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Introduction: Prior studies suggested that neuromuscular factors might be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS). The neuromuscular junction (NMJ) is the important pivot where the nervous system interacts with muscle fibers, but it has not been well characterized in the paraspinal muscles of AIS. This study aims to perform the quantitative morphological analysis of NMJs from paraspinal muscles of AIS. Methods: AIS patients who received surgery in our center were prospectively enrolled. Meanwhile, age‐matched congenital scoliosis (CS) and non‐scoliosis patients were also included as controls. Fresh samples of paraspinal muscles were harvested intraoperatively. NMJs were immunolabeled using different antibodies to reveal pre‐synaptic neuronal architecture and post‐synaptic motor endplates. A confocal microscope was used to acquire z‐stack projections of NMJs images. Then, NMJs images were analyzed on maximum intensity projections using ImageJ software. The morphology of NMJs was quantitatively measured by a standardized 'NMJ‐morph' workflow. A total of 21 variables were measured and compared between different groups. Results: A total of 15 AIS patients, 10 CS patients and 5 normal controls were enrolled initially. For AIS group, NMJs in the convex side of paraspinal muscles demonstrated obviously decreased overlap when compared with the concave side (34.27% ± 8.09% vs. 48.11% ± 10.31%, p = 0.0036). However, no variables showed statistical difference between both sides of paraspinal muscles in CS patients. In contrast with non‐scoliosis controls, both sides of paraspinal muscles in AIS patients demonstrated significantly smaller muscle bundle diameters. Conclusions: This study first elucidated the morphological features of NMJs from paraspinal muscles of AIS patients. The NMJs in the convex side showed smaller overlap for AIS patients, but no difference was found in CS. This proved further evidence that neuromuscular factors might contribute to the mechanisms of AIS and could be considered as a novel potential therapeutic target for the treatment of progressive AIS. [ABSTRACT FROM AUTHOR]

Candida lusitaniae vaginitis is clinically rare. This article reports a case of vaginitis caused by Candida lusitaniae in a 40-year-old female patient. The main clinical manifestations of the patient were pruritus vulvae before menstruation, increased vaginal discharge, and recurrent symptoms for six months. Vaginal secretions from the patient were directly smeared and subjected to immunofluorescence staining, Gram staining, and Giemsa staining, revealing fungal spores upon microscopic examination. Identification using CHROMagar chromogenic plates revealed the presence of Candida lusitaniae. The bioMérieux VITEK-2 Compact automated microbiology system identified the organism as Candida lusitaniae. The diagnostic and treatment process of this case suggests that laboratory diagnosis of Candida lusitaniae can easily be confused with other Candida species. To prevent underreporting or misreporting, it is necessary to consider the possibility of infection by rare strains in refractory cases.

This study investigates NADPH oxidase 4 (NOX4) involvement in iron-mediated astrocyte cell death in Alzheimer's Disease (AD) using single-cell sequencing data and transcriptomes. We analyzed AD single-cell RNA sequencing data, identified astrocyte marker genes, and explored biological processes in astrocytes. We integrated AD-related chip data with ferroptosis-related genes, highlighting NOX4. We validated NOX4's role in ferroptosis and AD in vitro and in vivo. Astrocyte marker genes were enriched in AD, emphasizing their role. NOX4 emerged as a crucial player in astrocytic ferroptosis in AD. Silencing NOX4 mitigated ferroptosis, improved cognition, reduced Aβ and p-Tau levels, and alleviated mitochondrial abnormalities. NOX4 promotes astrocytic ferroptosis, underscoring its significance in AD progression. [ABSTRACT FROM AUTHOR]

Abstract Introduction Prior studies suggested that neuromuscular factors might be involved in the pathogenesis of adolescent idiopathic scoliosis (AIS). The neuromuscular junction (NMJ) is the important pivot where the nervous system interacts with muscle fibers, but it has not been well characterized in the paraspinal muscles of AIS. This study aims to perform the quantitative morphological analysis of NMJs from paraspinal muscles of AIS. Methods AIS patients who received surgery in our center were prospectively enrolled. Meanwhile, age‐matched congenital scoliosis (CS) and non‐scoliosis patients were also included as controls. Fresh samples of paraspinal muscles were harvested intraoperatively. NMJs were immunolabeled using different antibodies to reveal pre‐synaptic neuronal architecture and post‐synaptic motor endplates. A confocal microscope was used to acquire z‐stack projections of NMJs images. Then, NMJs images were analyzed on maximum intensity projections using ImageJ software. The morphology of NMJs was quantitatively measured by a standardized ‘NMJ‐morph’ workflow. A total of 21 variables were measured and compared between different groups. Results A total of 15 AIS patients, 10 CS patients and 5 normal controls were enrolled initially. For AIS group, NMJs in the convex side of paraspinal muscles demonstrated obviously decreased overlap when compared with the concave side (34.27% ± 8.09% vs. 48.11% ± 10.31%, p = 0.0036). However, no variables showed statistical difference between both sides of paraspinal muscles in CS patients. In contrast with non‐scoliosis controls, both sides of paraspinal muscles in AIS patients demonstrated significantly smaller muscle bundle diameters. Conclusions This study first elucidated the morphological features of NMJs from paraspinal muscles of AIS patients. The NMJs in the convex side showed smaller overlap for AIS patients, but no difference was found in CS. This proved further evidence that neuromuscular factors might contribute to the mechanisms of AIS and could be considered as a novel potential therapeutic target for the treatment of progressive AIS.

Background/purpose: Dental pulp stem cells (DPSCs) exhibit versatile differentiation capabilities, including neural differentiation, prompting the hypothesis that they may be implicated in the neurodevelopment of teeth. This study aimed to explore the temporospatial dynamics between DPSCs and tooth innervation, employing immunofluorescence staining and fluorescent dye injections to investigate the distribution of DPSCs, neural stem cells (NSCs), nerve growth cones, and sensory nerves in developing mouse tooth germs at various stages. Materials and methods: Immunofluorescence staining targeting CD146, Nestin, and GAP-43, along with the injection of AM1-43 fluorescent dye, were utilized to observe the distribution of DPSCs, NSCs, nerve growth cones, and sensory nerves in mouse tooth germs at different developmental stages. Results: Positive CD146 immunostaining was observed in microvascular endothelial cells and pericytes within and around the tooth germ. The percentage of CD146-positive cells remained consistent between 4-day-old and 8-day-old second molar tooth germs. Conversely, Nestin expression in odontoblasts and their processes decreased in 8-day-old tooth germs compared to 4-day-old ones. Positive immunostaining for GAP-43 and AM1-43 fluorescence revealed the entry of nerve growth cones and sensory nerves into the pulp in 8-day-old tooth germs, while these elements were confined to the dental follicle in 4-day-old germs. No co-localization of CD146-positive DPSCs with nerve growth cones and sensory nerves was observed. Conclusion: DPSCs and NSCs were present in dental pulp tissue before nerves penetrated the pulp. The decline in NSCs after nerve entry suggests a potential role for DPSCs and NSCs in attracting neural growth and/or differentiation within the pulp.

Dental pulp stem cells (DPSCs) exhibit versatile differentiation capabilities, including neural differentiation, prompting the hypothesis that they may be implicated in the neurodevelopment of teeth. This study aimed to explore the temporospatial dynamics between DPSCs and tooth innervation, employing immunofluorescence staining and fluorescent dye injections to investigate the distribution of DPSCs, neural stem cells (NSCs), nerve growth cones, and sensory nerves in developing mouse tooth germs at various stages. Immunofluorescence staining targeting CD146, Nestin, and GAP-43, along with the injection of AM1-43 fluorescent dye, were utilized to observe the distribution of DPSCs, NSCs, nerve growth cones, and sensory nerves in mouse tooth germs at different developmental stages. Positive CD146 immunostaining was observed in microvascular endothelial cells and pericytes within and around the tooth germ. The percentage of CD146-positive cells remained consistent between 4-day-old and 8-day-old second molar tooth germs. Conversely, Nestin expression in odontoblasts and their processes decreased in 8-day-old tooth germs compared to 4-day-old ones. Positive immunostaining for GAP-43 and AM1-43 fluorescence revealed the entry of nerve growth cones and sensory nerves into the pulp in 8-day-old tooth germs, while these elements were confined to the dental follicle in 4-day-old germs. No co-localization of CD146-positive DPSCs with nerve growth cones and sensory nerves was observed. DPSCs and NSCs were present in dental pulp tissue before nerves penetrated the pulp. The decline in NSCs after nerve entry suggests a potential role for DPSCs and NSCs in attracting neural growth and/or differentiation within the pulp. [ABSTRACT FROM AUTHOR]

Introduction: This was an ambispective cohort study evaluating the prognostic significance of lymphocytic foci and its lymphoid composition in minor salivary gland biopsy (MSGB) for short-term disease flare and severity in Sjögren's syndrome (SS). Methods: The inclusion criteria comprised individuals meeting the ACR/EULAR 2016 criteria who underwent MSGB with an infiltration of more than 50 lymphocytes and received clinical diagnosis between September 2017 and December 2018. Patients with inadequate biopsy samples were excluded. The number of lymphocytic foci and their lymphoid composition in MSGB were assessed using immunofluorescence staining. Major organ damage and improvements in the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) were measured. Statistical analyses, including Cox and linear regressions, were conducted. Results: A total of 78 patients with at least one lymphocytic focus were included in the study. The presence of higher T-cell counts in lymphocytic foci in MSGB was associated with severe disease flare, and a logarithmic transformation of Tcell count indicated increased risk (HR 1.96, 95% CI 0.91-4.21). Improvements in the ESSDAI were associated with higher total lymphocyte count and T- and Bcell numbers in the lymphoid composition of the lymphocytic foci. Seropositive patients exhibited higher T CD4+ cell numbers. Correlation analysis showed negative associations between age and lymphocytic foci and the T-cell count. Positive correlations were observed between antinuclear antibody (ANA) titers and total lymphocyte numbers. Discussion: Patients with a higher number of T cells in the lymphocytic infiltrates of lymphocytic foci may have a two-fold risk of severe disease flare. The number of B cells and T CD4+ cells in the lymphocytic infiltrates of lymphocytic foci showed a weak but positive relation with the ESSDAI improvement during followup. Age and seropositivity appeared to influence the lymphoid composition of the lymphocytic foci. [ABSTRACT FROM AUTHOR]

Abstract Background Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. Methods We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. Results The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. Conclusion Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques.

Abstract Background Diabetic foot ulcers (DFU) are among the fastest-growing diseases worldwide. Recent evidence has emphasized the critical role of microRNA (miRNA)-mRNA networks in various chronic wounds, including DFU. In this study, we aimed to clarify the miRNA-mRNA axes associated with the occurrence of DFU. Methods Expression profiles of miRNAs and mRNAs were extracted from the Gene Expression Omnibus. Differentially expressed genes and differentially expressed miRNAs were identified, and miRNA-mRNA regulatory axes were constructed through integrated bioinformatics analyses. We validated the miRNA-mRNA axes using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays. We conducted an immune infiltration analysis and confirmed the bioinformatics results using immunofluorescence staining. Single-sample gene set enrichment analysis (ssGSEA) was used to analyze the metabolic mechanisms. Results miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 interactions were identified using in silico analysis. The qPCR results showed apparent dysregulation of these miRNA-mRNA axes in DFU. The dual-luciferase reporter assay confirmed that miR-182-5p targeted CHL1 and MITF, and miR-338-3p targeted NOVA1. We conducted an immune infiltration analysis and observed that key genes correlated with decreased infiltration of M1 macrophages and resting mast cells in DFU. Immunofluorescence staining verified the co-localization of CHL1 and tryptase, while MITF and CD68 showed weak positive correlations. Metabolic pathways related to these three genes were identified using ssGSEA. Conclusions In summary, the miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions and decreased infiltration of M1 macrophages and resting mast cells may provide novel clues to the pathogenesis of DFU. Trial registration The clinical trial included in this study was registered in the Chinese Clinical Trial Registry ( ChiCTR2200066660 ) on December 13, 2022.

Background: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. Methods: We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. Results: The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. Conclusion: Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques. [ABSTRACT FROM AUTHOR]

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo. [ABSTRACT FROM AUTHOR]

Background: Diabetic foot ulcers (DFU) are among the fastest-growing diseases worldwide. Recent evidence has emphasized the critical role of microRNA (miRNA)-mRNA networks in various chronic wounds, including DFU. In this study, we aimed to clarify the miRNA-mRNA axes associated with the occurrence of DFU. Methods: Expression profiles of miRNAs and mRNAs were extracted from the Gene Expression Omnibus. Differentially expressed genes and differentially expressed miRNAs were identified, and miRNA-mRNA regulatory axes were constructed through integrated bioinformatics analyses. We validated the miRNA-mRNA axes using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays. We conducted an immune infiltration analysis and confirmed the bioinformatics results using immunofluorescence staining. Single-sample gene set enrichment analysis (ssGSEA) was used to analyze the metabolic mechanisms. Results: miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 interactions were identified using in silico analysis. The qPCR results showed apparent dysregulation of these miRNA-mRNA axes in DFU. The dual-luciferase reporter assay confirmed that miR-182-5p targeted CHL1 and MITF, and miR-338-3p targeted NOVA1. We conducted an immune infiltration analysis and observed that key genes correlated with decreased infiltration of M1 macrophages and resting mast cells in DFU. Immunofluorescence staining verified the co-localization of CHL1 and tryptase, while MITF and CD68 showed weak positive correlations. Metabolic pathways related to these three genes were identified using ssGSEA. Conclusions: In summary, the miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions and decreased infiltration of M1 macrophages and resting mast cells may provide novel clues to the pathogenesis of DFU. Trial registration: The clinical trial included in this study was registered in the Chinese Clinical Trial Registry (ChiCTR2200066660) on December 13, 2022. [ABSTRACT FROM AUTHOR]

Although remarkable progress has been made, colorectal cancer remains a significant global health issue. One of the most challenging aspects of cancer treatment is the resistance of tumor cells to classical chemotherapy. Conventional therapy for colorectal cancer often involves the use of 5-fluorouracil as a chemotherapeutic agent. Aspirin, a drug used primarily to prevent cardiovascular complications, became a focus of attention due to its potential use as an antitumor agent. The purpose of the study was to evaluate the potential synergistic cytotoxic effects of aspirin and 5-fluorouracil on colorectal adenocarcinoma cells. The viability of cells, the impact on the morphology and nuclei of cells, the potential antimigratory effect, and the impact on the expression of the major genes associated with cell apoptosis (Bcl-2, Bax, Bad), as well as caspases 3 and 8, were evaluated. The results indicated that the two compounds exerted a synergistic effect, causing a reduction in cell viability accompanied by changes characteristic of the apoptosis process—the condensation of nuclei and the reorganization of actin filaments in cells, the reduction in the expression of the Bcl-2 gene, and the increase in the expression of Bax and Bad genes, along with caspases 3 and 8. Considering all these findings, it appears that aspirin may be investigated in depth in order to be used in conjunction with 5-fluorouracil to increase antitumor activity.

Vessel pathology such as increased permeability and blue discoloration is frequently observed with highly pathogenic PRRSV strains. However, data concerning the viral replication in the environment of blood vessels are absent.In the present study, ex vivo models with swine ear and hind leg vein explants were established to study the interaction of PRRSV-1 subtype 1 reference strain LV and highly pathogenic subtype 3 strain Lena with perivenousmacrophages. The replication characteristics of these two strainswere compared in vein explants by immunofluorescence analysis. The explants maintained a good viability during 48 hours of in vitro culture. We found that CD163- positive macrophages were mainly present around the veins and their number gradually decreased with increasing distance from the veins and longer incubation time. More CD163+ Sn- cells than CD163+ Sn+ cells (6.6 times more) were observed in the vein explants. The Lena strain demonstrated a higher replication level than the LV strain, with approximately 1.4-fold more infected cells in the surrounding areas of the ear vein and 1.1-fold more infected cells in the leg vein explants at 48 hours post inoculation. In both LV and Lena inoculated vein explants, most infected cells were identified as CD163+ Sn+ (> 94%). In this study, an ex vivo vein model was successfully established, and our findings will contribute to a better understanding of the vein pathology during viral infections (e.g., PRRS, classical and African swine fever). [ABSTRACT FROM AUTHOR]

Although remarkable progress has been made, colorectal cancer remains a significant global health issue. One of the most challenging aspects of cancer treatment is the resistance of tumor cells to classical chemotherapy. Conventional therapy for colorectal cancer often involves the use of 5-fluorouracil as a chemotherapeutic agent. Aspirin, a drug used primarily to prevent cardiovascular complications, became a focus of attention due to its potential use as an antitumor agent. The purpose of the study was to evaluate the potential synergistic cytotoxic effects of aspirin and 5-fluorouracil on colorectal adenocarcinoma cells. The viability of cells, the impact on the morphology and nuclei of cells, the potential antimigratory effect, and the impact on the expression of the major genes associated with cell apoptosis (Bcl-2, Bax, Bad), as well as caspases 3 and 8, were evaluated. The results indicated that the two compounds exerted a synergistic effect, causing a reduction in cell viability accompanied by changes characteristic of the apoptosis process—the condensation of nuclei and the reorganization of actin filaments in cells, the reduction in the expression of the Bcl-2 gene, and the increase in the expression of Bax and Bad genes, along with caspases 3 and 8. Considering all these findings, it appears that aspirin may be investigated in depth in order to be used in conjunction with 5-fluorouracil to increase antitumor activity. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the immunofluorescence patterns of autophagic markers: Light chain 3 beta (LC3B), Glucose regulating protein 78 (GRP78), Heat shock cognate 71 (HSC70) and Lysosomal-associated membrane protein 2A (LAMP2A) in the developing and postnatal kidneys of Dab1−/− (yotari) mice to those of wild-type samples. Embryos were obtained on gestation days 13.5 and 15.5 (E13.5 and E15.5), and adult animals were sacrificed at postnatal days 4, 11 and 14 (P4, P11, and P14). After fixation and dehydration, paraffin-embedded kidney tissues were sectioned and incubated with specific antibodies. Using an immunofluorescence microscope, sections were analyzed. For statistical analysis, a two-way ANOVA test and a Tukey's multiple comparison test were performed with a probability level of p < 0.05. A significant increase in GRP78 and LAMP2A expression was observed in the renal vesicles and convoluted tubules of yotari in embryonic stages. In postnatal kidneys, all observed proteins showed higher signal intensities in proximal and distal convoluted tubules of yotari, while a higher percentage of LC3B-positive cells was also observed in glomeruli. Our findings suggest that all of the examined autophagic markers play an important role in normal kidney development, as well as the potential importance of these proteins in renal pathology, where they primarily serve a protective function and thus may be used as diagnostic and therapeutic targets. [ABSTRACT FROM AUTHOR]

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence. [ABSTRACT FROM AUTHOR]

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0–4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at −20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved. [ABSTRACT FROM AUTHOR]

Pericentric heterochromatin (PCH) forms spatio-temporarily distinct compartments and affects chromosome organization and stability. Albeit some of its components are known, an elucidation of its proteome and how it differs between tissues in vivo is lacking. Here, we find that PCH compartments are dynamically organized in a tissue-specific manner, possibly reflecting compositional differences. As the mouse brain and liver exhibit very different PCH architecture, we isolated native PCH fractions from these tissues, analyzed their protein compositions using quantitative mass spectrometry, and compared them to identify common and tissue-specific PCH proteins. In addition to heterochromatin-enriched proteins, the PCH proteome includes RNA/transcription and membrane-related proteins, which showed lower abundance than PCH-enriched proteins. Thus, we applied a cut-off of PCH-unspecific candidates based on their abundance and validated PCH-enriched proteins. Amongst the hits, MeCP2 was classified into brain PCH-enriched proteins, while linker histone H1 was not. We found that H1 and MeCP2 compete to bind to PCH and regulate PCH organization in opposite ways. Altogether, our workflow of unbiased PCH isolation, quantitative mass spectrometry, and validation-based analysis allowed the identification of proteins that are common and tissue-specifically enriched at PCH. Further investigation of selected hits revealed their opposing role in heterochromatin higher-order architecture in vivo.

Background: Mesenchymal stem cells (MSCs) are beneficial for cell and gene therapy approaches. These cells are highly potent in producing various cells, including adipocytes, osteoblasts, hematopoietic cells, etc., particularly bone marrow-derived. High proliferation and regenerative capacity are important phenotypes of MSCs which contribute to the recovery process at the damaged sites. Various techniques have been used, which have inconvenient effects on stromal cell properties. The present study evaluates the stem cell markers in the isolated cells from the Murine mouse bone marrow. Methodology: The murine mesenchymal stem cells (mMSCs) were isolated from the bone marrow of murine Balb/c mice using a simple in vitro culture protocol. Trypan blue exclusion assay was performed for viable cell count, and cellular morphology was checked using an inverted microscope. The immunofluorescence staining was done to analyze the immunophenotypic features of mMSCs by treating cells with CD44, CD90, and CD45 fluorescent antibodies. Results: mMSCs with spindle-like cell appearance were attained within three weeks. Immunofluorescence staining of isolated mMSCs displayed a statistically significant (p

Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard “in-house” assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.

The tumor suppressor protein p53 is mutated in half of all cancers and has been described to form amyloid-like structures, commonly known from key proteins in neurodegenerative diseases. Still, the clinical relevance of p53 aggregates remains largely unknown, which may be due to the lack of sensitive and specific detection methods. The aim of the present study was to compare the suitability of four different methodologies to specifically detect p53 aggregates: co-immunofluorescence (co-IF), proximity ligation assay (PLA), co-immunoprecipitation (co-IP), and the p53-Seprion-ELISA in cancer cell lines and epithelial ovarian cancer tissue samples. In 7 out of 10 (70%) cell lines, all applied techniques showed concordance. For the analysis of the tissue samples co-IF, co-IP, and p53-Seprion-ELISA were compared, resulting in 100% concordance in 23 out of 30 (76.7%) tissue samples. However, Co-IF lacked specificity as there were samples, which did not show p53 staining but abundant staining of amyloid proteins, highlighting that this method demonstrates that proteins share the same subcellular space, but does not specifically detect p53 aggregates. Overall, the PLA and the p53-Seprion-ELISA are the only two methods that allow the quantitative measurement of p53 aggregates. On the one hand, the PLA represents the ideal method for p53 aggregate detection in FFPE tissue, which is the gold-standard preservation method of clinical samples. On the other hand, when fresh-frozen tissue is available the p53-Seprion-ELISA should be preferred because of the shorter turnaround time and the possibility for high-throughput analysis. These methods may add to the understanding of amyloid-like p53 in cancer and could help stratify patients in future clinical trials targeting p53 aggregation. [ABSTRACT FROM AUTHOR]

Craniopharyngiomas (CPs) are histologically benign tumors located in the sellar-suprasellar region. Although the transcriptome development in recent years have deepened our knowledge to the tumorigenesis process of adamantinomatous craniopharyngioma (ACP), the peritumoral immune infiltration of tumor is still not well understood. In this study, weighted gene coexpression network analysis (WGCNA) was applied to identify different gene modules based on clinical characteristics and gene expression, and then, the protein-protein interaction (PPI) network with the Cytohubba plug-in were performed to screen pivotal genes. In addition, immune cell infiltration (ICI) analysis was used to evaluate the immune microenvironment of ACP patients. In total, 8,568 differential expression genes were identified based on our datasets and two microarray profiles from the public database. The functional enrichment analysis revealed that upregulated genes were mainly enriched in immune-related pathways while downregulated genes were shown in the hormone and transduction of signaling pathways. The WGCNA investigated the most relevant modules, and 1,858 hub genes was detected, from which the PPI network identified 14 pivotal genes, and the Hypoxia-inducible factor 1-alpha (HIF-1α) pathway including four critical genes may be involved in the development of ACP. Moreover, naïve CD4+ and CD8+ T cells were decreased while specific subtypes of T cells were significantly increased in ACP patients according to ICI analysis. Validation by immunofluorescence staining revealed a higher expression of HIF-1α in ACP (ACP vs. control) and adult-subtype (adult vs. children), suggesting a possible state of immune system activation. Notably, children with low HIF-1α scores were related to the hypothalamus involvement and hydrocephalus symptoms. In this study, we successfully identified HIF-1α as a key role in the tumorigenesis and development of ACP through comprehensive integrated analyses and systematically investigated the potential relationship with immune cells in ACP. The results may provide valuable resources for understanding the underlying mechanisms of ACP and strengthen HIF-1α as a potential immunotherapeutic target in clinical application. [ABSTRACT FROM AUTHOR]

Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C. elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid–structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures.

The tumor suppressor protein p53 is mutated in half of all cancers and has been described to form amyloid-like structures, commonly known from key proteins in neurodegenerative diseases. Still, the clinical relevance of p53 aggregates remains largely unknown, which may be due to the lack of sensitive and specific detection methods. The aim of the present study was to compare the suitability of four different methodologies to specifically detect p53 aggregates: co-immunofluorescence (co-IF), proximity ligation assay (PLA), co-immunoprecipitation (co-IP), and the p53-Seprion-ELISA in cancer cell lines and epithelial ovarian cancer tissue samples. In 7 out of 10 (70%) cell lines, all applied techniques showed concordance. For the analysis of the tissue samples co-IF, co-IP, and p53-Seprion-ELISA were compared, resulting in 100% concordance in 23 out of 30 (76.7%) tissue samples. However, Co-IF lacked specificity as there were samples, which did not show p53 staining but abundant staining of amyloid proteins, highlighting that this method demonstrates that proteins share the same subcellular space, but does not specifically detect p53 aggregates. Overall, the PLA and the p53-Seprion-ELISA are the only two methods that allow the quantitative measurement of p53 aggregates. On the one hand, the PLA represents the ideal method for p53 aggregate detection in FFPE tissue, which is the gold-standard preservation method of clinical samples. On the other hand, when fresh-frozen tissue is available the p53-Seprion-ELISA should be preferred because of the shorter turnaround time and the possibility for high-throughput analysis. These methods may add to the understanding of amyloid-like p53 in cancer and could help stratify patients in future clinical trials targeting p53 aggregation.

Immunofluorescence staining is an important method for detecting the localization of proteins in the cell. It is also frequently used in the localization study of chloroplast-division proteins. Although this method has been improved before by using protoplasts, it still has some limitations. Now we developed a new method to make it much easier. We just broke the plant leaf tissue with a serrated blade, stained the samples directly, and simply lysed the tissue into separatable cells. The localization of the target protein can then be observed with a clear view. Since this method directly uses broken leaf pieces, it is very fast. It can also be applied to the plants in which protoplasts are difficult to prepare. We first used this method to observe the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis. A ring was clearly seen in the middle of chloroplasts. In addition, we used this method to analyze the localization of FtsZ1 in arc3 and pdv2 mutants, as well as in dozens of other species, including some woody plants. This new immunofluorescence staining method is not only easy to use, but also has a wide applicability in various plants. [ABSTRACT FROM AUTHOR]

Simple Summary: Cyromazine, an insect growth regulator, is used to control the Dipteran pest population. Previous findings observed that treatment with cyromazine increased the larval mortality, by interfering with the ecdysone signaling. In addition, the application of exogenous 20E significantly reduced the mortality caused by cyromazine. Many studies have also supported the role of ecdysone signaling in the maintenance of germline stem cells (GSCs), where mutations in ecdysone signaling-related genes significantly decreased the number of GSCs. However, to date, no study has reported the effect of cyromazine on the GSCs of Drosophila melanogaster. In the present study, we observed that cyromazine significantly reduced the number of both GSCs and cystoblasts (CBs) in the ovary of adult female. To further understand the effect of cyromazine on germ cells, we selected some key genes related to the ecdysone signaling pathway and evaluated their expression through RT-qPCR. Additionally, we measured the ecdysone titer from the cyromazine-treated ovaries. Our results indicated a significant decrease in the expression of ecdysone signaling-related genes and also in the ecdysone titer. These results further supported our findings that cyromazine reduced the number of germ cells by interfering with the ecdysone signaling pathway. In the present study, we observed a 58% decrease in the fecundity of Drosophila melanogaster, after treatment with the cyromazine. To further elucidate the effects of cyromazine on reproduction, we counted the number of both germline stem cells (GSCs) and cystoblasts (CBs) in the ovary of a 3-day-old adult female. The results showed a significant decrease in the number of GSCs and CBs as compared to the control group. The mode of action of cyromazine is believed to be through the ecdysone signaling pathway. To further support this postulate, we observed the expression of key genes involved in the ecdysone signaling pathway and also determined the ecdysone titer from cyromazine-treated ovaries. Results indicated a significant decrease in the expression of ecdysone signaling-related genes as compared to the control group. Furthermore, the titer of the ecdysone hormone was also markedly reduced (90%) in cyromazine-treated adult ovaries, suggesting that ecdysone signaling was directly related to the decrease in the number of GSCs and CBs. However, further studies are required to understand the mechanism by which cyromazine affects the GSCs and CBs in female adult ovaries. [ABSTRACT FROM AUTHOR]

Abstract Background Inappropriate matching of motor and sensory fibers after nerve repair or nerve grafting can lead to failure of nerve recovery. Identification of motor and sensory fibers is important for the development of new approaches that facilitate neural regeneration and the next generation of nerve signal-controlled neuro-prosthetic limbs with sensory feedback technology. Only a few methods have been reported to differentiate sensory and motor nerve fascicles, and the reliability of these techniques is unknown. Immunofluorescence staining is one of the most commonly used methods to distinguish sensory and motor nerve fibers, however, its accuracy remains unknown. Methods In this study, we aim to determine the efficacy of popular immunofluorescence markers for motor and sensory nerve fibers. We harvested the facial (primarily motor fascicles) and sural (primarily sensory fascicles) nerves in rats, and examined the immunofluorescent staining expressions of motor markers (choline acetyltransferase (ChAT), tyrosine kinase (TrkA)), and sensory markers [neurofilament protein 200 kDa (NF-200), calcitonin gene-related peptide (CGRP) and Transient receptor potential vanillic acid subtype 1 (TRPV1)]. Three methods, including the average area percentage, the mean gray value, and the axon count, were used to quantify the positive expression of nerve markers in the immunofluorescence images. Results Our results suggest the mean gray value method is the most reliable method. The mean gray value of immunofluorescence in ChAT (63.0 ± 0.76%) and TRKA (47.6 ± 0.43%) on the motor fascicles was significantly higher than that on the sensory fascicles (ChAT: 49.2 ± 0.72%, P

For proper mammalian brain development and functioning, the translation of many neuronal mRNAs needs to be repressed without neuronal activity stimulations. We have discovered that the expression of a subclass of neuronal proteins essential for neurodevelopment and neuron plasticity is co-regulated at the translational level by TDP-43 and the Fragile X Syndrome protein FMRP. Using molecular, cellular and imaging approaches, we show that these two RNA-binding proteins (RBP) co-repress the translation initiation of Rac1, Map1b and GluR1 mRNAs, and consequently the hippocampal spinogenesis. The co-repression occurs through binding of TDP-43 to mRNA(s) at specific UG/GU sequences and recruitment of the inhibitory CYFIP1-FMRP complex by its glycine-rich domain. This novel regulatory scenario could be utilized to silence a significant portion of around 160 common target mRNAs of the two RBPs. The study establishes a functional/physical partnership between FMRP and TDP-43 that mechanistically links several neurodevelopmental disorders and neurodegenerative diseases.

Immunofluorescence staining is an important method for detecting the localization of proteins in the cell. It is also frequently used in the localization study of chloroplast-division proteins. Although this method has been improved before by using protoplasts, it still has some limitations. Now we developed a new method to make it much easier. We just broke the plant leaf tissue with a serrated blade, stained the samples directly, and simply lysed the tissue into separatable cells. The localization of the target protein can then be observed with a clear view. Since this method directly uses broken leaf pieces, it is very fast. It can also be applied to the plants in which protoplasts are difficult to prepare. We first used this method to observe the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis. A ring was clearly seen in the middle of chloroplasts. In addition, we used this method to analyze the localization of FtsZ1 in arc3 and pdv2 mutants, as well as in dozens of other species, including some woody plants. This new immunofluorescence staining method is not only easy to use, but also has a wide applicability in various plants.

Lupus nephritis (LN) is most frequently associated with poor outcomes in patients with systemic lupus erythematosus (SLE). LN manifests as histopathological changes in the kidney caused by immune complex formation and deposition. In particular, immunoglobulin G (IgG) deposits are frequently observed by immunofluorescence staining, which helps to establish the diagnosis of LN. In this case report, we describe a 57-year-old woman with SLE who had been undergoing treatment on an outpatient basis for 11 years. Her first and second renal biopsies revealed class V LN with a coarsely granular pattern of IgG deposition in the peripheral capillary walls. However, her third renal biopsy showed no IgG deposition, which indicated histopathological resolution of her class V LN. We used low-vacuum scanning electron microscopy (LV-SEM) to examine the three-dimensional structural alterations in her glomerular basement membranes. In this report, we describe findings that indicated resorption of epithelial deposits, that is, resolution of LN. The results of repeated kidney biopsies confirmed by LV-SEM suggested the possibility of a state unrelated to LN.

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0–4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at −20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.

The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7-15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system.

Objective: The meiotic spindle controls chromosome movement and mediates various functions essential for fertilization and early postfertilization events. This study aimed to examine whether vitrification causes meiotic damage in vitro- matured metaphase II (MII) human oocytes, and whether the meiotic spindle morphology influences the subsequent developmental outcomes. Methods: The spindle characteristics of MII human oocytesin vitro matured were studied before and after vitrification using PolScope imaging and immunofluorescence staining. The developmental competence of oocytes was also examined. Results: A total of 419 human MII oocytes were obtained from 593 intracytoplasmic sperm injection cycles at our hospital. Of these oocytes, 54 were used for immunofluorescence staining, whereas the other oocytes were examined by PolScope imaging and classified into three groups according to the meiotic spindle morphology: (A) normal morphology, (B) weak refraction and short meiotic spindle, and (C) no detectable meiotic spindle. The three groups demonstrated statistically significant differences in terms of survival after vitrification. However, differences were not found in terms of oocyte chromosome structure and meiotic spindle morphology on immunofluorescence staining performed before and after vitrification. Oocyte survival, fertilization, and early embryonic development rates were significantly higher in Group A than in Groups B and C with or without vitrification. While vitrification had no effect on these metrics in Group A, Groups B and C demonstrated significantly lower fertilization and cleavage rates after vitrification/warming. Conclusions: Screening for normal meiotic spindle morphology and chromosome configuration before vitrification may increase the yield of healthy viable oocytes for various assisted reproductive technologies.

Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis.

Background: Inappropriate matching of motor and sensory fibers after nerve repair or nerve grafting can lead to failure of nerve recovery. Identification of motor and sensory fibers is important for the development of new approaches that facilitate neural regeneration and the next generation of nerve signal-controlled neuro-prosthetic limbs with sensory feedback technology. Only a few methods have been reported to differentiate sensory and motor nerve fascicles, and the reliability of these techniques is unknown. Immunofluorescence staining is one of the most commonly used methods to distinguish sensory and motor nerve fibers, however, its accuracy remains unknown.Methods: In this study, we aim to determine the efficacy of popular immunofluorescence markers for motor and sensory nerve fibers. We harvested the facial (primarily motor fascicles) and sural (primarily sensory fascicles) nerves in rats, and examined the immunofluorescent staining expressions of motor markers (choline acetyltransferase (ChAT), tyrosine kinase (TrkA)), and sensory markers [neurofilament protein 200 kDa (NF-200), calcitonin gene-related peptide (CGRP) and Transient receptor potential vanillic acid subtype 1 (TRPV1)]. Three methods, including the average area percentage, the mean gray value, and the axon count, were used to quantify the positive expression of nerve markers in the immunofluorescence images.Results: Our results suggest the mean gray value method is the most reliable method. The mean gray value of immunofluorescence in ChAT (63.0 ± 0.76%) and TRKA (47.6 ± 0.43%) on the motor fascicles was significantly higher than that on the sensory fascicles (ChAT: 49.2 ± 0.72%, P < 0.001; and TRKA: 29.1 ± 0.85%, P < 0.001). Additionally, the mean gray values of TRPV1 (51.5 ± 0.83%), NF-200 (61.5 ± 0.62%) and CGRP (37.7 ± 1.22%) on the motor fascicles were significantly lower than that on the sensory fascicles respectively (71.9 ± 2.32%, 69.3 ± 0.46%, and 54.3 ± 1.04%) (P < 0.001). The most accurate cutpoint occurred using CHAT/CRCP ratio, where a value of 0.855 had 100% sensitivity and 100% specificity to identify motor and sensory nerve with an area under the ROC curve of 1.000 (P < 0.001).Conclusions: A combination of ChAT and CGRP is suggested to distinguish motor and sensory nerve fibers. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) is most frequently associated with poor outcomes in patients with systemic lupus erythematosus (SLE). LN manifests as histopathological changes in the kidney caused by immune complex formation and deposition. In particular, immunoglobulin G (IgG) deposits are frequently observed by immunofluorescence staining, which helps to establish the diagnosis of LN. In this case report, we describe a 57-year-old woman with SLE who had been undergoing treatment on an outpatient basis for 11 years. Her first and second renal biopsies revealed class V LN with a coarsely granular pattern of IgG deposition in the peripheral capillary walls. However, her third renal biopsy showed no IgG deposition, which indicated histopathological resolution of her class V LN. We used low-vacuum scanning electron microscopy (LV-SEM) to examine the three-dimensional structural alterations in her glomerular basement membranes. In this report, we describe findings that indicated resorption of epithelial deposits, that is, resolution of LN. The results of repeated kidney biopsies confirmed by LV-SEM suggested the possibility of a state unrelated to LN. [ABSTRACT FROM AUTHOR]

Introduction: This was an ambispective cohort study evaluating the prognostic significance of lymphocytic foci and its lymphoid composition in minor salivary gland biopsy (MSGB) for short-term disease flare and severity in Sjögren's syndrome (SS)., Methods: The inclusion criteria comprised individuals meeting the ACR/EULAR 2016 criteria who underwent MSGB with an infiltration of more than 50 lymphocytes and received clinical diagnosis between September 2017 and December 2018. Patients with inadequate biopsy samples were excluded. The number of lymphocytic foci and their lymphoid composition in MSGB were assessed using immunofluorescence staining. Major organ damage and improvements in the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) were measured. Statistical analyses, including Cox and linear regressions, were conducted., Results: A total of 78 patients with at least one lymphocytic focus were included in the study. The presence of higher T-cell counts in lymphocytic foci in MSGB was associated with severe disease flare, and a logarithmic transformation of T-cell count indicated increased risk (HR 1.96, 95% CI 0.91-4.21). Improvements in the ESSDAI were associated with higher total lymphocyte count and T- and B-cell numbers in the lymphoid composition of the lymphocytic foci. Seropositive patients exhibited higher T CD4+ cell numbers. Correlation analysis showed negative associations between age and lymphocytic foci and the T-cell count. Positive correlations were observed between antinuclear antibody (ANA) titers and total lymphocyte numbers., Discussion: Patients with a higher number of T cells in the lymphocytic infiltrates of lymphocytic foci may have a two-fold risk of severe disease flare. The number of B cells and T CD4+ cells in the lymphocytic infiltrates of lymphocytic foci showed a weak but positive relation with the ESSDAI improvement during follow-up. Age and seropositivity appeared to influence the lymphoid composition of the lymphocytic foci., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Park, Martínez-Martínez, Magallares López, Castellví, Moya, Codes-Mendez, Hernandez Sosa, Diaz-Torne, Laiz, Sainz, Tandaipan, Mariscal, Franco-Leyva, Casademont, Juarez and Corominas.)

To investigate the effect of Myocardin-related transcription factor A (MRTF-A) on apoptosis induced by ischemic/reperfusion (I/R), middle cerebral artery occlusion/reperfusion (MCAO/R) in rats were applied to mimic I/R. The neurological deficit score, cerebral infarct size, cortical neuron apoptosis and cleaved caspase 3 level were evaluated to determine the effect and the level of apoptosis by TTC straining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) straining, Western blot and immunofluorescence staining. The myeloid cell leukemia-1 (Mcl-1) expression, release of cytochrome C (Cyt C) and its colocalization with apoptotic protease activating factor-1 (Apaf-1) were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. The results showed that MRTF-A overexpression could decrease the neurological deficit score and reduce cerebral infarct size (P

Here we investigated the morphology, quantity, distribution and gene expression of mechanoreceptors in the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), patellar tendon (PaT) and popliteal tendon (PoT) of the knee joint. Twelve 6-month-old rabbits were divided into two groups. In one group, the ACL, PCL, MCL, LCL, PaT and PoT were collected to observe the morphology, distribution and quantity of mechanoreceptors. In another group, the ACL, PCL, MCL, LCL, PaT and PoT of bilateral knee joints were used to determine S100B, CGRP and NEFM gene levels. Five types of mechanoreceptors were observed including Ruffini corpuscles, Pacinian corpuscles, Golgi-tendon bodies, atypical mechanoreceptors and free nerve endings. The total amount of mechanoreceptors was significantly lower in MCL, LCL, PaT and PoT as compared with ACL and PCL (P < 0.001). All examined mechanoreceptors were present in ACL, PCL and LCL. However, no Pacinian corpuscles and Golgi-tendon bodies were found in MCL and PoT as well as Pacinian corpuscles were not observed in PaT. The present study indicated that the levels of NEFM was significantly lower in PCL, MCL, LCL, PaT and PoT as compared with ACL (P < 0.05), but there was no significant difference in CGRP level between ACL and other ligaments except LCL (P > 0.05). Thus, the quantity, type and gene expression of mechanoreceptors are different in various ligaments. Thus, the quantity and distribution of mechanoreceptors may be related to ligament's function. [ABSTRACT FROM AUTHOR]

The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7-15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system. [ABSTRACT FROM AUTHOR]

Objective(s):We investigated the relationship between the expression of tumor necrosis factor-inducible gene 6 (TSG-6) with inflammation and integrity of the bladder epithelium in the bladder tissues of patients with bladder pain syndrome/interstitial cystitis (BPS/IC) and the mechanism of action using a rat model of BPS/IC. Materials and Methods: Expression of TSG-6 and uroplakin III was determined by immuno- histochemistry of bladder biopsy samples from control human subjects and patients with verified BPS/IC. Our rat model of BPS/IC was employed to measure the perfusion of bladders with hyaluronidase, and assessment of the effect of TSG-6 administration on disease progression. Treatment effects were assessed by measurement of metabolic characteristics, RT-PCR of TGR-6 and interleukin-6, bladder histomorphology, and immunohistochemistry of TGR-6 and uroplakin III. Results: The bladders of patients with BPS/IC had lower expression of uroplakin III and higher expression of TSG-6 than controls. Rats treated with hyaluronidase for 1 week developed the typical signs and symptoms of BPS/IC, and rats treated with hyaluronidase for 4 weeks had more serious disease. Administration of TSG-6 reversed the effects of hyaluronidase and protected against disease progression. Conclusion:Our results indicate that TSG-6 plays an important role in maintaining the integrity of the bladder epithelial barrier.

To investigate the effect of Myocardin-related transcription factor A (MRTF-A) on apoptosis induced by ischemic/reperfusion (I/R), middle cerebral artery occlusion/reperfusion (MCAO/R) in rats were applied to mimic I/R. The neurological deficit score, cerebral infarct size, cortical neuron apoptosis and cleaved caspase 3 level were evaluated to determine the effect and the level of apoptosis by TTC straining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) straining, Western blot and immunofluorescence staining. The myeloid cell leukemia-1 (Mcl-1) expression, release of cytochrome C (Cyt C) and its colocalization with apoptotic protease activating factor-1 (Apaf-1) were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. The results showed that MRTF-A overexpression could decrease the neurological deficit score and reduce cerebral infarct size (P < 0. 0 1 versus Sham). In the MRTF-A-I/R group, TUNEL-positive cells and apoptosis ratio (%) (5 1. 6 1 ± 6. 1 7 %) were significantly decreased compared to the Neg-I/R group (7 6. 4 5 ± 8. 7 7 %) at 24 h reperfusion. Meanwhile, the cleaved caspase 3 expression revealed a similar trend while the expression of Mcl-1 was the opposite. Moreover, MRTF-A overexpression significantly enhanced Mcl-1 fluorescence intensity, which up-regulated the mRNA and protein level (P < 0. 0 5 or P < 0. 0 1 versus Neg-I/R). Furthermore, MRTF-A overexpression markedly inhibited the release of Cyt C, and decreased the colocalization with Apaf-1 in the cytoplasm (P < 0. 0 5 or P < 0. 0 1 versus Neg-I/R). All the data indicated that MRTF-A overexpression could improve the neurological function against cerebral I/R-induced apoptosis since underlying mechanism might be involved in the Mcl-1/Cyt C/cleaved caspase 3 signaling pathway. [ABSTRACT FROM AUTHOR]

Ninety-five effusion samples were collected from cats with suspected feline infectious peritonitis in northern Taiwan; these samples showed a 47.4% (45/95) feline coronavirus (FCoV) positivity rate on immunofluorescence staining and RT-PCR. Young cats (≤24 months old) were found to have a significantly higher risk than cats >24 months old (odds ratio (OR) = 6.19, 95% confidence interval (CI) 2.54–16.00). No significant association was found between the positive rates and sex or breed. The A/G ratio in positive cases was significantly lower than the A/G ratio in negative cases. Genotyping and sequencing of the positive cases revealed 71.9% single infection with type I strains and 28.1% coinfection with types I and II. No single infections with type II strains were noted. The type I sequences had high diversity, while the type II sequences had high internal sequence identity and were more similar to CoVs from other species, such as dogs, pigs, and various small mammals. This study demonstrates the latest analysis of FCoV infection cases in northern Taiwan.

The peripheral nervous system communicates specifically with the immune system via local interactions. These interactions include the “hardwiring” of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to primary (e.g., thymus and bone marrow) and secondary (e.g., lymph node, spleen, and gut-associated lymphoid tissue) lymphoid tissue/organs. To gain a better understanding of this bidirectional interaction/crosstalk between the two systems, we have investigated the distribution of nerve fibres and PNS-immune cell associations in situ in the mouse lymph node by using immunofluorescent staining and confocal microscopy/ three-dimensional reconstruction. Our results demonstrate i) the presence of extensive nerve fibres in all compartments (including B cell follicles) in the mouse lymph node; ii) close contacts/associations of nerve fibres with blood vessels (including high endothelial venules) and lymphatic vessels/sinuses; iii) close contacts/associations of nerve fibres with various subsets of dendritic cells (e.g., B220+CD11c+, CD4+CD11c+, CD8a+CD11c+, and Mac1+CD11c+), Mac1+ macrophages, and B/T lymphocytes. Our novel findings concerning the innervation and nerve-immune cell interactions inside the mouse lymph node should greatly facilitate our understanding of the effects that the peripheral nervous system has on cellular- and humoral-mediated immune responses or vice versa in health and disease.

This study aimed to explore morphology changes in the kidneys of Dab1−/− (yotari) mice, as well as expression patterns of reelin, NOTCH2, LC3B, and cleaved caspase3 (CASP3) proteins, as potential determinants of normal kidney formation and function. We assumed that Dab1 functional inactivation may cause disorder in a wide spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). Animals were sacrificed at postnatal days P4, P11, and P14. Paraffin-embedded kidney tissues were sectioned and analyzed by immunohistochemistry using specific antibodies. Kidney specimens were examined by bright-field, fluorescence, and electron microscopy. Data were analyzed by two-way ANOVA and t-tests. We noticed that yotari kidneys were smaller in size with a reduced diameter of nephron segments and thinner cortex. TEM microphotographs revealed foot process effacement in the glomeruli (G) of yotari mice, whereas aberrations in the structure of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) were not observed. A significant increase in reelin expression, NOTCH2, LC3B and cleaved CASP3 proteins was observed in the glomeruli of yotari mice. Renal hypoplasia in conjunction with foot process effacement and elevation in the expression of examined proteins in the glomeruli revealed CAKUT phenotype and loss of functional kidney tissue of yotari.