observations have significant clinical and mechanistic implications.
microarray; tryptase; chymase; carboxypeptidase A3; bronchopulmonary dysplasia
Skates are widely consumed across the globe, but many large species are subject to considerable concern regarding their conservation and management. Within Europe such issues have recently driven policy changes so that, for the first time, reports of skate landings now have to be made under species-specific names. Total allowable catches have also been established for many groups, which have been set to zero for a number of the most vulnerable species (e.g., Dipturus batis, Raja undulata and Rostoraja alba). Whilst accurate species identification has become an important issue for landings, the sale of skates is still usually made under a blanket term of “skate” or “ray”. The matter of identifying species of skate is further complicated by their morphologically conservative nature and the fact that they are commercially valued for their wings. Thus, before sale their bodies are usually discarded (i.e., “winged”) and often skinned, making morphological identification impossible. For the first time, DNA barcoding (of the mitochondrial COI gene) was applied to samples of skate wings from retail outlets across the British Isles, providing insight into which species are sold for consumption. A total of 98 wing samples were analysed, revealing that six species were sold; blonde ray (Raja brachyura), spotted ray (Raja montagui), thornback ray (Raja clavata), cuckoo ray (Leucoraja naevus) small-eyed ray (Raja microocellata) and shagreen ray (Leucoraja fullonica). Statistical testing demonstrated that there were significant differences in the species sold in the distinct retail groups which suggests complex drivers behind the patterns of sale in skates. The results also indicate that endangered species are not commonly being passed on to consumers. In addition, the practice of selling skate wings under ambiguous labels is highlighted as it makes it extremely difficult for consumers to exercise a right to avoid species of conservation concern. Interestingly, a single retailer chain labelled their wings as originating from three smaller-growing species (generally to be considered of lower conservation concern); of the six samples analysed from this company a third were mislabelled and originated from the thornback ray (a larger species that is currently undergoing population declines).
Elasmobranch; Species identification; Forensically informative nucleotide sequencing; Mislabelling; DNA barcoding; Skate; Ray
Palliative care for people with dementia is often sub-optimal. This is partly because of the challenging nature of dementia itself, and partly because of system failings that are particularly salient in primary care and community services. There is a need to systematize palliative care for people with dementia, to clarify where changes in practice could be made.
To develop a model of palliative care for people with dementia that captures commonalities and differences across Europe, a technology development approach was adopted, using mixed methods including 1) critical synthesis of the research literature and policy documents, 2) interviews with national experts in policy, service organisation, service delivery, patient and carer interests, and research in palliative care, and 3) nominal groups of researchers tasked with synthesising data and modelling palliative care.
A generic model of palliative care, into which quality indicators can be embedded. The proposed model includes features deemed important for the systematisation of palliative care for people with dementia. These are: the division of labour amongst practitioners of different disciplines; the structure and function of care planning; the management of rising risk and increasing complexity; boundaries between disease-modifying treatment and palliative care and between palliative and end-of-life care; and the process of bereavement.
The co-design approach to developing a generic model of palliative care for people with dementia has placed the person needing palliative care within a landscape of services and professional disciplines. This model will be explored further in the intervention phase of the IMPACT project.
Injury is a public health problem in terms of mortality, morbidity and disability. The implementation of a regionalised trauma system has been proved to significantly reduce the social impact of severe trauma on population. A population-based registry may be useful to obtain reliable epidemiologic data.
To perform an exhaustive analysis of severe trauma patients hospitalised in Lombardia, a region of northern Italy.
Materials and methods
The regional Hospital Discharge Registry (HDR) was used to retrieve data of all patients who suffered from serious injuries from 2008 to 2010. ICD9-CM codes of discharge diagnoses were analysed and patients coded from 800.0 to 939.9 or from 950.0 to 959.9 have been retrieved. Femur fractures in elderly and patients with length of hospital stay less than 2 days were excluded. Patients have been considered seriously injured if discharged dead or any of followings: admission or transit in ICU, need of mechanical ventilation, tracheotomy, invasive hemodynamic monitoring. Average reimbursement based on DRG has been evaluated.
Student’s t test, ANOVA for continuous data, chi-square test for categorical data were used, and a p value less than 0.05 was considered significant.
The severely injured patients hospitalised in Lombardia in three years were 11704, 391 per million per year. Overall mortality was 24.17% and increased with age. Males aging from 18 to 64 years had more occupational injuries, trauma on the road and violence by others. Females were more susceptible to domestic injuries and self inflicted violence, mostly in older ages. Acute mortality was higher after traffic accidents, while late mortality was increased in domestic trauma. Pediatric cases were unusual. A significant increase (+10.18%) in domestic trauma, with a concomitant decrease (-17.76%) in road-related accidents was observed in the three years study period. Reimbursement paid to hospitals for seriously injured was insufficient with regard to estimated costs of care.
Serious injury requiring hospitalisation in Lombardia is still an healthcare problem, with a trend toward a decrease of traffic accidents, increase in domestic trauma and involvement of older people. These results may help to plan a new regionalised Trauma System.
Epidemiology; Major trauma; Population-based study; Trauma registry; Trauma system
Currently, no clearly superior management strategy exists for recurrent, platinum-resistant ovarian cancer. We tested the efficacy and safety of gemcitabine combined with oxaliplatin (GEMOX) in a multicentre phase II clinical trial.
Forty one patients with recurrent, platinum-resistant ovarian cancer were enrolled. Prior to study entry, all the participants had received at least one platinum-based regimen. Gemcitabine was administered at 1000 mg/m2 as protracted infusion (100 min) on day 1, and oxaliplatin at the dose of 100 mg/m2 on day 2 in a 2 hour infusion. Cycles were repeated every two weeks.
We observed an overall response rate of 37% [95% Confidence Interval (CI), 22.3–51.7]. Objective responses plus disease stabilization (clinical benefit) occurred in 78% of patients. Median progression-free survival was 6.8 months (95% CI, 5.8–7.8), and median overall survival was 16.5 months (95% CI, 12.2–20.8). Median time to self-reported symptom relief, which was described by 22 out of 27 symptomatic patients (81.5%), was 4 weeks (range, 2–8). Grade 4 neutropenia and febrile neutropenia were observed in 2 (5%) and 1 (2.5%) patients, while grade 3 anemia was encountered in 2 (5%) patients, respectively. The most common adverse effects of any grade were gastrointestinal symptoms, fatigue and neutropenia. Nine patients (22%) experienced mild allergic reaction to oxaliplatin, with no treatment discontinuation.
In our cohort of recurrent, platinum-resistant ovarian cancer patients, GEMOX showed encouraging activity and manageable toxicity. Under circumstances requiring a rapid disease control, this combination regimen may offer a particularly viable option, particularly in heavily pretreated patients.
Combination therapy; Gemcitabine; Ovarian cancer; Oxaliplatinum; Platinum-resistant