Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA

The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods

Нові явища у функціонально-стилістичному вживанні протиставних сполучників в українській літературній мові кінця ХХ — початку ХХІ століть

У статті досліджено зміни у функціонуванні найуживаніших протиставних сполучників у мові української преси та художньої літератури кінця ХХ — почат ку ХХІ століть, обґрунтовано слабку семантико-синтаксичну спеціалізацію протиставних сполучників та визначено їхні транспозиційні можливості.In the article the changes in the functioning of the most used adversative conjunctions in the language of Ukrainian press and artistic literature of the end of the XX — the beginning of the XXI centuries have been investigated, weak semantic-syntactic specialization of adversative conjunctions has been explained and their transisting resources have been determined

Microphysical explanation of the RH-dependent water affinity of biogenic organic aerosol and its importance for climate

This is the final version of the article. Available from American Geophysical Union via the DOI in this record.A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH-dependent SOA water-uptake with solubility and phase separation; (2) show that laboratory data on IP- and MT-SOA hygroscopicity are representative of ambient data with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single-parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources.The data presented in the paper will be available through the Bolin Centre database (http://bolin.su.se/data/). The EC H2020 European Research Council ERC (ERC-StGATMOGAIN-278277 and ERC-StG-QAPPA-335478) and integrated project 641816 CRESCENDO Svenska Forskningsrådet Formas (Swedish Research Council Formas) (2015-749), Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation Wallenberg Fellowship AtmoRemove), Academy of Finland (grants 272041 and 259005), Natural Environment Research Council (NERC grants NE/M003531/1 and NE/J02175X/1), Norwegian Research Council (EVA grant 229771), Natural Sciences and Engineering Research Council of Canada (NSERC, grant RGPIN/04315-2014), National Science Foundation (NSF, grants ATM-1242258, AGS-1242932, and AGS-1360834), U.S. Environmental Protection Agency (EPA, STAR grant R835410), National Oceanic and Atmospheric Administration (NOAA, CPO award 538NA10OAR4310102), Electric Power Research Institute (EPRI, grant 10004734), U.S. Department of Energy (DOE, grants BER/ASR DE-SC0016559 and DE-SC0012792), Georgia Institute of Technology, and NordForsk (Nordic Centre of Excellence eSTICC) are gratefully acknowledged for funding. The climate model simulations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the National Supercomputing Centre. Benjamin Murphy is acknowledged for useful discussions

A Gap Analysis Methodology for Collecting Crop Genepools: A Case Study with Phaseolus Beans

Background The wild relatives of crops represent a major source of valuable traits for crop improvement. These resources are threatened by habitat destruction, land use changes, and other factors, requiring their urgent collection and long-term availability for research and breeding from ex situ collections. We propose a method to identify gaps in ex situ collections (i.e. gap analysis) of crop wild relatives as a means to guide efficient and effective collecting activities. Methodology/Principal Findings The methodology prioritizes among taxa based on a combination of sampling, geographic, and environmental gaps. We apply the gap analysis methodology to wild taxa of the Phaseolus genepool. Of 85 taxa, 48 (56.5%) are assigned high priority for collecting due to lack of, or under-representation, in genebanks, 17 taxa are given medium priority for collecting, 15 low priority, and 5 species are assessed as adequately represented in ex situ collections. Gap “hotspots”, representing priority target areas for collecting, are concentrated in central Mexico, although the narrow endemic nature of a suite of priority species adds a number of specific additional regions to spatial collecting priorities. Conclusions/Significance Results of the gap analysis method mostly align very well with expert opinion of gaps in ex situ collections, with only a few exceptions. A more detailed prioritization of taxa and geographic areas for collection can be achieved by including in the analysis predictive threat factors, such as climate change or habitat destruction, or by adding additional prioritization filters, such as the degree of relatedness to cultivated species (i.e. ease of use in crop breeding). Furthermore, results for multiple crop genepools may be overlaid, which would allow a global analysis of gaps in ex situ collections of the world's plant genetic resource

Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

Loa loa is a filarial nematode that infects over 10 million people in Africa. Most infections cause no symptoms, but individuals with large numbers of blood-stage microfilariae are at risk for fatal reactions to ivermectin, an antiparasitic agent used to treat and prevent infections with Onchocerca volvulus, a related filarial parasite that may occur alongside L. loa. To address the urgent need for a point-of-care L. loa diagnostic assay, we screened a Loa microfilaria gene expression library and identified 18 Loa-specific DNA targets. From two targets, we developed a novel, rapid quantitative PCR assay for estimating L. loa microfilaria burden. The assay is highly sensitive (detects a single microfilaria in 20 µL of blood) and correlates well with microfilaria counts obtained with conventional microscopic techniques. The assay is species-specific for L. loa compared with related filarial parasites (including O. volvulus) and can be used in its current form in resource-rich areas as a diagnostic tool for L. loa infection. Although modifications will be required to make point-of-care use feasible, our assay provides a proof of concept for a potentially valuable tool to identify individuals at risk for adverse reactions to ivermectin and to facilitate the implementation of filarial control programs

HIV-associated bladder cancer: a case series evaluating difficulties in diagnosis and management

<p>Abstract</p> <p>Background</p> <p>Chronic human immunodeficiency virus (HIV) infection is associated with an increased incidence of Non-Acquired Immunodeficiency Syndrome (non-AIDS) defining cancers. To date, only a limited number of cases of bladder cancer have been linked with HIV infection. We sought to describe the clinical characteristics of HIV-associated bladder cancer.</p> <p>Methods</p> <p>A retrospective study was performed involving HIV-positive patients with bladder cancer, combining cases from multiple institutions with published case reports. Data regarding patient demographics, HIV status, clinical presentation, pathology, cancer treatment, and outcome were analyzed using descriptive statistics.</p> <p>Results</p> <p>Eleven patients were identified with a median age of 55 years (range, 33 - 67). The median CD4+ count at cancer diagnosis was 280 cells/mm³(range, 106 - 572 cells/mm³). Six patients (55%) had a known risk factor for bladder cancer, and nine (82%) presented with hematuria. Ten patients had transitional cell carcinoma, and most had superficial disease at presentation. Treatment included mainly transurethral resection of bladder tumor followed by a combination of local and systemic therapies. One patient received intravesical bacillus Calmette-Guèrin (BCG) without complication. Several patients (55%) were alive following therapy, although many (64%) suffered from local relapse and metastatic disease.</p> <p>Conclusion</p> <p>Bladder cancer is part of the growing list of cancers that may be encountered in patients living longer with chronic HIV-infection. Our patients presented at a younger age and with only mild immunosuppression, however, they experienced an expected course for their bladder cancer. Hematuria in an HIV-infected patient warrants a complete evaluation.</p

Enterococcal colonization of infants in a neonatal intensive care unit: associated predictors, risk factors and seasonal patterns

<p>Abstract</p> <p>Background</p> <p>During and shortly after birth, newborn infants are colonized with enterococci. This study analyzes predictors for early enterococcal colonization of infants in a neonatal intensive care unit and describes risk factors associated with multidrugresistant enterococci colonization and its seasonal patterns.</p> <p>Methods</p> <p>Over a 12-month period, we performed a prospective epidemiological study in 274 infants admitted to a neonatal intensive care unit. On the first day of life, we compared infants with enterococcal isolates detected in meconium or body cultures to those without. We then tested the association of enterococcal colonization with peripartal predictors/risk factors by using bivariate and multivariate statistical methods.</p> <p>Results</p> <p>Twenty-three percent of the infants were colonized with enterococci. The three most common enterococcal species were <it>E. faecium </it>(48% of isolates), <it>E. casseliflavus </it>(25%) and <it>E. faecalis </it>(13%). Fifty-seven percent of the enterococci found were resistant to three of five antibiotic classes, but no vancomycin-resistant isolates were observed. During winter/spring months, the number of enterococci and multidrug-resistant enterococci were higher than in summer/fall months (p = 0.002 and p < 0.0001, respectively). With respect to enterococcal colonization on the first day of life, predictors were prematurity (p = 0.043) and low birth weight (p = 0.011). With respect to colonization with multidrug-resistant enterococci, risk factors were prematurity (p = 0.0006), low birth weight (p < 0.0001) and prepartal antibiotic treatment (p = 0.019). Using logistic regression, we determined that gestational age was the only parameter significantly correlated with multidrug-resistant enterococci colonization. No infection with enterococci or multidrugresistant enterococci in the infants was detected. The outcome of infants with and without enterococcal colonization was the same with respect to death, necrotizing enterocolitis, intracerebral hemorrhage and bronchopulmonary dysplasia.</p> <p>Conclusion</p> <p>In neonatal intensive care units, an infant's susceptibility to early colonization with enterococci in general, and his or her risk for colonization with multidrug-resistant enterococci in particular, is increased in preterm newborns, especially during the winter/spring months. The prepartal use of antibiotics with no known activity against enterococci appears to increase the risk for colonization with multidrug-resistant enterococci.</p

Missing effects of zinc in a porcine model of recurrent endotoxemia

BACKGROUND: Chronic human sepsis often is characterised by the compensatory anti-inflammatory response syndrome (CARS). During CARS, anti-inflammatory cytokines depress the inflammatory response leading to secondary and opportunistic infections. Proved in vitro as well as in vivo, zinc's pro-inflammatory effect might overcome this depression. METHODS: We used the model of porcine LPS-induced endotoxemia established by Klosterhalfen et al. 10 pigs were divided into two groups (n = 5). Endotoxemia was induced by recurrent intravenous LPS-application (1.0 μg/kg E. coli WO 111:B4) at hours 0, 5, and 12. At hour 10, each group received an intravenous treatment (group I = saline, group II = 5.0 mg/kg elementary zinc). Monitoring included hemodynamics, blood gas analysis, and the thermal dilution technique for the measurement of extravascular lung water and intrapulmonary shunt. Plasma concentrations of IL-6 and TNF-alpha were measured by ELISA. Morphology included weight of the lungs, width of the alveolar septae, and rate of paracentral liver necrosis. RESULTS: Zinc's application only trended to partly improve the pulmonary function. Compared to saline, significant differences were very rare. IL-6 and TNF-alpha were predominately measured higher in the zinc group. Again, significance was only reached sporadically. Hemodynamics and morphology revealed no significant differences at all. CONCLUSION: The application of zinc in this model of recurrent endotoxemia is feasible and without harmful effects. However, a protection or restoration of clinical relevance is not evident in our setting. The pulmonary function just trends to improve, cytokine liberation is only partly activated, hemodynamics and morphology were not influenced. Further pre-clinical studies have to define zinc's role as a therapeutic tool during CARS

New Insights into the Evolution of Metazoan Tyrosinase Gene Family

Tyrosinases, widely distributed among animals, plants and fungi, are involved in the biosynthesis of melanin, a pigment that has been exploited, in the course of evolution, to serve different functions. We conducted a deep evolutionary analysis of tyrosinase family amongst metazoa, thanks to the availability of new sequenced genomes, assessing that tyrosinases (tyr) represent a distinctive feature of all the organisms included in our study and, interestingly, they show an independent expansion in most of the analyzed phyla. Tyrosinase-related proteins (tyrp), which derive from tyr but show distinct key residues in the catalytic domain, constitute an invention of chordate lineage. In addition we here reported a detailed study of the expression territories of the ascidian Ciona intestinalis tyr and tyrps. Furthermore, we put efforts in the identification of the regulatory sequences responsible for their expression in pigment cell lineage. Collectively, the results reported here enlarge our knowledge about the tyrosinase gene family as valuable resource for understanding the genetic components involved in pigment cells evolution and development