Characterization of cultivated fungi isolated from grape marc wastes through the use of amplified rDNA restriction analysis and sequencing

Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7×10⁶ per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.     

Raman spectroscopy of blood and urine liquid biopsies for ovarian cancer diagnosis: identification of chemotherapy effects

Blood plasma and serum Raman spectroscopy for ovarian cancer diagnosis has been applied in pilot studies, with promising results. Herein, a comparative analysis of these biofluids, with a novel assessment of urine, was conducted by Raman spectroscopy application in a large patient cohort. Spectra were obtained through samples measurements from 116 ovarian cancer patients and 307 controls. Principal component analysis identified significant spectral differences between cancers without previous treatment (n = 71) and following neo-adjuvant chemotherapy (NACT), (n = 45). Application of five classification algorithms achieved up to 73% sensitivity for plasma, high specificities and accuracies for both blood biofluids, and lower performance for urine. A drop in sensitivities for the NACT group in plasma and serum, with an opposite trend in urine, suggest that Raman spectroscopy could identify chemotherapy-related changes. This study confirms that biofluids' Raman spectroscopy can contribute in ovarian cancer's diagnostic work-up and demonstrates its potential in monitoring treatment response.     

Changing social awareness of the illegal killing of migratory birds in the Ionian Islands,western Greece

ABSTRACT

The current study aims at monitoring, measuring and evaluating the ‘Safe havens for wild birds’ campaign, implemented within the framework of the LIFE programme, in Greece and more specifically in the Ionian Islands. The study detects attitude changes that occurred in three target groups (pupils, local hunters and residents) on three islands where the phenomenon of illegal spring killing is more intense. A questionnaire-based survey was conducted in two phases, just before the launch (2013) and after the completion of the campaign (2015), to enable a comparison of answers and data. The results show that pupils and to a lesser extent residents, enhanced their knowledge and awareness of poaching, the migration of avifauna and the consequences of illegal killing on migratory birds, while this aspect of attitude change was not observed in the hunting community which still believes that spring poaching should be treated as a legal activity and part of local culture.     

Quantification of fibroblast growth factor 23 and N-terminal pro-B-type natriuretic peptide to identify patients with atrial fibrillation using a high-throughput platform: A validation study

BackgroundLarge-scale screening for atrial fibrillation (AF) requires reliable methods to identify at-risk populations. Using an experimental semi-quantitative biomarker assay, B-type natriuretic peptide (BNP) and fibroblast growth factor 23 (FGF23) were recently identified as the most suitable biomarkers for detecting AF in combination with simple morphometric parameters (age, sex, and body mass index [BMI]). In this study, we validated the AF model using standardised, high-throughput, high-sensitivity biomarker assays.Methods and findingsFor this study, 1,625 consecutive patients with either (1) diagnosed AF or (2) sinus rhythm with CHA₂DS₂-VASc score of 2 or more were recruited from a large teaching hospital in Birmingham, West Midlands, UK, between September 2014 and February 2018. Seven-day ambulatory ECG monitoring excluded silent AF. Patients with tachyarrhythmias apart from AF and incomplete cases were excluded. AF was diagnosed according to current clinical guidelines and confirmed by ECG. We developed a high-throughput, high-sensitivity assay for FGF23, quantified plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and FGF23, and compared results to the previously used multibiomarker research assay. Data were fitted to the previously derived model, adjusting for differences in measurement platforms and known confounders (heart failure and chronic kidney disease). In 1,084 patients (46% with AF; median [Q1, Q3] age 70 [60, 78] years, median [Q1, Q3] BMI 28.8 [25.1, 32.8] kg/m², 59% males), patients with AF had higher concentrations of NT-proBNP (median [Q1, Q3] per 100 pg/ml: with AF 12.00 [4.19, 30.15], without AF 4.25 [1.17, 15.70]; p < 0.001) and FGF23 (median [Q1, Q3] per 100 pg/ml: with AF 1.93 [1.30, 4.16], without AF 1.55 [1.04, 2.62]; p < 0.001). Univariate associations remained after adjusting for heart failure and estimated glomerular filtration rate, known confounders of NT-proBNP and FGF23. The fitted model yielded a C-statistic of 0.688 (95% CI 0.656, 0.719), almost identical to that of the derived model (C-statistic 0.691; 95% CI 0.638, 0.744). The key limitation is that this validation was performed in a cohort that is very similar demographically to the one used in model development, calling for further external validation.ConclusionsAge, sex, and BMI combined with elevated NT-proBNP and elevated FGF23, quantified on a high-throughput platform, reliably identify patients with AF.Trial registrationRegistry IRAS ID 97753 Health Research Authority (HRA), United Kingdom

Winnie Chua and colleagues identify and validate biomarkers for atrial fibrillation     

Pumpkin pectin: gel formation at unusually low concentration

The gel properties of high-methoxy pectin from pumpkins have been investigated to assess the potential of this material as a hard-currency export from the former Soviet Union. Comparison was made with commercial slow-set, medium-set and rapid-set pectins from citrus peel. Gels were formed by cooling pectin solutions (pH 3·0; 60% (w/w) sucrose; 5% (w/w) corn syrup) from 95°C to 25°C, and the time-temperature course of network formation was monitored by small-deformation oscillatory measurements of storage modulus (G′). At concentrations above 1% (w/w) the pumpkin pectin gave weaker gels than the other three samples, but its minimum critical gelling concentration (c₀) was found to be much lower (by at least a factor of five). Compression testing gave similar results, with pumpkin pectin giving useful breaking-stress (‘hardness’) at concentrations down to 0·5% (w/w), about a factor of two lower than for the citrus samples. Its gelation was also less rapid, giving G′ values below those of the other three samples at temperatures down to 60°C, but then setting sharply; this behaviour could be useful in avoiding ‘pregelation’ in commercial processing. The commercial slow-set pectin showed typical ‘weak gel’ properties in the solution state at 95°C, with systematic reduction in gel-like character with increasing ester content in the other samples. The rigidity of the final gels also decreased systematically through the series: rapid-set < medium-set < slow-set. These observations are tentatively ascribed to stable association of unesterified galacturonate chain segments at low pH, where electrostatic repulsion is suppressed.     

The structure of the 2[4Fe–4S] ferredoxin from Pseudomonas aeruginosa at 1.32-Å resolution: comparison with other high-resolution structures of ferredoxins and contributing structural features to reduction potential values

The structure of the 2[4Fe–4S] ferredoxin (PaFd) from Pseudomonas aeruginosa, which belongs to the Allochromatium vinosum (Alvin) subfamily, has been determined by X-ray crystallography at 1.32-Å resolution, which is the highest up to now for a member of this subfamily of Fds. The main structural features of PaFd are similar to those of AlvinFd. However, the significantly higher resolution of the PaFd structure makes possible a reliable comparison with available high-resolution structures of [4Fe–4S]-containing Fds, in an effort to rationalize the unusual electrochemical properties of Alvin-like Fds. Three major factors contributing to the reduction potential values of [4Fe–4S]^2+/+ clusters of Fds, namely, the surface accessibility of the clusters, the N–H···S hydrogen-bonding network, and the volume of the cavities hosting the clusters, are extensively discussed. The volume of the cavities is introduced in the present work for the first time, and can in part explain the very negative potential of cluster I of Alvin-like Fds.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Implementation of Novel Design Features for qPCR-Based eDNA Assessment

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.     

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna–Matthews–Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.     

The polyisoprenoid chain length influences the interaction of ubiquinones with phospholipid bilayers

The interaction between ubiquinone homologues with polyisoprenoid chain lengths varying from 3 to 10 units and dipalmitoylphosphatidylcholine bilayers has been examined by differential scanning calorimetry and wide angle X-ray diffraction analysis. Decreasing the polyisoprenoid chain lengths of ubiquinone in mixed dispersions with phospholipid in mol ratios of about 10 mol% caused a decrease in the gel-liquid crystalline phase transition temperature of the phospholipid and a broadening of the transition. Enthalpy measurements showed that most of the phospholipid (greater than 92%) was involved in the transition endotherm and the formation of a gel phase was also confirmed by the presence of a sharp X-ray reflection of 0.42 nm. These results are consistent with a model in which all of the ubiquinone homologous ultimately undergo a phase separation from phospholipid molecules entering a gel phase on cooling below the phase transition temperature. Reducing the length of the polyisoprenoid chain alters the amphipathic balance of the ubiquinone molecules and is reflected in the tendency of shorter chain ubiquinones to intercalate between the phospholipid molecules upon reheating through the main phase transition.     

Prior expectations evoke stimulus-specific activity in the deep layers of the primary visual cortex

The way we perceive the world is strongly influenced by our expectations. In line with this, much recent research has revealed that prior expectations strongly modulate sensory processing. However, the neural circuitry through which the brain integrates external sensory inputs with internal expectation signals remains unknown. In order to understand the computational architecture of the cortex, we need to investigate the way these signals flow through the cortical layers. This is crucial because the different cortical layers have distinct intra- and interregional connectivity patterns, and therefore determining which layers are involved in a cortical computation can inform us on the sources and targets of these signals. Here, we used ultra-high field (7T) functional magnetic resonance imaging (fMRI) to reveal that prior expectations evoke stimulus-specific activity selectively in the deep layers of the primary visual cortex (V1). These findings are in line with predictive processing theories proposing that neurons in the deep cortical layers represent perceptual hypotheses and thereby shed light on the computational architecture of cortex.

The way we perceive the world is strongly influenced by our expectations, but the neural circuitry through which the brain achieves this remains unknown. A study using ultra-high field fMRI reveals that prior expectations evoke stimulus-specific signals in the deep layers of the primary visual cortex.