In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River.

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

Analysing urban resilience through alternative stormwater management options: application of the conceptual Spatial Decision Support System model at the neighbourhood scale.

Recent changes in cities and their environments, caused by rapid urbanisation and climate change, have increased both flood probability and the severity of flooding. Consequently, there is a need for all cities to adapt to climate and socio-economic changes by developing new strategies for flood risk management. Following a risk paradigm shift from traditional to more integrated approaches, and considering the uncertainties of future urban development, one of the main emerging tasks for city managers becomes the development of resilient cities. However, the meaning of the resilience concept and its operability is still not clear. The goal of this research is to study how urban engineering and design disciplines can improve resilience to floods in urban neighbourhoods. This paper presents the conceptual Spatial Decision Support System (DS3) model which we consider a relevant tool to analyse and then implement resilience into neighbourhood design. Using this model, we analyse and discuss alternative stormwater management options at the neighbourhood scale in two specific areas: Rotterdam and New Orleans. The results obtained demonstrate that the DS3 model confirmed in its framework analysis that stormwater management systems can positively contribute to the improved flood resilience of a neighbourhood.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours.

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

In vitro antiseptic properties of an ammonium compound combined with denture base acrylic resin.

BACKGROUND: Denture base acrylic resin is easily colonised by oral endogenous bacteria and Candida spp., and eventually by extra-oral species such as Staphylococcus spp., Pseudomonadaceae or members of Enterobacteriaceae. This microbial reservoir can be responsive for denture related stomatitis and aspiration pneumonia, a life-threatening infection especially in geriatric patients. However, the oral and denture hygiene of dependent elderly individuals is extremely poor. OBJECTIVE: This in vitro study aimed to determine the per cent of a quaternary ammonium compound heat-polymerised in acrylic resin necessary to obtain denture base displaying antiseptic properties. DESIGN: Acrylic resin discs containing 2-50% ammonium polymer (Poly 202063A; 0% control) were soaked in artificial saliva for 4 weeks. Resin discs were incubated for 24 hours with Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa [37 degrees C, brain-heart infusion (BHI) broth and phosphate-buffered saline (PBS) buffer] and Candida albicans (30 degrees C, Schaedler broth), in 15 ml (168 discs) and 600 microl (168 discs) of inoculum. Microbial growth was verified at t 0 hours and t 24 hours. Data were recorded as the mean of three colony forming unit (CFU) numerations. The borderline of antimicrobial effect was determined at 0.1% viable cells. RESULTS: In 600 microl of PBS inoculum, resin specimens had a bactericidal effect (E. coli and S. aureus: 2%; P. aeruginosa: 10%) and a fungicidal effect (C. albicans: 50%). Long-term stability and toxicity in vivo studies are now required. CONCLUSION: A 2% quaternary ammonium compound polymerised with a denture acrylic resin displayed antiseptic properties after a 4-week soaking period in artificial saliva. Such antiseptic denture base could help geriatric patients to improve their oral health.

Nuclear insertions help and hinder inference of the evolutionary history of gorilla mtDNA.

Numts are fragments of mitochondrial DNA (mtDNA) that have been translocated to the nucleus, where they can persist while their mitochondrial counterparts continue to rapidly evolve. Thus, numts represent 'molecular fossils' useful for comparison with mitochondrial variation, and are particularly suited for studies of the fast-evolving hypervariable segment of the mitochondrial control region (HV1). Here we used information from numts found in western gorillas (Gorilla gorilla) and eastern gorillas (Gorilla beringei) to estimate that these two species diverged about 1.3 million years ago (Ma), an estimate similar to recent calculations for the divergence of chimpanzee and bonobo. We also describe the sequence of a gorilla numt still possessing a segment lost from all contemporary gorilla mtDNAs. In contrast to that sequence, many numts of the HV1 are highly similar to authentic mitochondrial organellar sequences, making it difficult to determine whether purported mitochondrial sequences truly derive from that genome. We used all available organellar HV1 and corresponding numt sequences from gorillas in a phylogenetic analysis aimed at distinguishing these two types of sequences. Numts were found in several clades in the tree. This, in combination with the fact that only a limited amount of the extant variation in gorillas has been sampled, suggests that categorization of new sequences by the indirect means of phylogenetic comparison would be prone to uncertainty. We conclude that for taxa such as gorillas that contain numerous numts, direct approaches to the authentication of HV1 sequences, such as amplification strategies relying upon the circularity of the mtDNA molecule, remain necessary.

In vitro new dialysis protocol to assay the antiseptic properties of a quaternary ammonium compound polymerized with denture acrylic resin.

AIMS: To develop an in vitro protocol in order to assess the antiseptic properties of a quaternary ammonium compound polymerized with acrylic denture resin base, using experimental resin discs and dialysis membranes. METHODS AND RESULTS: Experimental acrylic resin discs were polymerized with Poly 202063A, an ammonium compound (2-50%). Antiseptic properties were assayed against two reference strains (Escherichia coli, Staphylococcus aureus) and a laboratory strain (Candida albicans), using three different conditions (test A, B and C). In test A, according to classical protocols the resin discs were first soaked in large volumes of microbial inoculum (45 ml). An original dialysis protocol was then designed to recreate the small biofilm volume on the prosthetic surface. In test B, discs and bacterial inoculum (600 microl) were introduced in a dialysis bag and dialysed against a sterile buffer. A bactericidal effect was observed against E. coli and Staph. aureus (<0.1% viable cells in initial bacterial suspension). A dose-dependent fungistatic effect was observed against C. albicans. Finally, in test C discs and sterile buffer (600 microl) were introduced in a dialysis bag and dialysed against microbial inoculum. Reduced activity was found outside the dialysis bag, demonstrating that free ammonium was able to diffuse through the dialysis membrane, displaying antiseptic properties. CONCLUSIONS: The present protocol demonstrated that a quaternary ammonium compound remains efficient after heat polymerization with an acrylic denture base resin, both in immediate and distant microbial environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Such removable prosthetic devices with intrinsic antiseptic properties would contribute to improve the long-term management of denture stomatitis.

Mutations induced by ancient DNA extracts?

We have investigated whether some factor in ancient DNA extracts induces site-specific mutations in modern DNA. We find no evidence for higher mutation rates when extracts from three different Pleistocene mammals are added to modern DNA than when water or extraction blanks are added. We also fail to find evidence that any such factor affects ancient DNA sequences determined from the same extracts. This as well as the patterns of nucleotide substitutions seen in DNA sequences determined from hundreds of other specimens leads us to doubt that a previously unknown mutagenic factor can be a general feature of extracts from old tissues.

DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.

Ancient DNA.

DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.

Multiple juvenile xanthogranulomas without foam cells and giant cells.

A 4-month-old boy had numerous nodular lesions. Microscopic findings revealed a histiocytic proliferation without foam cells and giant cells. Immunohistochemical studies showed that the histiocytes belonged to the monocyte-macrophage system and not to the Langerhans cell system. This case is an unusual variant of juvenile xanthogranuloma that, when congenital, may be difficult to differentiate from Hashimoto-Pritzker disease.

[Immunohistochemical study of cells of invasive cancer of the vaginal part of the cervix uteri]. / Immunogistokhimicheskoe issledovanie kletok invazivnogo raka vlagalishchnoi chasti sheiki matki.

Immunohistochemical study of cervical carcinoma used EE21-06d monoclonal antibodies which identify five cytokeratin polypeptides inherent in the squamous epithelium. PAP-method and reaction of immunofluorescence were employed. Initial stages of squamous cell carcinoma invasion were characterized by bleaching or complete cell discoloring of most tumor cells. However, in deeply invading tumors, the share of intensively stained cells was markedly increased. The results point to expression of different cytokeratins or cell clones replacement with tumor progression. The peculiarities of cytokeratin distribution may serve to determine the degree of invasion and differentiation of tumor cells.

[Immunohistochemical identification of reserve cells of the endocervical canal by monoclonal antibodies EE21-06d]. / Immunohistokhimicheskaia identifikatsiia rezervnykh kletok éndotservikal'nogo kanala monoklonal'nymi antitelami EE21-06d.

Expression of cytokeratin polypeptides characteristic of squamous epithelium was studied in reserve cells of cervical canal obtained from 8 women by the immunofluorescence method with the help of monoclonal antibodies EE21-06d (MAB). MAB EE21-06d were shown to detect individual reserve cells as well as their hyperplasia foci without staining columnar cells.