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.     

DNA ligase-AMP adducts: Identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [α-³²P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.     

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.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

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

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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.     

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.