Pub Date : 2025-05-06DOI: 10.1016/j.acthis.2025.152248
Bing Gao , Maogong Xi , Ying Cui , Kai Wang , Hui Zhang , Yiyong Wang
Atherosclerosis (AS) significantly impacts both cardiovascular and cerebrovascular health, making it an important area of research for potential therapeutic interventions. This study investigates the role of lncRNA SNHG16 in macrophage polarization and its effects on the progression of AS. We assessed the expression of SNHG16 in macrophages and vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (ox-LDL) using qPCR. Cell proliferation was evaluated via EdU assay and western blotting, while flow cytometry and immunofluorescence were employed to analyze the polarization of macrophages. Foam cell formation was examined using Oil Red O staining. In a co-culture system, VSMCs treated with ox-LDL were cultured alongside macrophages pretreated with sh-SNHG16, and VSMC viability, migration, and motility were assessed using CCK-8, migration, and scratch assays. The levels of inflammatory cytokines, including IL-6, IL-10, TGFβ, and TNFα, were quantified by ELISA. Our results show that SNHG16 expression is upregulated in ox-LDL-treated cells, which correlates with enhanced macrophage proliferation. Inhibition of SNHG16 promoted M1-to-M2 macrophage polarization, reducing foam cell formation and inflammation. Furthermore, SNHG16 knockdown limited VSMC viability and motility, while attenuating ox-LDL-induced inflammatory responses. In conclusion, suppression of SNHG16 favors M2 macrophage polarization and presents a potential therapeutic target for AS management.
{"title":"SNHG16 suppression enhances M2 macrophage polarization and inhibits VSMC migration in atherosclerosis","authors":"Bing Gao , Maogong Xi , Ying Cui , Kai Wang , Hui Zhang , Yiyong Wang","doi":"10.1016/j.acthis.2025.152248","DOIUrl":"10.1016/j.acthis.2025.152248","url":null,"abstract":"<div><div>Atherosclerosis (AS) significantly impacts both cardiovascular and cerebrovascular health, making it an important area of research for potential therapeutic interventions. This study investigates the role of lncRNA SNHG16 in macrophage polarization and its effects on the progression of AS. We assessed the expression of SNHG16 in macrophages and vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (ox-LDL) using qPCR. Cell proliferation was evaluated via EdU assay and western blotting, while flow cytometry and immunofluorescence were employed to analyze the polarization of macrophages. Foam cell formation was examined using Oil Red O staining. In a co-culture system, VSMCs treated with ox-LDL were cultured alongside macrophages pretreated with sh-SNHG16, and VSMC viability, migration, and motility were assessed using CCK-8, migration, and scratch assays. The levels of inflammatory cytokines, including IL-6, IL-10, TGFβ, and TNFα, were quantified by ELISA. Our results show that SNHG16 expression is upregulated in ox-LDL-treated cells, which correlates with enhanced macrophage proliferation. Inhibition of SNHG16 promoted M1-to-M2 macrophage polarization, reducing foam cell formation and inflammation. Furthermore, SNHG16 knockdown limited VSMC viability and motility, while attenuating ox-LDL-induced inflammatory responses. In conclusion, suppression of SNHG16 favors M2 macrophage polarization and presents a potential therapeutic target for AS management.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152248"},"PeriodicalIF":2.3,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143906975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ambystoma mexicanum, also known as the axolotl, is a paedomorphic urodele. Metamorphosis can be induced experimentally, and the most significant changes occur in the skin. These include thinning of the epidermis, increased keratinization of the stratified squamous epithelium, and loss of Leydig cells (LCs). Similar epidermal changes are observed in other metamorphic urodeles. Epidermal cells are responsible for the secretory function of the skin in juvenile amphibians, whereas dermal glands perform this function in adults after metamorphosis. In the axolotl, this occurrence is still partially understood. The only recognized epidermal secretory cells in juvenile A. mexicanum are the LCs, whose specific secretion products have not yet been characterized from the histochemical standpoint. Additionally, the persistence of LCs in adulthood, when mucous and serous (granular-protein secretion) glands are abundant, remains a matter of debate. The present study aims to describe the morphological and histochemical changes in the epidermis of 10 cutaneous regions from juvenile (4 months old) and adult (24 and 48 months old) non-metamorphic A. mexicanum, with a particular focus on the amount and histochemical characteristics of LCs. Results indicate that the juvenile epidermis is a stratified cuboidal epithelium formed by three strata: basal, spinosum (containing the LCs), and apical. The most superficial layer contains cuboidal cells that lack the characteristics of a true stratum corneum. In adults, the stratum apical is also formed by squamous cells, suggesting a transition to a cornified and squamous layer as age increases. Histochemical methods demonstrated that LCs are most likely serous and not mucous cells. On the other hand, cuboidal cells of the juvenile apical stratum would be responsible for producing mucous secretion components. Morphometric analysis revealed a significant decrease in both LCs and the epidermal thickness in the 24-month-old adult axolotl compared to the juvenile. While LC count and epidermal thickness in the 48-month-old adult showed a slight increase compared to the 24-month-old adult, these differences were not statistically significant and far lower than those observed in the juvenile axolotl, which exhibited the highest number of LCs and a thicker epidermis. These natural axolotl epidermal changes indicate a gradual transition toward a morphology resembling metamorphic skin as age advances. The decreased number of LCs and the transition from cuboid cells to squamous cells in the stratum apical suggest that both cell types may naturally disappear entirely at some point during development.
{"title":"Histological, histochemical, and morphometric analysis of epidermal Leydig cells and histochemical characterization of epidermal apical cells in juvenile and adult axolotls (Ambystoma mexicanum)","authors":"Omar Betancourt-León , Verónica Rodríguez-Mata , Antonieta Martínez-Guerrero , Armando Pérez-Torres","doi":"10.1016/j.acthis.2025.152255","DOIUrl":"10.1016/j.acthis.2025.152255","url":null,"abstract":"<div><div><em>Ambystoma mexicanum</em>, also known as the axolotl, is a paedomorphic urodele. Metamorphosis can be induced experimentally, and the most significant changes occur in the skin. These include thinning of the epidermis, increased keratinization of the stratified squamous epithelium, and loss of Leydig cells (LCs). Similar epidermal changes are observed in other metamorphic urodeles. Epidermal cells are responsible for the secretory function of the skin in juvenile amphibians, whereas dermal glands perform this function in adults after metamorphosis. In the axolotl, this occurrence is still partially understood. The only recognized epidermal secretory cells in juvenile <em>A. mexicanum</em> are the LCs, whose specific secretion products have not yet been characterized from the histochemical standpoint. Additionally, the persistence of LCs in adulthood, when mucous and serous (granular-protein secretion) glands are abundant, remains a matter of debate. The present study aims to describe the morphological and histochemical changes in the epidermis of 10 cutaneous regions from juvenile (4 months old) and adult (24 and 48 months old) non-metamorphic <em>A. mexicanum</em>, with a particular focus on the amount and histochemical characteristics of LCs. Results indicate that the juvenile epidermis is a stratified cuboidal epithelium formed by three strata: basal, spinosum (containing the LCs), and apical. The most superficial layer contains cuboidal cells that lack the characteristics of a true stratum corneum. In adults, the stratum apical is also formed by squamous cells, suggesting a transition to a cornified and squamous layer as age increases. Histochemical methods demonstrated that LCs are most likely serous and not mucous cells. On the other hand, cuboidal cells of the juvenile apical stratum would be responsible for producing mucous secretion components. Morphometric analysis revealed a significant decrease in both LCs and the epidermal thickness in the 24-month-old adult axolotl compared to the juvenile. While LC count and epidermal thickness in the 48-month-old adult showed a slight increase compared to the 24-month-old adult, these differences were not statistically significant and far lower than those observed in the juvenile axolotl, which exhibited the highest number of LCs and a thicker epidermis. These natural axolotl epidermal changes indicate a gradual transition toward a morphology resembling metamorphic skin as age advances. The decreased number of LCs and the transition from cuboid cells to squamous cells in the stratum apical suggest that both cell types may naturally disappear entirely at some point during development.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152255"},"PeriodicalIF":2.3,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143864230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tissue observation has traditionally been limited to obtaining two-dimensional information from thinly sliced tissues due to issues with light transmission and antibody penetration. In recent years, three-dimensional tissue observation methods combining tissue clearing and deep immunostaining methods have been reported. However, due to the significantly different procedures in these methods from conventional immunostaining methods and the requirement for an expensive and specialized light-sheet microscope for tissue observation, the widespread adoption of these methods has been limited. To promote the shift from the current two-dimensional tissue observation to three-dimensional tissue observation using a combination of tissue clearing and immunostaining, it is essential to establish a simple and easy-to-implement protocol that is compatible with observation using a confocal microscope, which is available in many facilities. In this study, we first examined the effects of tissue clearing and staining conditions of immunostaining with thin tissue slices. We showed that CUBIC-L enhances immunolabeling without diminishing the immunoreactivity of antigens. We also showed that high detergent concentrations enhance the intensity of immunoreactivity and that a two-step staining procedure is suitable for our proposed protocol. Based on the results, we propose a simple protocol that can be easily adapted from conventional methods and is compatible with confocal microscopes. The results of this study are expected to facilitate a shift from traditional methods to three-dimensional tissue observation techniques that combine tissue clearing and immunostaining, contributing to the broader adoption of three-dimensional tissue observation.
{"title":"Proposal for a simple and easy-to-implement protocol for three-dimensional tissue imaging that is compatible with observation using a confocal microscope","authors":"Takuto Matano , Kiyotada Naitou , Jannatul Ferdous , Takahiko Shiina , Mitsuya Shiraishi","doi":"10.1016/j.acthis.2025.152257","DOIUrl":"10.1016/j.acthis.2025.152257","url":null,"abstract":"<div><div>Tissue observation has traditionally been limited to obtaining two-dimensional information from thinly sliced tissues due to issues with light transmission and antibody penetration. In recent years, three-dimensional tissue observation methods combining tissue clearing and deep immunostaining methods have been reported. However, due to the significantly different procedures in these methods from conventional immunostaining methods and the requirement for an expensive and specialized light-sheet microscope for tissue observation, the widespread adoption of these methods has been limited. To promote the shift from the current two-dimensional tissue observation to three-dimensional tissue observation using a combination of tissue clearing and immunostaining, it is essential to establish a simple and easy-to-implement protocol that is compatible with observation using a confocal microscope, which is available in many facilities. In this study, we first examined the effects of tissue clearing and staining conditions of immunostaining with thin tissue slices. We showed that CUBIC-L enhances immunolabeling without diminishing the immunoreactivity of antigens. We also showed that high detergent concentrations enhance the intensity of immunoreactivity and that a two-step staining procedure is suitable for our proposed protocol. Based on the results, we propose a simple protocol that can be easily adapted from conventional methods and is compatible with confocal microscopes. The results of this study are expected to facilitate a shift from traditional methods to three-dimensional tissue observation techniques that combine tissue clearing and immunostaining, contributing to the broader adoption of three-dimensional tissue observation.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152257"},"PeriodicalIF":2.3,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859605","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-21DOI: 10.1016/j.acthis.2025.152254
Fuping Gao , Xiaohua Zhou , Jin Wei , Qiong Sun , Jiapeng Wang , Qing Li
Objective: The chorionic gonadotropin (CG) subunit beta 5 (CGB5) gene is a member of the glycoprotein hormone β chain family, encoding the β5 subunit of CG, which has been shown to promote tumorigenesis and induce proliferation in various types of cancer including gastric cancer (GC). However, the mechanistic role of CGB5 in GC has not been fully elucidated. Therefore, this study investigated relevant genes that regulate GC through bioinformatics analysis. Methods: Immunohistochemistry, immunofluorescence, and western blot (WB) detection methods were appropriately used to evaluate the expression pattern and clinical significance of CGB5 in 100 Chinese GC patients that were recruited from the Gaochun People's Hospital. The effect of small interfering ribonucleic acid (siRNA) on apoptosis, migration, and invasion of GC cells was investigated in vitro. Three-dimensional tumor spheres of these two types of GC cells (NCI-N87 cells and MKN45 cells) were constructed before investigation of the Calcein acetoxymethyl ester (AM)/ Propidium iodide (PI) staining, flow cytometric apoptosis, and apoptotic-related protein content of the tumor spheres after siRNA inhibition of CGB5 expression. Results: It was observed that compared with adjacent normal gastric tissue, expression of CGB5 was significantly upregulated in GC tissue. The siRNA inhibited CGB5 expression in two GC cell lines (NCI-N87 cells and MKN45 cells). Also, it was discovered that CGB5 highly correlated with microsatellite instability (MSI) and immune cell activity in GC, thus revealing the greater research value of CGB5 gene. More importantly, CGB5 siRNA could inhibit invasion and migration of tumor cells, induce apoptosis of GC cells and GC tumor spheres, as well as the mechanism relating to regulation of apoptosis associated gene expression. Overall, the findings suggest that CGB5 may play a crucial role in the development of GC carcinogenesis. Thus, this research may contribute to design of potential drug targets for treatment of GC.
{"title":"Expression characteristics and biological functions of CGB5 gene in gastric cancer","authors":"Fuping Gao , Xiaohua Zhou , Jin Wei , Qiong Sun , Jiapeng Wang , Qing Li","doi":"10.1016/j.acthis.2025.152254","DOIUrl":"10.1016/j.acthis.2025.152254","url":null,"abstract":"<div><div>Objective: The chorionic gonadotropin (CG) subunit beta 5 (CGB5) gene is a member of the glycoprotein hormone β chain family, encoding the β5 subunit of CG, which has been shown to promote tumorigenesis and induce proliferation in various types of cancer including gastric cancer (GC). However, the mechanistic role of CGB5 in GC has not been fully elucidated. Therefore, this study investigated relevant genes that regulate GC through bioinformatics analysis. Methods: Immunohistochemistry, immunofluorescence, and western blot (WB) detection methods were appropriately used to evaluate the expression pattern and clinical significance of CGB5 in 100 Chinese GC patients that were recruited from the Gaochun People's Hospital. The effect of small interfering ribonucleic acid (siRNA) on apoptosis, migration, and invasion of GC cells was investigated in vitro. Three-dimensional tumor spheres of these two types of GC cells (NCI-N87 cells and MKN45 cells) were constructed before investigation of the Calcein acetoxymethyl ester (AM)/ Propidium iodide (PI) staining, flow cytometric apoptosis, and apoptotic-related protein content of the tumor spheres after siRNA inhibition of CGB5 expression. Results: It was observed that compared with adjacent normal gastric tissue, expression of CGB5 was significantly upregulated in GC tissue. The siRNA inhibited CGB5 expression in two GC cell lines (NCI-N87 cells and MKN45 cells). Also, it was discovered that CGB5 highly correlated with microsatellite instability (MSI) and immune cell activity in GC, thus revealing the greater research value of CGB5 gene. More importantly, CGB5 siRNA could inhibit invasion and migration of tumor cells, induce apoptosis of GC cells and GC tumor spheres, as well as the mechanism relating to regulation of apoptosis associated gene expression. Overall, the findings suggest that CGB5 may play a crucial role in the development of GC carcinogenesis. Thus, this research may contribute to design of potential drug targets for treatment of GC.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152254"},"PeriodicalIF":2.3,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143852056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Approximately 90 % of humans are infected with the Epstein-Barr virus (EBV); however, most do not develop neoplastic lesions. Despite various investigations, the underlying reasons remain largely unknown. Therefore, we aimed to address this question through morphological observations to identify the ultrastructural alterations occurring in EBV-infected cells. EBV-positive cells from legacy lymph node specimens obtained from patients with HIV and modern fresh specimens of aberrantly proliferating human EBV-positive lymphocytes in a patient-derived xenograft (PDX) of immunodeficient mouse were examined. By utilizing a special technique that allowed us to observe exactly the same specimen using both optical and electron microscopy, we were able to detect a peculiar phenomenon in EBV-infected cells. EBV-infected lymphocytes occasionally exhibited nuclear cavities, a finding that was confirmed in multiple specimens from both patients with HIV and the PDX model. It was suggested that EBV-infected cells may activate cell death pathways, based on the protein expression patterns of p53 and FAS. Taken together, these results indicated that nuclear cavity formation appeared to be a characteristic morphological alteration associated with EBV infection. Further research could clarify the potential relationship between nuclear cavities and cellular biology in EBV-infected cells, possibly shedding light on the mechanisms that prevent EBV-infected cells from progressing to tumor formation.
{"title":"Discovery of nuclear cavities in Epstein-Barr virus-infected cells","authors":"Shinji Shimada , Hideya Kawasaki , Hiroto Katoh , Shumpei Ishikawa","doi":"10.1016/j.acthis.2025.152253","DOIUrl":"10.1016/j.acthis.2025.152253","url":null,"abstract":"<div><div>Approximately 90 % of humans are infected with the Epstein-Barr virus (EBV); however, most do not develop neoplastic lesions. Despite various investigations, the underlying reasons remain largely unknown. Therefore, we aimed to address this question through morphological observations to identify the ultrastructural alterations occurring in EBV-infected cells. EBV-positive cells from legacy lymph node specimens obtained from patients with HIV and modern fresh specimens of aberrantly proliferating human EBV-positive lymphocytes in a patient-derived xenograft (PDX) of immunodeficient mouse were examined. By utilizing a special technique that allowed us to observe exactly the same specimen using both optical and electron microscopy, we were able to detect a peculiar phenomenon in EBV-infected cells. EBV-infected lymphocytes occasionally exhibited nuclear cavities, a finding that was confirmed in multiple specimens from both patients with HIV and the PDX model. It was suggested that EBV-infected cells may activate cell death pathways, based on the protein expression patterns of p53 and FAS. Taken together, these results indicated that nuclear cavity formation appeared to be a characteristic morphological alteration associated with EBV infection. Further research could clarify the potential relationship between nuclear cavities and cellular biology in EBV-infected cells, possibly shedding light on the mechanisms that prevent EBV-infected cells from progressing to tumor formation.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152253"},"PeriodicalIF":2.3,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143848641","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-18DOI: 10.1016/j.acthis.2025.152256
Gabriella Meier Bürgisser, Pietro Giovanoli, Maurizio Calcagni, Johanna Buschmann
Collagen I and III distribution is not only crucial to assess the status of healing wounds, but also to characterise healthy connective tissue and pathological extracellular matrix composition. In this technical note, we have therefore compared the dual-coloured Herovici staining, indicating pink collagen I and blue collagen III in serial sections with immunohistochemistry (IHC) labellings for collagen I and III, respectively. Furthermore, we used, chromogenic DAB for IHC labelling. Seven different organs of a healthy New Zealand white rabbit were collected for this purpose, including kidney, liver, tonsil, tongue, duodenum, heart, and brain, respectively. A dual-coloured staining like Herovici turned out to be as good as two single-colour labellings utilising IHC. In some cases, co-localisation and extent of collagen I and III expression could be qualitatively visualised better using Herovici, with gradients of blue-violet-pink, than by mere comparison of labelling intensities side by side in two different sections, although taken at the same place as serial sections. Nevertheless, a quantitative analysis of the Collagen I-to-III ratio revealed no significant differences between these two approaches to assess the extracellular matrix composition. From these comparisons, we conclude that a Herovici staining is recommended as a valuable alternative staining to collagen I and III IHC; and it may act as a fast and cheap preliminary staining method. These findings encourage researchers focusing on ECM composition of the experimental rabbit tissue to use Herovici staining to determine the ratio of the extracellular collagen I and III expression.
{"title":"Comparison of collagen I and collagen III immunohistochemistry with Herovici staining in various rabbit organs","authors":"Gabriella Meier Bürgisser, Pietro Giovanoli, Maurizio Calcagni, Johanna Buschmann","doi":"10.1016/j.acthis.2025.152256","DOIUrl":"10.1016/j.acthis.2025.152256","url":null,"abstract":"<div><div>Collagen I and III distribution is not only crucial to assess the status of healing wounds, but also to characterise healthy connective tissue and pathological extracellular matrix composition. In this technical note, we have therefore compared the dual-coloured Herovici staining, indicating pink collagen I and blue collagen III in serial sections with immunohistochemistry (IHC) labellings for collagen I and III, respectively. Furthermore, we used, chromogenic DAB for IHC labelling. Seven different organs of a healthy New Zealand white rabbit were collected for this purpose, including kidney, liver, tonsil, tongue, duodenum, heart, and brain, respectively. A dual-coloured staining like Herovici turned out to be as good as two single-colour labellings utilising IHC. In some cases, co-localisation and extent of collagen I and III expression could be qualitatively visualised better using Herovici, with gradients of blue-violet-pink, than by mere comparison of labelling intensities side by side in two different sections, although taken at the same place as serial sections. Nevertheless, a quantitative analysis of the Collagen I-to-III ratio revealed no significant differences between these two approaches to assess the extracellular matrix composition. From these comparisons, we conclude that a Herovici staining is recommended as a valuable alternative staining to collagen I and III IHC; and it may act as a fast and cheap preliminary staining method. These findings encourage researchers focusing on ECM composition of the experimental rabbit tissue to use Herovici staining to determine the ratio of the extracellular collagen I and III expression.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152256"},"PeriodicalIF":2.3,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143843744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Uracil-DNA glycosylase (UDG) plays a key role in the base repair system, and detecting its enzymatic activity is crucial for early disease diagnosis. A rapid method for detecting UDG was developed, utilizing amplification initiated by a ligation reaction. A DNA probe modified with uracil was utilized to ligate two free DNA strands to form a newly generated DNA strand. This triggers a nicking enzyme-assisted amplification reaction, resulting in the production of single-stranded DNA (ssDNA). Then, the amplified ssDNA triggered the molecular beacons to emit fluorescence. However, the addition of UDG results in the removal of uracil from the DNA probe strand, leaving abasic site (AP site). After heat denaturation, this site was destroyed, preventing subsequent ligation or amplification reactions, resulting in the absence of fluorescence. The findings of our study indicate that the addition of UDG at concentrations exceeding 0.5 U/mL resulted in complete suppression of fluorescence intensity, reaching a value of 0. Conversely, in the absence of the UDG enzyme or upon the addition of other enzymes and proteins such as HAAG, EndoIV and BSA, the fluorescence intensity of the system remains unaffected, achieving 100 % intensity within 5–20 min. This study presents a rapid method for assessing UDG activity that could be valuable for early disease diagnosis in the future.
{"title":"Fluorescent strategy for detection of uracil-DNA glycosylase activity based on isothermal amplification triggered by ligase","authors":"Pansong Zhang , Fangfang He , Xin Chang , Chenxia Ren","doi":"10.1016/j.acthis.2025.152252","DOIUrl":"10.1016/j.acthis.2025.152252","url":null,"abstract":"<div><div>Uracil-DNA glycosylase (UDG) plays a key role in the base repair system, and detecting its enzymatic activity is crucial for early disease diagnosis. A rapid method for detecting UDG was developed, utilizing amplification initiated by a ligation reaction. A DNA probe modified with uracil was utilized to ligate two free DNA strands to form a newly generated DNA strand. This triggers a nicking enzyme-assisted amplification reaction, resulting in the production of single-stranded DNA (ssDNA). Then, the amplified ssDNA triggered the molecular beacons to emit fluorescence. However, the addition of UDG results in the removal of uracil from the DNA probe strand, leaving abasic site (AP site). After heat denaturation, this site was destroyed, preventing subsequent ligation or amplification reactions, resulting in the absence of fluorescence. The findings of our study indicate that the addition of UDG at concentrations exceeding 0.5 U/mL resulted in complete suppression of fluorescence intensity, reaching a value of 0. Conversely, in the absence of the UDG enzyme or upon the addition of other enzymes and proteins such as HAAG, EndoIV and BSA, the fluorescence intensity of the system remains unaffected, achieving 100 % intensity within 5–20 min. This study presents a rapid method for assessing UDG activity that could be valuable for early disease diagnosis in the future.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152252"},"PeriodicalIF":2.3,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143838018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Endometrioid adenocarcinoma (EA) has been on the increase in recent years in developed countries. Early detection of endometrioid adenocarcinoma in the endometrial corpus is crucial for patient prognosis and early treatment, although their distinction can sometimes be challenging. In this study, we focused on DNA replication-related proteins through immunohistochemical analysis and investigated whether the discrimination between EA and their precursor lesions is achievable using machine learning techniques. The research utilized tissue specimens from 100 cases, including EA of different grades (Grade 1; G1, Grade 2; G2, Grade 3; G3) and their precursor lesions (endometrial hyperplasia without atypia; EH, endometrial atypical hyperplasia: AH). Immunohistochemical analysis of DNA replication-related proteins, such as ORC1, Cdt1, Cdc6, MCM7, Cdc7, and Geminin, was conducted for each case, measuring the Labeling Index (LI) and optical density (OD) of protein expression. Furthermore, we performed statistical significance tests and machine learning -discriminant analysis using LI and OD as inputs, employing non-linear Support Vector Machines (NSVM). The NSVM discriminant analysis demonstrated the accuracy of over 85 % between EH and each differentiation grade of EA, the accuracy is also similar for AH and each differentiation grade of EA. In addition, changing the combination of DNA replication-related proteins used for discrimination resulted in a high accuracy (95–100 %). A discriminant analysis with NSVM using the LI and OD of DNA replication-related proteins may enable the differentiation of EA from its precursor lesions.
{"title":"Immunohistochemistry and machine learning study of DNA replication-associated proteins in uterine epithelial tumors and precursor lesions","authors":"Takumi Urata , Fumikazu Kimura , Kengo Ohshima , Koyo Ikehata , Masahiro Yamaguchi , Keiko Ishii","doi":"10.1016/j.acthis.2025.152251","DOIUrl":"10.1016/j.acthis.2025.152251","url":null,"abstract":"<div><div>Endometrioid adenocarcinoma (EA) has been on the increase in recent years in developed countries. Early detection of endometrioid adenocarcinoma in the endometrial corpus is crucial for patient prognosis and early treatment, although their distinction can sometimes be challenging. In this study, we focused on DNA replication-related proteins through immunohistochemical analysis and investigated whether the discrimination between EA and their precursor lesions is achievable using machine learning techniques. The research utilized tissue specimens from 100 cases, including EA of different grades (Grade 1; G1, Grade 2; G2, Grade 3; G3) and their precursor lesions (endometrial hyperplasia without atypia; EH, endometrial atypical hyperplasia: AH). Immunohistochemical analysis of DNA replication-related proteins, such as ORC1, Cdt1, Cdc6, MCM7, Cdc7, and Geminin, was conducted for each case, measuring the Labeling Index (LI) and optical density (OD) of protein expression. Furthermore, we performed statistical significance tests and machine learning -discriminant analysis using LI and OD as inputs, employing non-linear Support Vector Machines (NSVM). The NSVM discriminant analysis demonstrated the accuracy of over 85 % between EH and each differentiation grade of EA, the accuracy is also similar for AH and each differentiation grade of EA. In addition, changing the combination of DNA replication-related proteins used for discrimination resulted in a high accuracy (95–100 %). A discriminant analysis with NSVM using the LI and OD of DNA replication-related proteins may enable the differentiation of EA from its precursor lesions.</div></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":"127 2","pages":"Article 152251"},"PeriodicalIF":2.3,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143816633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-05DOI: 10.1016/j.acthis.2025.152250
Tania M. Cortázar , Nohora A. Vega , Jinneth Acosta , Edgar A. Reyes-Montaño , Manuel A. Ballen-Vanegas , Orlando Ricaurte
Galactia lindenii lectin type-II (GLL-II) belongs to the group of the legume lectins. The present study investigated the GLL-II staining patterns in histological sections of neoplastic and non-neoplastic thyroid tissues. Besides, hemagglutination assays (HA) using the GLL-II on red blood cells of different glycomic profiles were performed, complementing previous results. The differential staining in Papillary Thyroid Cancer, Invasive Encapsulated Follicular Variant Papillary Thyroid Carcinoma, Hashimoto's thyroiditis, and non-neoplastic thyroid with goiter changes, together with the HA results, allowed us to propose the potential utility of GLL-II as part of lectin platforms used to discriminate between human thyroid pathological samples from normal ones. The present study shed light on potential applications of GLL-II in determining alterations of glycosylation patterns in specific cells, tissues, or body fluids, as well as glycotopes biomarkers of healthy or pathological conditions.
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