Comparative chromosome studies in species of subtribe Orchidinae (Orchidaceae)

In our study, FISH mapping using 18S-5.8S-25S rDNA and 5S rDNA sequences was performed for the first time on Ophrys tenthredinifera Willdenow, 1805, Serapias vomeracea (Burman f., 1770) Briquet, 1910 and Himantoglossum hircinum (Linnaeus, 1753) Sprengel, 1826. A detailed study was also performed on O. tenthredinifera using Giemsa-staining, silver-staining, CMA fluorescence banding and fluorescence in situ hybridisation (FISH) with rDNA probes. We analysed two subspecies, i.e. O. tenthredinifera subsp. neglecta (Parlatore, 1860) E.G. Camus, 1908 and O. tenthredinifera subsp. grandiflora (Tenore, 1819) Kreutz, 2004 by the traditional Feulgen method and constructed the karyotype. The cytotaxonomic implications for both taxa are also discussed. In Himantoglossum hircinum, FISH and silver staining highlighted differences in the number of two rDNA families (35S and 5S) with respect to Barlia robertiana (Loiseleur-Deslongchamps, 1807) Greuter, 1967. In addition, fluorescence in situ hybridisation was also applied to diploid (2n = 2x = 36) and triploid (2n = 3x = 54) Anacamptis morio (Linnaeus, 1753) R.M. Bateman, Pridgeon et M.W. Chase, 1997. As far as we are aware, this is the first case of autotriploidy observed in A. morio.

Staining Sodium Dodecyl Sulfate–Polyacrylamide Gels with Silver Salts

This protocol describes silver staining procedures to detect low-abundance proteins in sodium dodecyl sulfate-polyacrylamide gels.

BMAA, Methylmercury, and Mechanisms of Neurodegeneration in Dolphins: A Natural Model of Toxin Exposure

Dolphins are well-regarded sentinels for toxin exposure and can bioaccumulate a cyanotoxin called β-N-methylamino-l-alanine (BMAA) that has been linked to human neurodegenerative disease. The same dolphins also possessed hallmarks of Alzheimer’s disease (AD), suggesting a possible association between toxin exposure and neuropathology. However, the mechanisms of neurodegeneration in dolphins and the impact cyanotoxins have on these processes are unknown. Here, we evaluate BMAA exposure by investigating transcription signatures using PCR for dolphin genes homologous to those implicated in AD and related dementias: APP, PSEN1, PSEN2, MAPT, GRN, TARDBP, and C9orf72. Immunohistochemistry and Sevier Münger silver staining were used to validate neuropathology. Methylmercury (MeHg), a synergistic neurotoxicant with BMAA, was also measured using PT-GC-AFS. We report that dolphins have up to a three-fold increase in gene transcription related to Aβ+ plaques, neurofibrillary tangles, neuritic plaques, and TDP-43+ intracytoplasmic inclusions. The upregulation of gene transcription in our dolphin cohort paralleled increasing BMAA concentration. In addition, dolphins with BMAA exposures equivalent to those reported in AD patients displayed up to a 14-fold increase in AD-type neuropathology. MeHg was detected (0.16–0.41 μg/g) and toxicity associated with exposure was also observed in the brain. These results demonstrate that dolphins develop neuropathology associated with AD and exposure to BMAA and MeHg may augment these processes.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

The Contribution of Polysaccharide Antigens From Clinical Proteus spp. and Klebsiella spp. Isolates to the Serological Cross-Reactions

Klebsiella spp. and Proteus spp. cause hospital-acquired urinary tract infections (UTIs), which are often related to the use of catheters. To create a vaccine preventing UTI, immunogenic bacterial antigens with common epitopes are still being looked for. In this work, the role of polysaccharide antigens of four Klebsiella spp. and eight Proteus spp. strains in serological cross-reactions with specific antisera was examined. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and silver staining by Tsai method were performed. The Klebsiella and Proteus spp. LPSs and cells were used as antigens. Polyclonal rabbit sera specific to Klebsiella oxytoca 0.023 and 0.062 strains and four Klebsiella spp. LPSs were obtained. The ELISA and Western blotting results showed the strongest cross-reactions occurring between lipopolysaccharides (LPSs) from four Klebsiella strains and P. vulgaris O42 antiserum. The silver-staining procedure revealed the patterns typical of both slow- and fast-migrating mass species of the Klebsiella LPSs. The Klebsiella spp. antigens also cross-reacted with four P. penneri antisera, and most of the reactions were observed as low-migrating patterns. From two K. oxytoca antisera obtained in this work, only one, the K. oxytoca 0.062 antiserum, cross-reacted with satisfactory strength with P. penneri LPSs (19, 22, and 60). Obtaining cross-reactions between the antigens of Klebsiella strains and Proteus antisera and in the opposite systems is important for proving the immunogenic role of polysaccharide antigens in triggering the immunological response.

Investigation of the direct and indirect mechanisms of primary blast insult to the brain

The interaction of explosion-induced blast waves with the head (i.e., a direct mechanism) or with the torso (i.e., an indirect mechanism) presumably causes traumatic brain injury. However, the understanding of the potential role of each mechanism in causing this injury is still limited. To address this knowledge gap, we characterized the changes in the brain tissue of rats resulting from the direct and indirect mechanisms at 24 h following blast exposure. To this end, we conducted separate blast-wave exposures on rats in a shock tube at an incident overpressure of 130 kPa, while using whole-body, head-only, and torso-only configurations to delineate each mechanism. Then, we performed histopathological (silver staining) and immunohistochemical (GFAP, Iba-1, and NeuN staining) analyses to evaluate brain-tissue changes resulting from each mechanism. Compared to controls, our results showed no significant changes in torso-only-exposed rats. In contrast, we observed significant changes in whole-body-exposed (GFAP and silver staining) and head-only-exposed rats (silver staining). In addition, our analyses showed that a head-only exposure causes changes similar to those observed for a whole-body exposure, provided the exposure conditions are similar. In conclusion, our results suggest that the direct mechanism is the major contributor to blast-induced changes in brain tissues.

Whole-organism 3D Quantitative Characterization of Zebrafish Melanin by Silver Deposition Micro-CT

Melanin-rich zebrafish melanophores are used to study pigment development, human skin color, and as a large-scale screening phenotype. To facilitate more detailed whole-body, computational analyses of melanin content and morphology, we have combined X-ray microtomography (micro-CT), a non-destructive, full-volume imaging modality, with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, for both dramatic and subtle phenotypes not previously described. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, three-dimensional computational phenomic analysis of a particular cell type at cellular resolution, with potential applications in other model organisms and human melanoma biopsies. Whole-organism, high-resolution phenotyping is a challenging ideal, but provides superior context for functional studies of mutations, diseases, and environmental influences.