Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

Felid sex identification based on noninvasive genetic samples

We developed two tests for sex identification of felids using y‐chromosome deletions in the zinc‐finger and amelogenin regions. These tests provide positive results for both males and females, while reducing the need to co‐amplify microsatellites to test for DNA quality in hair and scat samples. Furthermore, the y‐chromosome deletions are absent in a wide‐range of prey species; thus, when these tests are used on scat samples, potential contamination caused by prey DNA incidentally extracted, is minimized.     

A new microcomputer-controlled neutron activation and analysis system

A microcomputer-controlled irradiation and measurement system and a microprocessor-controlled sample changer have been installed at the SLOWPOKE-2 Facility at the Royal Military College of Canada (RMC). These systems can provide the gamut of instrumental neutron activation analysis (INAA) techniques for the analyst. Custom software has been created for system control, data acquisition, and off-line spectral analysis using programs that incorporate Gaussian peak-fitting methods of analysis. The design and use of the equipment is discussed, and the performance is illustrated with results obtained from the analysis of marine sediment and biological reference materials.     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

Detection of Naegleria fowleri cysts in environmental samples by using a DNA probe

Abstract In this study we tried to detect DNA Naegleria fowleri in artificially contaminated environmental samples, with or without sediments, containing 10⁴ cysts of this pathogenic amoeba. We used two assays to extract DNA from samples: first, direct DNA extraction, which gave positive results only for water samples without sediment; second, DNA extraction after sample incubation on agar plates, which allowed us to remove amoeba growing out of the sediments, and which gave positive results for all samples, even those initially with sediments (5, 500 or 500 mg). Thus, this molecular identification appears as a powerful tool to investigate N. fowleri growth in environmental samples.     

Biological samples taken from Native American Ancestors are human remains under NAGPRA

In the United States, the Native American Graves Protection and Repatriation Act (NAGPRA) provides a specific framework for the disposition of Native American Ancestral remains within its purview. However, samples such as a bone fragment, tooth, or other biological tissue taken from the remains of these Ancestors have been treated by institutions and researchers as independent of the individual from whom they were removed and used in destructive research such as paleogenomic and other archaeometric analyses without consultation, consent, and collaboration from Native American communities; are not cared for in keeping with the current best practices for Indigenous Ancestors; and are not likely to be repatriated to their communities. Here, we demonstrate that any biological samples removed from Ancestors who are covered under NAGPRA must also be handled according to the stipulations defined for “human remains” within the legislation. As such, we are not proposing a change to existing legislation, but rather best practices, specific to the context of the United States and NAGPRA, relating to the use of and care for biological samples taken from Native American Ancestors.     

The concentrations of essential/toxic elements in serum of COVID-19 patients are not directly related to the severity of the disease

BackgroundIn recent months, the current COVID-19 pandemic has generated thousands of studies directly or indirectly related with this disease and/or the coronavirus SARS-CoV-2 causing the infection. On August 22, 2022, the database PUBMED included 287,639 publications containing the term COVID-19. However, in spite of the importance of trace elements in human health, including the immune system, data on the levels of metals/metalloids in COVID-19 patients is very limited.MethodsThe concentrations of As, Cd, Cr, Cu, Hg, Fe, Mg, Mn, Pb, Se, V and Zn were determined by inductively coupled plasma-mass spectrometry (ICP-MS) in 126 serum samples of individuals infected with SARS-CoV-2, as well as in 88 samples of non-infected individuals. Participants were divided into four groups: i) individuals COVID-19 positive (COVID-19 +) with an asymptomatic infection course; ii) individuals suffering mild COVID-19; iii) individuals suffering severe COVID-19, and iv) individuals COVID-19 negative (COVID-19-) (control group). The occurrence of the analyzed metals/metalloids was evaluated along with the biochemical profile, including blood cell counts, lipids, proteins and crucial enzymes.ResultsSerum levels of Mg, V, Cr, Cu, Cd, and Pb were higher in COVID-19 positive patients than those in the control group. Although no significant differences were observed between the different groups of patients, the concentrations of Cd, Pb, V and Zn showed a tendency to be higher in individuals with severe COVID-19 than in those showing mild symptoms or being asymptomatic. Arsenic and Hg were rarely detected, regardless if the subjects were infected by SARS-CoV-2, or not. The current results did not show significant differences in the levels of the rest of analyzed elements according to the severity of the disease (asymptomatic, mild and severe).ConclusionsIn spite of the results here obtained, we highlight the need to reduce the exposure to Cd, Pb and V to minimize the potential adverse health outcomes after COVID-19 infection. On the other hand, although a protective role of essential elements was not found, Mg and Cu concentrations were higher in severe COVID-19 patients than in non-infected individuals.     

Mutagenicity testing of benomyl, methyl-2-benzimidazole carbamate, streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium in vitro and in rodent host-mediated assays.

The fungicide benomyl and its commercial preparations Fundazol 50WP and Benlate 50WP and the benomyl metabolite methyl-2-benzimidazole carbamate and its commercial preparation MBC 50WP were tested for mutagenicity in in vitro spot tests, in microsomal plate assay, in liquid-culture treatments, or in rodent host-mediated assay. The base-pair substitution Salmonella typhimurium mutant hisG46 and the hisG46-bearing uvrB excision-repair-deficient mutants TA100, TA1530, TA1535 or TA1950 were used as test organisms. Complete genotypic information of these mutants is given in Ames et al. [2]. Captain 50WP, streptozotocin (SZN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-aminopurine and N-acetylaminofluorene were used as positive control compounds. In nonoverlay spot tests Benlate 50WP was not mutagenic over a dose range of 50-5000 microgram/spot in hisG46 and TA1535. In overlay spot tests 50 or 100 microgram/spot Benomyl, MBC, Fundazol 50WP, Benlate 50WP and MBC 50WP were tested in hisG46, TA1530 or TA1950. Only a non-commercial MBC sample at 100 microgram/spot showed weak mutagenic activity in hisG46. In microsomal activation plate assay MBC, benomyl, Fundazol 50WP and Benlate 50WP were tested in TA100 over a dose range of 50-2000 microgram/plate. None of the compounds showed mutagenicity. In a 20-h liquid-culture treatment 10, 100, 1000 and 10 000 microgram/ml Fundazol 50WP were not mutagenic in TA 30. In 1-h liquid-culture treatments benomyl, Benlate 50WP or Fundazol 50WP failed to induce mutations in hisG46, TA100 or TA1950 over a dose range of 0.25-1000 microgram/ml. Appropriate positive controls were mutagenic in each experiment. The consistently negative results in this study with commercial MBC and benomyl preparations are contrary to positive results reported earlier with similar methods and similar commercial preparations. Possible reasons to explain the different results are presented. The alkylating agents SZN and MNNG induced fewer mutations in TA1530 and TA1950 uvrB excision-repair-deficient strains than in the hisG46 excision-proficient strain, indicating that with these mutagens excision-repair is also a mutation-prone process. In rodent host-mediated assays with Fundazol 50WP in mice 3 consecutive subcutaneous hourly doses of 500 mg/kg in hisG46 and TA1950 and in rats or mice an oral dose of 4000 mg/kg in TA1950 were not mutagenic. The positive control SZN was mutagenic.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.