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Abstract: Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.
Keywords: immunohistochemistry, chromogens, aminoethyl carbazole, AEC, 3, 3'-diaminobenzidine, DAB, antigen retrieval, tonsil
Published in DiRROS: 24.07.2024; Views: 235; Downloads: 174
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Abstract: Hazelnut (Corylus avellana) is cultivated on 118 ha and ranks eighth in Slovenian fruit growing production, representing 2.8% of the total area of fruit plantations in the country. However, decline of some of the trees appeared in 2012 in two plantations located in eastern Slovenia. Together these orchards cover 5 ha, with around 1600 trees planted 12 to 15 years ago. By October 2018, ~12% of these trees had died, and an additional 12% showed decay symptoms. The dead and dying trees were scattered throughout both orchards, with no apparent pattern. The most affected cultivar was ‘Istrska dolgoplodna leska’. Using molecular diagnostic methods, we showed infection of symptomatic trees with three unrelated phytoplasmas: ‘Candidatus Phytoplasma fragariae’, of the 16SrXII-E phytoplasma subgroup, and phytoplasma of the 16SrV and 16SrIX groups. In 2018, the presence of ‘Ca. P. fragariae’ and/or phytoplasma of 16SrV group were confirmed in decayed hazelnut trees in eastern, north-eastern, central, south-eastern and western Slovenia. ‘Ca. P. fragariae’ has also been detected in a forest in south-western Slovenia, for Acer campestre, Carpinus betulus, Crataegus laevigata, Fraxinus ornus and Quercus petraea. All infected forest trees showed unusual dense proliferation of sprouts from roots and/or trunks. Molecular characterisations of partial 16S rRNA, secY, map and ribosomal protein genetic locus of hazelnut 16SrV phytoplasma isolates show that they are identical to isolates that can cause grapevine flavescence dorée disease. Here, the results of our recent study and the open questions on this burning issue for hazelnut production are presented.
Keywords: Corylus avellana, ʼCandidatus Phytoplasma fragariaeʼ, 16SrV phytoplasma group, 16SrIX phytoplasma group, decline
Published in DiRROS: 24.07.2024; Views: 261; Downloads: 183
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Abstract: Our understanding of spider reproductive biology is hampered by the vast anatomical diversity and difficulties associated with its study. Although authors agree on the two general types of female spider genitalia, haplogyne (plesiomorphic) and entelegyne (apomorphic), our understanding of variation within each group mostly concerns the external genital part, while the internal connections with the reproductive duct are largely unknown. Conventionally and simplistically, the spermathecae of haplogynes have simple two-way ducts, and those of entelegynes have separate copulatory and fertilization ducts for sperm to be transferred in and out of spermathecae, respectively. Sperm is discharged from the spermathecae directly into the uterus externus (a distal extension of the oviduct), which, commonly thought as homologous in both groups, is the purported location of internal fertilization in spiders. However, the structural evolution from haplo- to entelegyny remains unresolved, and thus the precise fertilization site in entelegynes is ambiguous. We aim to clarify this anatomical problem through a widely comparative morphological study of internal female genital system in entelegynes. Our survey of 147 epigyna (121 examined species in 97 genera, 34 families) surprisingly finds no direct connection between the fertilization ducts and the uterus externus, which, based on the homology with basal-most spider lineages, is a dead-end caecum in entelegynes. Instead, fertilization ducts usually connect with a secondary uterus externus, a novel feature taking over the functional role of the plesiomorphic uterus externus. We hypothesize that the transition from haplo- to entelegyny entailed not only the emergence of the two separate duct systems (copulatory, fertilization), but also involved substantial morphological changes in the distal part of the oviduct. Thus, the common oviduct may have shifted its distal connection from the uterus externus to the secondary uterus externus, perhaps facilitating discharge of larger eggs. Our findings suggest that the conventional model of entelegyne reproduction needs redefinition.
Keywords: spiders, comparative anatomy, genital anatomy
Published in DiRROS: 23.07.2024; Views: 451; Downloads: 230
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Abstract: Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Keywords: plant pathogens, potato virus Y, viral RNA
Published in DiRROS: 23.07.2024; Views: 293; Downloads: 223
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Abstract: One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
Keywords: absolute quantification, AAV, gene therapy, electron microscopy, digital PCR, real-time PCR
Published in DiRROS: 23.07.2024; Views: 332; Downloads: 162
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