Immunofluorescence Method Research Articles

SS31 alleviates LPS-induced acute lung injury by inhibiting inflammatory responses through the S100A8/NLRP3/GSDMD signaling pathway

BackgroundAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an acute, diffuse, inflammatory lung injury caused by various endogenous or exogenous factors. It is currently widely recognized that an excessive inflammatory response resulting from immune imbalance constitutes a crucial pathogenic mechanism in ALI/ARDS. SS31 is a novel mitochondria-targeted antioxidant peptide. This article validates the role of SS31 in lipopolysaccharide (LPS)-induced ALI.MethodsThe study applied transcriptome sequencing, immunofluorescence, PCR, immunofluorescence and other methods to explore the mechanism of SS31 in LPS induced ALI.ResultsTranscriptome sequencing results indicate that LPS-induced ALI is closely associated with immune regulatory processes, the Toll-like receptor pathway, and the NF-κB signaling pathway. The role of SS31 in acute lung injury is closely related to biological processes, such as immune regulation and cell death. This study demonstrated that SS31 can inhibit the expression of inflammatory factors IL-6, IL-1β, IL-18, and TNF-α, and reduce the expression of pyroptosis-related proteins NLRP3, and GSDMD-N. Further analysis revealed that S100A8 may be a key gene in the effect of SS31. LPS stimulation leads to increased expression of S100A8, while SS31 decreases its expression. Recombinant protein S100A8 can attenuate the inhibitory effect of SS31 on IL-1β, IL-18, NLRP3, and GSDMD-N.ConclusionsThe research results indicate that SS31 may inhibit the activation of the NLRP3 inflammasome and suppress inflammatory responses by regulating S100A8, thereby alleviating LPS-induced ALI in mice; this process may be related to pyroptosis.

Detection of human coronavirus-OC43 in nasopharyngeal swab specimens via immunofluorescence staining using human serum and an anti-human antibody.

Immunofluorescence methods to detect seasonal human coronavirus (HCoV)-OC43 in nasopharyngeal swab specimens using cell lines have not yet been established. A human rectal adenocarcinoma cell line (HRT-18) was exposed to the specimens obtained from patients with upper respiratory tract infections. Immunofluorescence staining was conducted with the combination of human serum containing the HCoV-OC43 anti-spike protein antibody and a fluorescence-labeled anti-human antibody. Positive staining in HRT-18 cells was detected after exposure to specimens obtained from nine of the eleven patients in which HCoV-OC43 RNA was detected using the FilmArray method. Increased supernatant viral RNA levels were also detected in HRT-18 cells exposed to specimens obtained from four of five patients. In contrast, positive staining was not detected in HRT-18 cells exposed to six patient specimens that tested negative for RNA from seventeen types and subtypes of respiratory viruses, including HCoV-OC43. The cells inoculated with the established strain HCoV-OC43 (ATCC VR-759) also showed positive staining. These findings suggest that the replication-competent HCoV-OC43 in the specimens could be detected via immunofluorescence staining of HRT-18 cells with human serum. It may be possible to obtain positive staining for viruses other than HCoV-OC43 using this method.

Sinapic acid alleviates glutamate-induced excitotoxicity by inhibiting neuroinflammation and endoplasmic reticulum stress pathway in C6 glioma cells

Sinapic acid (SA) is a polyphenol compound derived from hydroxycinnamic acid found in various foods such as cereals and vegetables and has antioxidant, anti-inflammatory and neuroprotective properties. However, its effects on glutamate-induced excitotoxicity, which is important in neurodegenerative diseases, have not been fully elucidated. This study aimed to investigate the effect of SA on glutamate excitotoxicity and the possible role of proinflammatory cytokines and the endoplasmic reticulum (ER) stress pathway. In the study, C6 rat glioma cell line was used and the cells were divided into 4 groups: control, glutamate, SA and glutamate+SA. Cells were treated with 10 mM glutamate for 24 h to induce excitotoxicity. Additionally, SA was applied to cells at concentrations of 12.5 to 100 μM to examine its effects on glutamate excitotoxicity. XTT test was used for cell viability, and apoptotic cells were determined by immunofluorescence and flow cytometry methods. Proinflammatory cytokines (tumor necrosis factor-alpha, TNF-α and interleukin-beta, IL-1β), ER stress markers (glucose regulatory protein 78, GRP78; C/EBP homologous protein, CHOP and activating transcription factor-4, ATF-4) and caspase-3 was used to measure ELISA method. Findings indicated that SA (50 μM) significantly increased cell viability against glutamate-induced excitotoxicity (p < 0.05). Also, SA caused a significant decrease in TNF-α, IL-1β, GRP78, CHOP, ATF-4 and caspase-3 levels in glutamate-treated cells (p < 0.05). Flow cytometry and immunofluorescence staining results showed that SA reduced apoptosis in C6 glioma cells. In conclusion, our findings suggested that SA attenuated glutamate-induced excitotoxicity by preventing apoptosis through reducing proinflammatory cytokines and ER stress protein levels.

Impairing Ferroptosis Through the PI3K/Akt/Nrf2 Pathway: The Way for Nerve Growth Factor to Mitigate Hypoxia-induced Cardiomyocyte Damage.

Myocardial infarction (MI) is an acute cardiovascular diseases, distinguished primarily by cardiomyocyte damage due to ischemia and hypoxia. Nerve growth factor (NGF) is paramount in ischemic heart disease, it contributes to maintaining heart function and protecting the heart. Nonetheless, the effects of NGF on cardiomyocyte damage induced by hypoxia and the precise mechanisms involved are still to be elucidated. Utilizing western blot and immunofluorescence methods to quantify the NGF levels in cardiomyocytes (H9C2) of rats after hypoxia. Cell Counting Kit-8 (CCK-8) assay was employed to monitor the dynamic changes in cells vitality. The lactate dehydrogenase (LDH), Fe2+, malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) levels were evaluated by different kits. Moreover, the PI3K/Akt/Nrf2 pathway and ferroptosis-linked protein levels were analyzed using western blotting. In H9C2 cells, exposure to hypoxia for 24 h led to weakened NGF level, as well as lowered cell vitality and SOD activity, but elevated levels of LDH, Fe2+, MDA, and ROS, triggering ferroptosis. Overexpression NGF alleviated the ferroptosis in H9C2 cells caused by hypoxia, while NGF knockdown intensified this process. Additionally, overexpression NGF reinforced heme oxygenase-1 (HO-1) and Nrf2 levels, and Akt and PI3K phosphorylation, whereas NGF silencing produced contrary outcomes. Furthermore, the PI3K/Akt pathway inhibitor negated the elevation in HO-1 and Nrf2 levels mediated by NGF amplification. In contrast, the pathway activator reversed the lowering in Nrf2 and HO-1 levels caused by silencing NGF. This suggested that NGF mediates the activation of Nrf2 through the PI3K/Akt axis. Overall, by mediating the activation of Nrf2 through the PI3K/Akt axis, NGF reduced the damage to H9C2 cells caused by hypoxia and thus hindered ferroptosis.

Analysis of antinuclear antibody in 9 528 pregnant women during early pregnancy in a hospital in Qingdao City

To analyze the positivity rate and titer of antinuclear antibody (ANA), as well as nuclear pattern and target antigen of ANA in healthy pregnant women during early pregnancy in Qingdao area. A prospective cohort study design was used to include a total of 9 528 healthy pregnant women registered at the Women and Children's Hospital Affiliated to Qingdao University from March 2023 to June 2024.Indirect immunofluorescence assay (IIF) was used to detect ANA, its titer and cell staining pattern. Fifteen specific antibodies were tested using the magnetic bar code immunofluorescent luminescence method. Logistic regression model was used to analyze the risk factors of pregnancy with autoimmune disease(AID). The results showed that among 9 528 pregnant women in early pregnancy, 1 346 cases (14.1%) were positive of ANA, including 1 011 cases with a titer of 1∶100 (10.6%), 236 cases (2.5%) with a titer of 1∶320, and 99 cases (1.0%) were detected at a titer >1∶320. Among the 1 346 ANA-positive pregnant women, nuclear granular type accounted for the highest proportion (483 cases, 35.9%), followed by speckled type (347 cases, 25.8%) and cytoplasmic type (176 cases, 13.1%).Then, pregnant women with ANA titers ≥1∶100 were detected 15 specific antibodies.Anti-SSA was tested in 121 cases accounted for the majority, followed by 110 cases with anti-Ro-52, 56 cases with anti-SSB, 51 cases with anti-mitochondrial M2 subtype antibodies and 37 cases with anti-centromere B. In conclusion,in healthy pregnant women in Qingdao area, ANA positivity rate was 14.1%, and the titer of ANA was mainly at 1∶100.The predominant nuclear patterns were nuclear granular and speckled types.The specific autoantibodies were mainly anti-SSA antibodies and anti-Ro-52 antibodies.The detection of ANA and specific autoantibodies is of great significance for early prediction, diagnosis, and intervention of autoimmune diseases during pregnancy.

Mechanism of intestinal flora affecting SLC2A9 transport function to promote the formation of hyperuricemia

ObjectiveTo investigate the structural characteristics of the intestinal flora in obese-hyperuricemic (HUA-W) patients and the mechanisms by which they promote the formation of hyperuricemia. Methods120 human fecal samples (60 cases in HC, 30 cases in HUA-N, and 30 cases in HUA-W) and 40 cases in the colonic tissues (20 cases in HC, 10 cases in HUA-N, and 10 cases in HUA-W) were collected. The intestinal flora of faeces was detected by 16s rRNA method; and the expression of SLC2A9 on human colon tissues was detected by RT-qPCR method and immunofluorescence method. 40 obese-hyperuricemia rat models were established (10 rats in Model, 10 rats in HC-FT, 10 rats in HUA-N-FT, and 10 rats in HUA-W-FT), and 10 rats were set up in Control; and the level of uric acid in rat serum, the levels of xanthine oxidase (XOD) activity and uric acid in intestinal fluid were examined. SLC2A9+ Caco-2 cells were produced to simulate the Transwell uric acid transport model, and the Caco-2 cells and SLC2A9+ Caco-2 cells were grown in five different culture media (Blank, Germ-free, HC-germ, HUA-N-germ and HUA-W-germ), and the uric acid levels in the upper and lower layers of the chambers were detected. ResultsThe HUA-W intestinal flora showed significant specificity, with a decrease in Bacteroidota and Bacteroidia and an increase in Escherichia and Ruminococcus. There were no significant differences in the fluorescence intensity of the SLC2A9 protein and the SLC2A9 mRNA levels in the colon tissues of the HUA-N and HUA-W (P = 0.447, P = 0.152, P = 0.4799 and P = 0.965, respectively). In rat animal experiments, uric acid levels were significantly higher (P < 0.05) and XOD activity was significantly higher (P < 0.05) in intestinal fluid of HUA-W-FT. In Transwell experiments with SLC2A9+ Caco-2 cells, uric acid levels were increased in the upper compartment and decreased in the lower compartment of HUA-W-germ. ConclusionHUA-W intestinal flora may increase XOD activity in the intestinal tract and improve the ability of uric acid transporter protein SLC2A9 to reabsorb uric acid, providing a new theoretical basis for the pathogenesis of hyperuricemia.

Hepatic Stellate Cell-mediated Increase in CCL5 Chemokine Expression after X-ray Irradiation Determined In Vitro and In Vivo.

Radiation exposure causes hepatitis which induces hepatic steatosis and fibrosis. Although hepatic stellate cells (HSCs) have been considered potential pathological modulators for the development of hepatitis due to viral and microbial infections, their involvement in radiation-induced hepatitis is yet to be determined. This study aimed to clarify the relationship between radiation exposure and expressions of inflammatory cytokines and chemokines in HSCs in vitro and invivo. HSCs were obtained from 1-week-old mice, known to be highly sensitive to radiation-induced hepatocellular carcinoma, using a newly established method combining liver perfusion, cell dissociation, and density gradient centrifugation, followed by magnetic negative selection of hematopoietic and endothelial cells with anti-CD45.2 and CD146 antibodies. The isolated HSCs were confirmed by the expression of desmin and glial fibrillary acidic protein (GFAP). We demonstrated that primary cultured HSCs, exposed to X-ray irradiation (0, 1.9, and 3.8 Gy) and cultured for 3 and 7 days, produced elevated levels of C-C motif chemokine ligand 5 (CCL5, also known as RANTES) inflammatory chemokine in a dose-dependent manner. An invivo immunofluorescence method confirmed that increased CCL5 signals were observed in GFAP-positive HSCs in mouse livers 7 days after whole-body X-ray irradiation (1.9 and 3.8 Gy). Adequate expression of C-C motif chemokine receptor 5 (Ccr5), a receptor for CCL5, was also detected using real-time PCR in the liver of both irradiated and non-irradiated mice. Taken together, our data suggest that HSCs may drive hepatitis via CCL5/CCR5 axis in the liver under radiation-induced stress. Furthermore, this newly established experimental protocol can help evaluate the expression of other inflammatory cytokines in primary cultures of HSCs isolated from infant mice.

Microglial DBP Signaling Mediates Behavioral Abnormality Induced by Chronic Periodontitis in Mice.

Several lines of evidence implicate that chronic periodontitis (CP) increases the risk of mental illnesses, such as anxiety and depression, yet, the associated molecular mechanism for this remains poorly defined. Here, it is reported that mice subjected to CP exhibited depression-like behaviors and hippocampal memory deficits, accompanied by synapse loss and neurogenesis impairment in the hippocampus. RNA microarray analysis disclosed that albumin D-site-binding protein (DBP) is identified as the most prominently upregulated target gene following CP, and invivoand invitro immunofluorescence methods showed that DBP is preferentially expressed in microglia but not neurons or astrocytes in the hippocampus. Interestingly, it is found that the expression of DBP is significantly increased in microglia after CP, and knockdown of microglial DBP ameliorated the behavioral abnormality, as well as reversed the synapse loss and hippocampalneurogenesis damage induced by CP. Furthermore, DBP knockdown improved the CP-induced hippocampal inflammation and microglial polarization. Collectively, these results indicate a critical role of DBP in orchestrating chronic periodontitis-related behavioral abnormality, hippocampal synapse loss andneurogenesis deficits, in which the microglial activation may be indispensably involved.

Wuzi-Yanzong-Wan Prevents the Defect of Cell-Cell Junctions between Sertoli-Germ Cells by Up-Regulating the Expression of TAp73.

The TAp73 gene is an anti-cancer gene that also affects the junction between Sertoli and germ cells. Inhibition of this gene causes infertility in male mice. Our previous research proved that Wuzi-Yanzong-Wan (WZYZW) can protect spermatogenesis and maturation by preventing TAp73 inhibition. This study aimed to investigate the effect of drug-containing serum of WZYZW on the defect of cell-cell junctions in the Sertoli-germ cells co-culture system in vitro. LC-HRMS was used to analyze the content of active ingredients in WZYZWmedicated serum. Then, primary extraction and co-culture of germ cells and Sertoli cells were carried out. Co-cultured cells were added with PFT-α to induce the TAp73 inhibition model, with WZYZW-medicated serum at 2.5%, 5%, and 10% treated in parallel. Sloughing of germ cells from Sertoli cells was calculated. Transmission electron microscopy (TEM), Immunofluorescence, qRT-PCR, and western blot methods were employed. The drug-containing serum of WZYZW contained schisandrin, hyperoside, geniposidic acid, ellagic acid, and quercetin. Using TEM assay, we observed restoration of the desmosomelike (Des), tight junctions (TJ), and basal ectoplasmic specialization (ES) structure following WZYZW treatment. WZYZW caused inhibition of peptidase and protease inhibitors (tissue inhibitor of metalloproteinase-1 (TIMP1), Serpina3n) by immunofluorescence analysis. Western blot and qRT-PCR analysis revealed that WZYZW was able to ameliorate the expressions of peptidase and protease inhibitors and cell adhesion factors, such as TAp73, TIMP1, Serpina3n, Desmocollin-3, N-cadherin, and Nectin-2. WZYZW-medicated serum could prevent the defect of cell-cell junctions between Sertoli-germ cells co-culture system in vitro by up-regulating the expression of TAp73.

Proteomic analysis and experimental validation reveal the blood–brain barrier protective of Huanshaodan in the treatment of SAMP8 mouse model of Alzheimer’s disease

BackgroundHuanshaodan (HSD) is a Chinese Herbal Compound which has a definite clinical effect on Alzheimer’s disease (AD), however, the underlying mechanism remains unclear. The aim of this study is to preliminarily reveal the mechanism of HSD in the treatment of AD model of SAMP8 mice.MethodsChemical composition of HSD and its drug-containing serum were identified by Q-Orbitrap high resolution liquid mass spectrometry. Six-month-old SAMP8 mice were treated with HSD and Donepezil hydrochloride by gavage for 2 months, and Wogonin for 28 days. Behavioral test was performed to test the learning and memory ability of mice. Immunofluorescence (IF) or Western-blot methods were used to detect the levels of pSer404-tau and β-amyloid (Aβ) in the brain of mice. Hematoxylin–eosin (H&E) staining and Transmission electron microscopy (TEM) assay was applied to observe the pathological changes of neurons. Proteomic technology was carried out to analyze and identify the protein network of HSD interventions in AD. Then the pathological process of the revealed AD-related differential proteins was investigated by IF, Q-PCR, Western-blot, Fluorescence in situ hybridization (FISH) and 16S rRNA sequencing methods.ResultsThe results showed that HSD and Wogonin, one of the components in its drug-containing serum, can effectively improve the cognitive impairments of SAMP8 mice, protect hippocampal neurons and synapses, and reduce the expression of pSer404-tau and Aβ. HSD and Wogonin reduced the levels of fibrinogen β chain (FGB) and γ chain (FGG), the potential therapeutic targets revealed by proteomics analysis, reduced the colocalization of FGB and FGG with Aβ, ionized calcium binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), increased level of and myelin basic protein (MBP). Meanwhile, HSD and Wogonin increased ZO-1 and Occludin levels, improved brain microvascular injury, and reduced levels of bacteria/bacterial DNA and lipopolysaccharide (LPS) in the brain of mice. In addition, 16S rRNA sequencing indicated that HSD regulated the structure of intestinal microbiota of mice.ConclusionThe effects of HSD on AD may be achieved by inhibiting the levels of fibrinogen and the interactions on glia cells in the brain, and by modulating the structure of intestinal microbiota and improving the blood–brain barrier function.

B-074 Comparative Evaluation of Clinical Utility between Indirect Immunofluorescence and Immunoassay Methods for Antineutrophil Cytopalsmic Antibody Detection

Abstract Background ANCA testing is essential for the diagnosis of vasculitis, commonly using indirect immunofluorescence(IIF) and immunoassay(IA) methods. Recent guidelines recommend IA as the primary test for suspected GPA and MPA, but actual practice varies among physicians. Here, we retrospectively analyzed ANCA test results, focusing on new cases of ANCA-associated vasculitis, and compared the utility of IIF and IA to guide appropriate test selection. Methods From January 2022 to July 2023, 24 new cases of ANCA-associated vasculitis were diagnosed. We checked whether ANCA testing was performed and the results, and compared the turnaround time(TAT) and costs. IIF ANCA tests were performed on a computer-based automated instrument(Helios), and IA ANCA tests were performed on automated instruments(Alegria, Phadia 250) with MPO antigen and PR3 antigen tested simultaneously in a bundled prescription. Results Among the 24 cases, there were 6 GPA, 7 MPA, 7 EGPA, and 4 ANCA-associated GN, with varying diagnostic sensitivities. GPA and EGPA had different ANCA positivity rates than previously reported, suggesting potential ethnic variations in ANCA expression and positivity rates. The test sequence and results, summarized in Table 1, showed 21 out of 22 cases had identical concordance between methods, with an excellent concordance rate (kappa value=0.83). TAT for IIF was 3,452 minutes, and for IA was 1,491 minutes for the whole period, but IA TAT increased to 2,748 minutes after implementing a days-of-the-week testing system. Test costs, with a relative value score of 118.99 for IIF and 156.05 for IA, indicated IA cost approximately 2.6 times higher, considering the bundled prescription of both antigens for IA ANCA testing. Conclusions Evaluating the utility of both methods for ANCA detection, our study highlights high inter-method agreement in ANCA-associated vasculitis. Considering overall cost and turnaround time, IIF ANCA testing remains effective as a screening test, demonstrating standalone effectiveness.

M protein ectodomain-specific immunity restrains SARS-CoV-2 variants replication.

The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear. We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay. Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30. The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections.

Characterization of Non-Small Cell Lung Cancer Tissue by Quantitative Assessment of Class III β-Tubulin Expression

Background. The cytoskeletal protein β-tubulin class III (Tubb3) is associated with tumor resistance to taxanes and vinca alkaloids, as well as with the metastatic potential of neoplasm, however, data from immunohistochemical analysis of Tubb3 expression in non-small cell lung cancer (NSCLC) tissue are few and contradictory. Purpose. Characterization of the level and intensity of Tubb3 expression in NSCLC tissue and analysis of the identified parameters correlation with clinically significant characteristics of the disease. Methods. Quantitative assessment of the level and intensity of Tubb3 expression in 120 surgical samples of NSCLC was carried out by immunofluorescence method associated with flow cytometry. Primary rabbit monoclonal antibodies specific to Tubb3 and secondary anti-rabbit antibodies conjugated with fluorescent dye DyLight650 (ab98510, UK) were used. The expression of the marker was assessed by two parameters: the level of expression measured as the percentage of the cells expressing Tubb3 and the intensity of expression in conventional units (CU) represented as the ratio of the geometric mean fluorescence intensity in the experimental and control samples (cells incubated with secondary antibodies only). Results. 1. Tubb3 expression was detected in all NSCLC samples studied. The median level and intensity of Tubb3 expression was 30.5% and 2.0 CU with significant differences (up to 10 times) in the quantitative values of both parameters in different patients. 2. The distribution of the studied tumors in terms of the level and intensity of Tubb3 expression differs from normal (P&lt;0.001), the associative relationship between the assessed parameters is very strong (Spearman's rank correlation coefficient was 0.91; P&lt;0.0001). 3. Statistical analysis did not reveal correlations between the level of Tubb3 expression and the gender and smoking status of the patients, with the degree of tumor differentiation, as well as with the stage of NSCLC. 4. In the group of lung adenocarcinomas, the median level of Tubb3 expression is higher compared with squamous cell lung cancer in male and female patients (P=0.01). Conclusion. High heterogeneity of Tubb3 expression level in NSCLC tissue in the patients and differences in the parameters between the tumors of various histotypes indicate the importance of further correlation analysis of Tubb3 expression level with the patients' life span in order to identify the prognostic value of the marker.

A method for quantitative spatial analysis of immunolabeled fibers at regenerative electrode interfaces

BackgroundRegenerative electrodes are being explored as robust peripheral nerve interfaces for neuro-prosthetic control and sensory feedback. Current designs differ in electrode number, spatial arrangement, and porosity which impacts the regeneration, activation, and spatial distribution of fibers at the device interface. Knowledge of sensory and motor fiber distributions are important in optimizing selective fiber activation and recording. New MethodWe use confocal microscopy and immunofluorescence methods to conduct spatial analysis of immunolabeled fibers across whole nerve cross sections. ResultsThis protocol was implemented to characterize motor fiber distribution within 3 macro-sieve electrode regenerated (MSE), 3 silicone-conduit regenerated, and 3 unmanipulated control rodent sciatic nerves. Total motor fiber counts were 1485 [SD: +/- 50.11], 1899 [SD: +/- 359], and 5732 [SD: +/- 1410] for control, MSE, and conduit nerves respectively. MSE motor fiber distributions exhibited evidence of deviation from complete spatial randomness and evidence of dispersion and clustering tendencies at varying scales. Notably, MSE motor fibers exhibited clustering within the central portion of the cross section, whereas conduit regenerated motor fibers exhibited clustering along the periphery. Comparison with Existing MethodsPrior exploration of fiber distributions at regenerative interfaces was limited to either quadrant-based density analysis of randomly sampled subregions or qualitative description. This method extends existing sample preparation and microscopy techniques to quantitatively assess immunolabeled fiber distributions within whole nerve cross-sections. ConclusionsThis approach is an effective way to examine the spatial organization of fiber subsets at regenerative electrode interfaces, enabling robust assessment of fiber distributions relative to electrode arrangement.

Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM.

Expansion microscopy (ExM) has significantly reformed the field of super-resolution imaging, emerging as a powerful tool for visualizing complex cellular structures with nanoscale precision. Despite its capabilities, the epitope accessibility, labeling density, and precision of individual molecule detection pose challenges. We recently developed an iterative indirect immunofluorescence (IT-IF) method to improve the epitope labeling density, improving the signal and total intensity. In our protocol, we iteratively apply immunostaining steps before the expansion and exploit signal processing through noise estimation, denoising, and deblurring (NEDD) to aid in quantitative image analyses. Herein, we describe the steps of the iterative staining procedure and provide instructions on how to perform NEDD-based signal processing. Overall, IT-IF in ExM-laser scanning confocal microscopy (LSCM) represents a significant advancement in the field of cellular imaging, offering researchers a versatile tool for unraveling the structural complexity of biological systems at the molecular level with an increased signal-to-noise ratio and fluorescence intensity. Key features • Builds upon the method developed by Mäntylä et al. [1] and introduces the IT-IF method and signal-processing platform for several nanoscopy imaging applications. • Retains signal-to-noise ratio and significantly enhances the fluorescence intensity of ExM-LSCM data. • Automatic estimation of noise, signal reconstruction, denoising, and deblurring for increased reliability in image quantifications. • Requires at least seven days to complete.

Study on pro-inflammatory effect and mechanism of galectin-9 (LGALS9) in osteoarthritis: Exacerbating inflammatory response by activating JNK and ERK1/2 pathways

Galectin-9 (LGALS9) plays an important role in the occurrence and development of many diseases, including immunity, infection, cancer, etc. Studies have found that LGALS9 can phosphorylate ERK1/2 in the MAPK pathway. However, there is currently no clear conclusion on the role of LGALS9 in OA, and it is worth further exploring the regulatory role and mechanism of LGALS9 in OA in this study. In the initial stage, we collected 6 cases of hip joint soft tissue from normal individuals and 6 cases from OA patients clinically to analyze the differential expression of LGALS9 between normal individuals and OA patients; Subsequently, RNAi technology was used to preliminarily clarify the regulatory role of LGALS9 in an in vitro OA model; Then, lentivirus was used to knock down and overexpress LGALS9, and in vivo and in vitro OA models were constructed. QRT-PCR, western blot, safranin fast green staining (SO), immunofluorescence and other experimental methods were used to quantitatively analyze inflammatory and signaling pathway indicators, further improving the regulatory effect of LGALS9 on inflammation and the pathogenesis of OA.

Capsaicin mitigates ventilator-induced lung injury by suppressing ferroptosis and maintaining mitochondrial redox homeostasis through SIRT3-dependent mechanisms

BackgroundVentilator-induced lung injury (VILI) is one of the severe complications in the clinic concerning mechanical ventilation (MV). Capsaicin (CAP) has anti-inflammatory and inhibitory effects on oxidative stress, which is a significant element causing cellular ferroptosis. Nevertheless, the specific role and potential mechanistic pathways through which CAP modulates ferroptosis in VILI remain elusive.MethodsVILI was established in vivo, and the pulmonary epithelial cell injury model induced by circulation stretching (CS) was established in vitro. Both mice and cells were pretreated with CAP. Transmission electron microscopy, ELISA, Western blot, immunofluorescence, RT-PCR, fluorescent probes, and other experimental methods were used to clarify the relationship between iron death and VILI in alveolar epithelial cells, and whether capsaicin alleviates VILI by inhibiting iron death and its specific mechanism.ResultsFerroptosis was involved in VILI by utilizing in vivo models. CAP inhibited ferroptosis and alleviated VILI's lung damage and inflammation, and this protective effect of CAP was dependent on maintaining mitochondrial redox system through SITR3 signaling. In the CS-caused lung epithelial cell injury models, CAP reduced pathological CS-caused ferroptosis and cell injury. Knockdown SIRT3 reversed the role of CAP on the maintaining mitochondria dysfunction under pathological CS and eliminated its subsequent advantageous impacts for ferroptosis against overstretching cells.ConclusionThe outcomes showed that CAP alleviated ferroptosis in VILI via improving the activity of SITR3 to suppressing mitochondrial oxidative damage and maintaining mitochondrial redox homeostasis, illustrating its possibility as a novel therapeutic goal for VILI.Graphical

Investigation of the potential teratogenic effects of fructose on the embryo using the rat whole embryo culture model

Excessive consumption of fructose-sweetened foods and beverages is a growing concern worldwide. Studies have demonstrated that fructose consumption before and during pregnancy can result in adverse outcomes such as decreased decidualization, increased fetal losses, and low birth weight. The study investigated the teratogenic effects of fructose on rat embryos during organogenesis using whole embryo culture. Within the scope of the study, 4 groups were formed as control, low, medium, and high-dose fructose (HDF) with 10 embryos in each group. The 9.5-day-old rat embryos were cultured with different concentrations of fructose (1, 5 and 10 mM) for 48 h and the possible effects of fructose were examined using morphological scoring, histochemistry, immunofluorescence, and TUNEL methods. According to the analyses, protein synthesis and proliferation were decreased, vascular formation was suppressed, and apoptosis was increased in embryos exposed to fructose, especially at concentrations of 5 mM and above. According to the morphological scoring results, it was determined that heart, hind limb, and somite development were retarded in all experimental groups compared to the control group, developmental retardation increased in direct proportion to fructose concentration, and also significant malformations were observed in all parameters examined in the HDF group. In addition, analysis of yolk sac diameter, head length, crown rump length and somite numbers showed that these parameters were significantly decreased in all experimental groups. End of the study, it was concluded that fructose at concentrations of 1 mM and above may induce embryonic development retardation and other anomalies by decreasing protein synthesis and cell proliferation, suppressing vascular formation, and increasing apoptosis in embryonic tissues.

The effects of brown rice as functional food on Lee Index, adipose tissues and PRDM16 levels in obesity model &lt;i&gt;Rattus norvegicus&lt;/i&gt;

Brown rice is a functional food known to improve the Lee Index, influencing PRDM16 levels in obesity conditions. Therefore, this study aims to determine the differences in Lee Index, Brown Adipose Tissue (BAT) weight, White Adipose Tissue (WAT) weight, BeAT weight, total body fat, and PRDM16 levels of obese model rats with the intervention of brown and white rice, as well as ℽ-oryzanol. A true experimental method was used with a post-test-only control group in vivo design. The obesity model was constructed with male Sprague Dawley rats (Rattus norvegicus), divided into five diet groups namely standard and HFHF diet control, as well as HFHF + brown rice, HFHF + white rice, and HFHF + ℽ-oryzanol combination diet. The experiment was carried out for 26 weeks, with details of 14 weeks to form an obese model and 12 weeks for the intervention. Before statistical correlation was tested, Lee index values, adipose tissues, and PRDM16 were analyzed. The anthropometric data collection method was carried out by weighing before and after the intervention, while adipose tissue was collected by weighing after sacrifice. The immunofluorescence method was used to collect the expression of PRDM16 and the mean of PRDM16 levels was analyzed in the ImageJ application. After the data collection process, analysis was performed using SPSS to determine possible differences in each group. Normally distributed data were analyzed using One-Way ANOVA, while those without normal distribution were assessed using the Kruskall-Walis method and the Mann Whitney-U advanced test, with a p-value of &lt;0.05 considered significantly different.The result showed that there were differences among several groups regarding total body fat (p=0.012), WAT (p=0.026), and BAT (p=0.025). However, no differences were found between all groups regarding the Lee index (p=0.275), BeAT (p=0.079), and PRDM16 level (p=0.292). In conclusion, brown rice intervention did not significantly affect Lee index values, the expression of PRDM16, and adipose tissue weights at the end of the intervention.

Specifically Targeting Capture and Photoinactivation of Viruses through Phosphatidylcholine-Ganglioside Vesicles with Photosensitizer.

Herein, we performed a simple virus capture and photoinactivation procedure using visible light on phosphatidylcholine vesicles. l-α-Phosphatidylcholine vesicles were enriched by viral receptors, GT1b gangliosides, and the nonpolar photosensitizer 5,10,15,20-tetraphenylporphyrin. These vesicles absorb in the blue region of visible light with a high quantum yield of antiviral singlet oxygen, O2 (1Δg). Through the successful incorporation of gangliosides into the structure of vesicles and the encapsulation of photosensitizers in their photoactive and monomeric state, the photogeneration of O2(1Δg) was achieved with high efficiency on demand; this process was triggered by light, and specifically targeting/inactivating viruses were captured on ganglioside receptors due to the short lifetime (3.3 μs) and diffusion pathway (approximately 100 nm) of O2(1Δg). Time-resolved and steady-state luminescence as well as absorption spectroscopy were used to monitor the photoactivity of the photosensitizer and the photogeneration of O2(1Δg) on the surface of the vesicles. The capture of model mouse polyomavirus and its inactivation were achieved using immunofluorescence methods, and loss of infectivity toward mouse fibroblast 3T6 cells was detected.