Micro-computed tomography (micro-CT) scanners allow for the three-dimensional analysis of bone structure to be visualized and numerous bone structural parameters to be quantified with a high degree of accuracy. Nucleic acid-based immunohistochemistry (IHC) allows for characterization of the presence, amounts and different types of DNA in paraffin-embedded fixed tissue sections. Micro-CT scanners (i.e., Bruker SkyScan 1173 µCT system), along with immunohistochemically processed tissue sections were used to examine a variety of different fixatives in order to characterize the effects of fixation on bone morphology (i.e., adult chicken), and preservation of different types of intact, non-denatured nucleic acids (e.g., B-DNA, Z-DNA). Several different types of bone (e.g., femur, tibia, and vertebra) were characterized. Fixation was performed using 10% Neutral Buffered Formalin (NBF), and Formalin-Alcohol-Acetic Acid (FAA) for 24, 48, 72 and 96 hours at room temperature. Both decalcified and non-decalcified bone tissues were processed. Molecular biological grade fixatives and water were used in order to ensure the best histotechnological results. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact B-DNA and Z-DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA. Tissues processed in 10% NBF required antigen retrieval. Our group is correlating morphological differences observed with different fixatives using a microCT scanner, relative to DNA identification employing immunohistochemical staining of bone tissue sections with anti-B-DNA and anti-Z-DNA monoclonal antibodies. Our data demonstrates that FAA results in easier histotechnological processing of bone tissue, better morphological results and superior IHC staining. Research project was supported by an NSF grant, Award ID No: 1828305. .

Terminal differentiation (i.e., denucleation) is a type of cell death, similar to apoptosis and necrosis, and is considered an alternative cell death pathway. Denucleating secondary crystalline lens fiber cells, keratinocytes of the epidermis and erythrocytes use this cell death mechanism to maintain their normal cellular physiology. In all three cases, the overall process of organelle removal is somewhat understood morphologically and biochemically; however, the molecular biology is not fully known. Structural transitions of the canonical A‐RNA into alternative structures such as left‐handed Z‐RNA, and the formation of multistranded DNA's (e.g., quadruplex DNA) are important in cells. We are examining how structural polymorphisms play a role in regulating cell death in the three above mentioned tissues, namely, denucleation, and how it differs from apoptosis and necrosis. Immunohistochemistry was performed to study the distribution and specific amounts of single‐stranded (ss−) DNA, A‐RNA, Z‐RNA and quadruplex DNA during denucleation. Immunohistochemical staining of tissue sections were performed with anti‐ss‐DNA, anti‐A‐RNA, anti‐Z‐RNA and anti‐quadruplex DNA antibodies. We observed ss‐DNA, A‐RNA, Z‐RNA and quadruplex DNA immunoreactivity. Both negative, positive and isotype controls were used. ELISA data correlates well with our immunohistochemical results. Immunohistochemical staining of tissues for the demonstration and quantification of ss‐DNA, A‐RNA, Z‐RNA and G4‐quadruplex DNA, reveals different levels of decreasing and increasing immunoreactivity as dying cells progress thru denucleation. Immunohistochemistry shows different levels of gradually decreasing quadruplex DNA as cells progress thru denucleation, with precipitous declines in certain tissue zones at specific times [i.e., this correlates well with decrease in B‐DNA (conrtols)]. A‐RNA and mainly Z‐RNA content decreases during denucleation. A decline in the presence of quadruplex DNA also reveals how denucleation is eradicating different DNA structures. This gradual decline in quadruplex DNA represents chromatin fragmentation. A precipitous decline in quadruplex DNA occurs towards the end of denucleation, depending on tissue type. We believe that specific types of canonical (e.g. B‐DNA), alternative (e.g., Z‐DNA) and multistranded DNAs (e.g., triplex DNA and quadruplex DNA) regulate pro‐ and anti‐cell death factors (e.g., DNase IIβ, CDK1, and cyclins in lens fiber cells, respectively), which control programmed elimination of cellular organelles and DNA. Pro‐cell death factors allows the denucleated cells to remain viable for a certain period of time. Denucleating keratinocytes, secondary crystalline lens fibers and erythrocytes share some common initial characteristics with apoptosis and necrosis, however, the latter part of denucleation is completely different. Based on our research we believe that pro‐apoptotic proteins are involved in the early stages of denucleation, and then other mechanisms (e.g., anti‐apoptotic) take over the remaining processes of terminal differentiation. The gradual decrease in A‐RNA, Z‐RNA and quadruplex DNA during denucleation may play a role in regulating this alternative cell death pathway. Our results suggest that dissimilar nucleic acid structures cause nucleic acid fragmentation in denucleating cells, which differs from apoptosis and necrosis, in the sequence of events and the time course.Support or Funding InformationProject was supported by several NYIT ISRC and TriBeta Biology Honor Society grants.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Medicinal treatments for this pathology are limited and therefore new approaches need to be taken, namely, new classes of drugs and/or biologicals. Tissues were processed in formalin-fixed and non-formalin-fixed paraffin-embedded tissue sections. Proper fixation of melanoma tissue samples is critical for the correct preservation of tissue morphology, and especially tissue-bound nucleic acids. Improper fixation will lead to regions of single-stranded (ss-) DNA that can interfere with the correct characterization of the tissue-bound components. Our group has developed a histotechnological procedure to preserve undamaged nucleic acids (1). Right-handed double-stranded (ds-) B-DNA is the conventional structure of DNA. In the past we have examined the epidermis of normal human skin for the presence of ds-B-DNA, ds-Z-DNA as it undergoes destruction due to the normal process of cell death [apoptosis and terminal differentiation (denucleation)]. Our research team has examined the distribution and intensity of anti-B-DNA antibody and anti-melanoma antibody binding in human melanoma (Ia and I B). Our results show that B-DNA is located in all cells of the melanoma tissue; however, the intensity of immunohistochemical staining is different within certain regions of the cancerous growth (an increased amount of ds-DNA content the vertical growth phase zone: papillary dermis). Using enhanced histotechnological processing procedures we were able to better preserve the tissue-bound ds-B-DNA, which was not damaged from tissue processing (i.e., ds-DNA converting to ss-DNA). This resulted in intact ds-B-DNA. Being able to locate intact ds-B-DNA in the cells of cancer will allow for the identification of specific target sites. 1. Gagna C.E., et al., (2007) Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-Stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry. Journal of Histochemistry and Cytochemistry. 55: 999-1014. Supported by a 2011 ISRC grant.

Stage II melanoma is a localized tissue tumor. Proper fixation of melanoma tissue samples is extremely important for the accurate preservation of tissue morphology, and mainly nucleic acids. Incorrect histotechnological fixation will lead to regions of denatured single-stranded DNA that can interfere with the correct characterization of the nucleic acid tissue-bound components. We have developed a procedure to preserve intact nucleic acids (1). Right-handed double-stranded (ds-) B-DNA is the conventional structure of DNA. Our team has examined the epidermis of normal human skin for the presence of ds-B-DNA as it undergoes cell death [apoptosis and terminal differentiation]. Our group has now examined the distribution and intensity of anti-B-DNA antibody, anti-melanoma antibody, and anti-single-stranded (ss-) DNA binding in Stage II human melanoma. Our results show that B-DNA is located in all cells of the melanoma tissue; however, the intensity of immunohistochemical staining is different within certain regions of the cancerous growth. Less immunohistochemical staining was found in the lateral regional and more in the vertical areas (i.e., papillary dermis). Using enhanced histotechnological processing procedures we were able to better preserve the tissue-bound ds-B-DNA. Consequently, the DNA was not damaged from tissue processing, and did not result in denatured ss-DNA that would interfere with the characterization of ds-B-DNA. We are also looking at differences between the DNA of ulcerated and non-ulcerated melanomas. Being able to differentiate between ds-B-DNA and ss-DNA in the cancer tissue will allow for the identification of specific nucleic acid target sites. 1. Gagna C.E., et al., (2007) Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-Stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry. Journal of Histochemistry and Cytochemistry. 55: 999-1014. Supported by an ISRC grant.

Stage 0 melanoma is an early pathology referred to as melanoma in situ. This stage of tumor growth is limited to the epidermis. We investigated the differences in DNA structure within tumor initiation and tumor promotion sites. Right-handed double-stranded (ds-) B-DNA is the conventional structure of DNA that results in the majority of DNA. As part of our past work we examined the epidermis of normal human skin for the presence of ds-B-DNA as it undergoes destruction due to normal cell death processes [i.e., apoptosis and terminal differentiation (denucleation)]. Our research team has examined the distribution and intensity of anti-B-DNA antibody binding in human melanoma; formalin-fixed paraffin-embedded tissue sections (1 micron). We also employed a variety of different anti-melanoma antibody probes [e.g., (HMB45) (ab787)]. Using enhanced histotechnological processing procedures we were able to better preserve the melanoma tissue-bound B-DNA (i.e., intact, unaltered and non-denatured nucleic acids). Superior preservation of tissue-bound components resulted in improved characterization of the immunostaining data (1). We characterized the lateral and vertical margins of the epidermal tumor growth to see if any changes were occurring in the non-cancerous areas of the epidermis next to the tumor sites. We found that B-DNA is located in all cells, and that the binding intensity [mean optical density] of immunohistochemistry is similar in all regions of the cancerous growth. Being able to locate hyperactive regions of B-DNA in the tumor growth will allow for new drug target sites. disease. 1. Gagna C.E., et al., (2007) Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-Stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry. Journal of Histochemistry and Cytochemistry. 55: 999-1014. Supported by a NYIT-ISRC grant.

Stage III melanoma refers to tissue tumors that have spread to regional lymph nodes, or have developed in transit metastasis (regional). With treatment this pathology is considered intermediate to high risk for recurrence locally or for distant metastasis. Consequently, new approaches towards treating melanoma need to be developed. Right-handed double-stranded (ds-) B-DNA is the most common structure that makes up the majority of DNA. Tissue samples were preserved in several different tissue fixatives (molecular grade), in order to better characterize DNA and DNA-protein complex interactions (10% formalin, 10% neutral buffered formalin, Clarke's solution, Carnoy's, solution, and zinc formalin fixative). Previously, we have characterized the epidermis of human skin for the presence of ds-B-DNA as it undergoes cell death [i.e., apoptosis and terminal differentiation]. Our data reveals the distribution and intensity of anti-B-DNA, anti-single-stranded DNA, anti-Z-RNA antibody binding, and a variety of different anti-melanoma antibody, in human melanoma (IIIA, IIIB, and IIIC). We carefully observed the differences in DNA structure within the papillary dermis. The intensity of immunohistochemical staining is different within certain regions of the cancerous growth, namely, less immunohistochemical staining in the lateral regional and much more in the vertical areas. Less ss-DNA was seen in the vertical areas (reticular dermis). Employing novel histotechnological processing procedures we were able to better preserve the tissue-bound ds-B-DNA as intact, unaltered and non-denatured molecules (1). This has resulted in improvements involving laser capture dissection techniques for the isolation of genetic materials. 1. Gagna CE, et al., (2007) Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-Stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry. Journal of Histochemistry and Cytochemistry. 55: 999-1014. This research project was supported in part by a 2011 ISRC grant.

The degree of cell proliferation in a tumor is often associated with metastatic risk and mortality. Proliferating cell nuclear antigen (PCNA) and Ki-67 are proliferation markers that can be used to assess malignant potential in cutaneous lesions and pathological cell proliferation in psoriasis. These markers are elevated during periods of cell proliferation; however, they are also upregulated following UV irradiation. This upregulation may be problematic, as many skin lesions are subject to sun exposure in an everyday setting.

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