Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

Regularity of Daily Activities in Stroke

Various studies have been performed using the Social Rhythm Metric (SRM), though none has been developed with stroke patients. Stroke is a pathology that provokes a strong physical and social impact caused by an abnormality in cerebral circulation. Consequently, we performed two studies to validate the SRM and translate it into Portuguese, and to evaluate the regularity of the daily activities of stroke patients. Both healthy individuals and patients with unilateral cerebral lesions were evaluated. Subjects were of both sexes and between 45 and 65 yrs of age. Participants underwent clinical evaluation and recorded the time of 17 daily activities on the SRM for two weeks. Data were analyzed by the Pearson correlation and Fisher tests. After conceptual translation into Portuguese, corrections were made to arrive at the final version. Normative SRM scores varied from 3.2 to 7.0, suggesting that the activities presented in SRM adequately represented the daily routines of the patients. A correlation was found in SRM between the weeks (r=0.84; p=0.0001), indicating instrument reliability. The mean (±SD) score of the stoke patients was 4.8 (±1.0), and the correlation between the SRM and level of neurological damage showed that patients with lower SRM values were more physically compromised (r=?0.29; p=0.04), suggesting that SRM may be a clinical predictor. Activities related to eating and the sleep‐wake cycle were rated by most patients. In all, 71% of the patients did not work, while 84% of healthy individuals did (p=0.001). Only 64% of patients left home compared to 90% of the healthy subjects (p=0.001), and 59% of patients recorded the activity of going home compared to 82% of healthy individuals (p=0.001). According to the results, there is evidence of the validity and reliability of the SRM, enabling it to be reliably used in chronobiological studies of stroke patients. Given that a less regular lifestyle may be associated with neurological compromise and a decrease in social activities, we suggest new studies with the repeated application of this instrument over the clinical evolution of the disease to better define improvement or worsening of the patient's condition in terms of their social and health aspects.     

Therapeutic Vaccination against the Rhesus Lymphocryptovirus EBNA-1 Homologue,rhEBNA-1, Elicits T Cell Responses to Novel Epitopes in Rhesus Macaques

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC–rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.     

Morphometric and molecular variation in concert: taxonomy and genetics of the reticulate Pyrenean and Iberian alpine spiny fescues (Festuca eskia complex,Poaceae)

The Iberian mountain spiny fescues are a reticulate group of five diploid grass taxa consisting of three parental species and two putative hybrids: F. × souliei (F. eskia × F. quadriflora) and F. × picoeuropeana (F. eskia × F. gautieri). Phenotypic and molecular studies were conducted with the aim of determining the taxonomic boundaries and genetic relationships of the five taxa and disentangling the origins of the two hybrids. Statistical analyses of 31 selected phenotypic traits were conducted on individuals from 159 populations and on nine type specimens. Molecular analyses of random amplified polymorphic DNA (RAPD) markers were performed on 29 populations. The phenotypic analyses detected significant differences between the five taxa and demonstrated the overall intermediacy of the F. × picoeuropeana and F. × souliei between their respective parents. The RAPD analysis corroborated the genetic differentiation of F. eskia, F. gautieri and F. quadriflora and the intermediate nature of the two hybrids; however, they also detected genetic variation within F. × picoeuropeana. These results suggest distinct origins for F. × picoeuropeana in the Cantabrian and Pyrenean mountains, with the sporadic Pyrenean populations having potentially resulted from recent hybridizations and the stabilized Cantabrian ones from older events followed by potential displacements of the parents. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 676–706.     

Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.     

Blowflies (Diptera: Calliphoridae) activity in sun exposed and shaded carrion in Portugal

The first forensic entomological study performed in Portugal is presented. Two piglet (Sus scrofa L.) carcasses were used to determine adult Calliphoridae activity on carrion over a period of 121 days, all along the end of spring and the summer, both in a shaded and a sunny site. Five decomposition stages were observed and a total of 10723 adult Calliphoridae, belonging to 11 species, were collected. Calliphora vicina, Calliphora vomitoria, Chrysomya albiceps and Lucilia caesar were the dominant species in this study. Decomposition was faster on the carcass exposed to the sun and the number of Calliphoridae specimens was higher there than in the shaded site. It was found a significant effect of the decomposition stage in the number of specimens attracted to the carcass, as well as a significant effect of the interaction between the decomposition stage and insolation regime. Calliphora and Lucilia species did not show preference for sunny or shaded areas. Important differences in the Calliphoridae community structure were found compared to other regions of the Iberian Peninsula, reinforcing the need of further studies in different environments and regions of this geographical area in order to collect information about the local necrophagous fauna used in forensic practice.