Development of species-specific primers for rapid identification by PCR of the ecologically important pathogen Fusarium keratoplasticum from isolated and environmental samples

The genus Fusarium contains many fungal species known to be pathogenic to animals and plants alike. One species complex within this genus, the Fusarium solani species complex (FSSC), is of particular concern due to its high numbers of pathogenic members. FSSC members are known to contribute significantly to plant, human and other animal fungal disease. One member of the FSSC, Fusarium keratoplasticum, is of particular ecological concern and has been implicated in low hatching success of endangered sea turtle eggs, as well as contribute to human and other animal Fusarium pathogenesis. Species-specific primers for molecular identification of F. keratoplasticum currently do not exist to our knowledge, making rapid identification, tracking and quantitation of this pathogenic fungus difficult. The objective of this study was to develop primers specific to F. keratoplasticum that could be applied to DNA from isolated cultures as well as total (mixed) DNA from environmental samples. RPB2 sequence from 109 Fusarium isolates was aligned and analysed to determine nucleotide polymorphisms specific to F. keratoplasticum useful for primer design. A set of primers were generated and found to be effective for identification of F. keratoplasticum from total DNA extracted from sand surrounding sea turtle nesting sites.     

Use of autonomous audio recordings for the rapid inventory of birds in the white‐sand forests of the Peruvian Amazon

White‐sand forests are patchily distributed ecosystems covering just 5% of Amazonia that host many specialist species of birds not found elsewhere, and these forests are threatened due to their small size and human exploitation of sand for construction projects. As a result, many species of birds that are white‐sand specialists are at risk of extinction, and immediate conservation action is paramount for their survival. Our objective was to evaluate current survey methods and determine the relative effect of the size of patches of these forests on the presence or absence of white‐sand specialists. Using point counts and autonomous recorders, we surveyed avian assemblages occupying patches of white‐sand forest in the Peruvian Amazon in April 2018. Overall, we detected 126 species, including 21 white‐sand forest specialists. We detected significantly more species of birds per survey point with autonomous recorders than point counts. We also found a negative relationship between avian species richness and distance from the edge of patches of white‐sand forest, but a significant, positive relationship when only counting white‐sand specialists. Although we detected more species with autonomous recorders, point counts were more effective for detecting canopy‐dwelling passerines. Therefore, we recommend that investigators conducting surveys for rare and patchily distributed species in the tropics use a mixed‐method approach that incorporates both autonomous recorders and visual observation. Finally, our results suggest that conserving large, continuous patches of white‐sand forest may increase the likelihood of survival of species of birds that are white‐sand specialists.     

Biosensors based on peptide exposure show single molecule conformations in live cells

1. Download : Download high-res image (175KB)
2. Download : Download full-size image

     

Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Mechanisms and development of self–other distinction in dyads and groups

This opinion piece offers a commentary on the four papers that address the theme of the development of self and other understanding with a view to highlighting the important contribution of developmental research to understanding of mechanisms of social cognition. We discuss potential mechanisms linking self–other distinction and empathy, implications for grouping motor, affective and cognitive domains under a single mechanism, applications of these accounts for joint action and finally consider self–other distinction in group versus dyadic settings.     

Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.