Asia Pacific Academy of Science Pte. Ltd.
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Assessing the Practical Application of Imaging Mass Cytometry for Visualizing Tumor Immune Microenvironment Heterogeneity
No full textBackground: Analyses of immune cell subsets and immune indices are becoming increasingly important. By interacting molecular probe and immunohistochemistry (IHC) analysis with laser ablation, imaging mass cytometry (IMC) offers high-dimensional in situ measurements in tissue slides with a spatial resolution of 1 μm. One of the most significant challenges is to implement stringent quality control strategies during the application of IMC to facilitate reproducible data analysis. We have developed a comprehensive protocol for clinical and experimental use by comparing IMC to standard clinical immunohistochemical approaches. Methods: We discussed the multi-step experimental processing procedures of IMC, including specimen preparation, panel design, antibody selection, lanthanide metal labelling, and data pre-processing workflows by IHC and fluorescent multiplex immunohistochemistry (mIHC) analysis. Based on IMC, we developed a standard operation and a well-established agreement for 29 breast cancer (BC) patients to identify a structured tumor immune microenvironment. Results: Metal labelling has no effect on the specificity of the antibodies target (p > 0.05). The 3 μm-thick formalin-fixed and paraffin-embedded (FFPE) and fresh-frozen slides exhibited the least blur (p < 0.01) and the lowest nonspecific adsorption of antibodies (p < 0.01). A detailed analysis of human breast cancer tissues revealed a wide range of differences in the composition and frequency of immune cells within the tumor microenvironment. Conclusions: This study presents optimized proposals on procedures for IMC analysis and a standardized quality control strategy
Quantum Dots as Functional Nanosystems for Enhanced Biomedical Applications
No full textQuantum Dots (QDs) have emerged as promising nanomaterials with unique optical and physical properties, making them highly attractive for various applications in biomedicine. This review provides a comprehensive overview of the types, modes of synthesis, characterization, applications, and recent advances of QDs in the field of biomedicine, with a primary focus on bioimaging, drug delivery, and biosensors. The unique properties of QDs, such as tunable emission spectra, long-term photostability, high quantum yield, and targeted drug delivery, hold tremendous promise for advancing diagnostics, therapeutics, and imaging techniques in biomedical research. However, several significant hurdles remain before their full potential in the biomedical field, like bioaccumulation, toxicity, and short-term stability. Addressing these hurdles is essential to effectively incorporate QDs into clinical use and enhance their influence on healthcare outcomes. Furthermore, the review conducts a critical analysis of potential QD toxicity and explores recent progress in strategies and methods to mitigate these adverse effects, such as surface modification, surface coatings, and encapsulation. By thoroughly examining current research and recent advancements, this comprehensive review offers invaluable insights into both the future possibilities and the challenges that lie ahead in fully harnessing the potential of QDs in the field of biomedicine, promising a revolution in the landscape of medical diagnostics, therapies, and imaging technologies
DKK1 and TNF-α Affect the Promotion Effect of Berberine on Osteogenic Differentiation of Dental Pulp Stem Cells in Peri-Implantitis
No full textBackground: Dental pulp stem cells (DPSCs) can improve periodontal tissue regeneration and have significant clinical application value in treating peri-implantitis. The Wnt signaling pathway is crucial for osteogenic differentiation of DPSCs. Berberine (BBR) has been found to promote the viability and differentiation of DPSC, whereas tumor necrosis factor-alpha (TNF-α) is a key factor affecting bone resorption. Additionally, Dickkopf 1 (DKK1) inhibits the Wnt canonical pathway. This study aims to explore how DKK1/TNF-α affects BBR-regulated osteogenic differentiation of DPSCs via the Wnt signaling pathway. Methods: We first evaluated the impact of varying doses of BBR, DKK1, and TNF-α on the viability of DPSCs through Cell Counting Kit-8 (CCK-8) experiments. Subsequently, we assessed the influence of DKK1/TNF-α/BBR on the osteogenic differentiation of DPSCs using alkaline phosphatase (ALP) staining, Alizarin Red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot techniques. Finally, we investigated the effects of DKK1/TNF-α/BBR on Wnt signaling-related genes and proteins during DPSC osteogenic differentiation using qRT-PCR and Western blot. Results: The results showed that moderate to low concentrations of BBR, DKK1, and TNF-α enhanced the activity of DPSCs, while high concentrations of BBR, DKK1, and TNF-α inhibited DPSC proliferation. BBR promoted the osteogenic differentiation of DPSC by activating the Wnt/β-catenin pathway. DKK1 or TNF-α alone inhibited the promotional effect of BBR, while the combination of DKK1 and TNF-α reduced the inhibitory effect. TNF-α up-regulated the expression of the classical Wnt pathway and down-regulated the expression of the non-classical Wnt pathway. On the other hand, DKK1 inhibited the classical pathway and promoted the non-classical pathway. Conclusions: DKK1 reduced the inhibitory effect of TNF-α on BBR and promoted the osteogenic differentiation of DPSC through the Wnt signaling pathway. These findings provided a reference value for the treatment of bone resorption caused by peri-implantitis
Asiaticoside Attenuates Burn Serum-induced Human Dermal Microvascular Endothelial Cell Injury via Regulating ACTN4-mediated p38-MAPK/p53 Signaling Pathway
No full textBackground: Asiaticoside has been affirmed to enhance the healing process of burn wounds, but the underlying molecular mechanism needs to be further elucidated. Therefore, this study aims to investigate the mechanism by which asiaticoside affects the burn serum-induced human dermal microvascular endothelial cell (HDMVEC) injury. Methods: Sprague-Dawley (SD) rats (n = 20) were divided into two groups: the burn group and the Sham group. Rats in the burn group were exposed to an electrically heated brass rod. HDMVECs were transfected with small interfering RNA targeting alpha-actinin-4 (ACTN4) (siACTN4) and negative control of siACTN4 (siNC). Burn serum-induced HDMVEC injury was used to mimic microvascular injury in vivo. Furthermore, HDMVECs were treated with asiaticoside and SB203580 (p38 mitogen-activated protein kinase (MAPK) specific inhibitor). The monolayer permeability in HDMVECs was evaluated using transendothelial electrical resistance, and cell viability was assessed using cell counting kit-8 (CCK-8) assay. The localization of zonula occludens-1 (ZO-1) and filamentous actin (F-actin) was determined through immunofluorescence staining. Moreover, the mRNA/protein expression levels of ACTN4, phosphorylated (p)-p53, p53, p-p38, and p38 in treated HDMVECs were evaluated using quantitative real-time PCR (qRT-PCR) and Western blot analysis. Results: Asiaticoside negated the inhibitory impacts of burn serum on viability and F-actin levels in HDMVECs (p < 0.05) as well as the promotive impacts of burn serum on monolayer permeability and ZO-1 levels. The expression of ACTN4 at both mRNA and protein was downregulated in burn serum-induced HDMVECs and upregulated by asiaticoside (p < 0.05). Asiaticoside reduced the protein expression levels of p-p53/p53 and p-p38/p38 and level of ZO-1, suppressed the monolayer permeability as well as promoted F-actin level and viability in burn serum-induced HDMVECs, all of which could be reversed by ACTN4 silencing (p < 0.05). Furthermore, SB203580 further offset the impacts of ACTN4 silencing on the protein expression levels of p-p53/p53 and p-p38/p38, levels of ZO-1 and F-actin as well as the monolayer permeability in burn serum-induced HDMVECs (p < 0.05). Conclusions: These findings suggest that asiaticoside enhances the healing process in burn serum-induced HDMVEC injury by regulating the ACTN4-mediated p38-MAPK/p53 signaling pathway
TNNC2 is a Microsatellite Instability-Related Gene for Response to Immune Checkpoint Blocking Therapy in Colorectal Cancer and Contributes to Tumor Progression
No full textBackground: Microsatellite instability (MSI) status may alter the tumor microenvironment (TME) and can affect the effectiveness of immune checkpoint blocking (ICB) immunotherapy in colorectal cancer (CRC) patients. This study aimed to determine the MSI-related genes as biomarkers for response to ICB therapy in CRC. Methods: The role and signature of MSI-related genes in CRC were analyzed through the data acquirement (LinkedOmics database), copy number alteration (CNA) assay (cBioPortal database), Association immune cells and Troponin C2 (TNNC2) levels analysis (Immune Cell Abundance Identifier (immuCellAI)), Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA). Silencing of TNNC2 was constructed in vitro and used for cell count kit 8 (CCK-8). CCK-8 was used to detect cell growth after relevant treatments. The cell apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit. The wound healing assay was used to determine cell migration. The enrichment of the TNNC2 pathways was performed using Gene Set Enrichment Analysis (GSEA). Results: The results showed that a total of 15 MSI-related genes were identified, including TNNC2 and ACSL6. Then, TNNC2 high-frequency amplification was found in the CRC. The expression of TNNC2 was lower in MSI-high group than MSI-low or microsatellite stability (MSS) group (p < 0.05). Compared to the high expression of TNNC2, the low expression of TNNC2 is more conducive to ICB response (p < 0.05). Additionally, TNNC2 regulated immune cell infiltration and ICB immunotherapy-related genes. Furthermore, TNNC2 was upregulated in CRC tissues and cell lines (p < 0.05). Cellular function assays showed that silence of TNNC2 decreased cell proliferation and migration, and induced apoptosis in CRC cells. Biological analysis also showed that TNNC2 plays a critical role in the activation of Wnt/β-catenin and PPAR in CRC. Conclusion: Hence, it can be concluded that TNNC2 is an MSI-related gene in response to ICB therapy in colorectal cancer. This study provided a better understanding of potential biomarker of ICB response and underlying mechanism in CRC, which is beneficial for CRC diagnosis and treatment research
Knockdown of Opsin 3 (OPN3) Enhances G6PD Autophagic Degradation and Aggravates Oxidative Stress Damage to Radiosensitize Cervical Cancer Cells
Get PDFBackground: Cervical cancer (CC) is the fourth most prevalent cancer among women worldwide, imposing a significant burden. While radiotherapy is a cornerstone in CC treatment, radioresistance remains a challenge, necessitating the exploration of mechanisms and novel targets for overcoming this challenge. Opsin 3 (OPN3) has been implicated in cancer treatment resistance, and the glucose-6-phosphate dehydrogenase (G6PD)-mediated glucose metabolism, along with oxidative stress, has been associated with radioresistance. This study aimed to explore the impact of OPN3 knockdown on the radioresistance of CC cells and the involvement of G6PD and oxidative stress in the underlying mechanisms. Methods: The HeLa cell line, a representative of CC, was utilized in this study. OPN3 knockdown was established through cell transfection. The subsequent effects on HeLa cell viability and apoptosis under ionizing radiation (IR) were assessed using cell counting kit-8 (CCK-8) assay, colony forming assay, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and western blot analysis to detect apoptosis-related proteins. Additionally, proteins associated with oxidative stress, DNA damage, and G6PD protein levels and activity were measured using enzyme-linked immunosorbent assay (ELISA) and western blot. Acquired radioresistant HeLa cells (RR-HeLa) were generated using IR. OPN3 knockdown was performed in RR-HeLa cells, and subsequent effects were assessed as described for HeLa cells. Reactive oxygen species (ROS) scavenger and G6PD inhibitor were employed to investigate the role of OPN3 in regulating oxidative stress and G6PD during radioresistance. Furthermore, in the G6PD degradation, autophagy inhibitor and proteasome pathway inhibitor were used to confirm the regulatory role of OPN3 on G6PD through autophagy. Results: OPN3 was upregulated in RR-HeLa cells (p < 0.001). OPN3 knockdown decreased the proliferation (p < 0.001), DNA damage (p < 0.001), and oxidative stress (p < 0.001) while reducing cell apoptosis (p < 0.001), glucose metabolism (p < 0.001), G6PD protein levels (p < 0.001), and activity (p < 0.001) in HeLa and RR-HeLa cells following IR treatment. OPN3 overexpression in HeLa cells increased proliferation post-IR treatment. The post-IR cell proliferation reduction induced by OPN3 knockdown was reversed by ROS inhibitor and G6PD overexpression (p < 0.001), while further decreased by G6PD inhibitor (p < 0.001). The OPN3 knockdown-induced decrease in G6PD was unchanged by the proteasome pathway inhibitor (p > 0.05) but was reversed by the autophagy inhibitor (p < 0.001), thereby reversing the post-IR cell proliferation (p < 0.001). Conclusion: OPN3 knockdown renders HeLa cells more radiosensitive and mitigates radioresistance by promoting the autophagic degradation of G6PD and exacerbating oxidative stress within the cells
Causal Associations between Genetically Determined Blood Metabolites and Ankylosing Spondylitis: A Two-Sample Mendelian Randomization Study
No full textBackground: Ankylosing spondylitis (AS) is a common inflammatory arthritis. The complex etiology of AS involves genetic, environmental, and immunological factors. However, prior research has associated blood metabolites with the onset and progression of AS. Therefore, in this study, a Mendelian randomization (MR) approach was utilized to assess the causal effects of blood metabolites on AS. Methods: The exposure factor data were retrieved from the Metabolite Genome-Wide Association Study (mGWAS) Summary data (IEU OpenGWAS project: https://gwas.mrcieu.ac.uk/datasets/). Whereas, the outcome factor data were accessed from the FinnGen consortium (ID: M13_ANKYLOSPON), a collaboration between Finnish biobanks and pharmaceutical companies (https://r9.finngen.fi/pheno/M13_ANKYLOSPON). A two-sample MR study was conducted using the inverse variance weighted model as the primary method. Several sensitivity analyses were performed to assess the validity of the outcomes. This included assessing heterogeneity to determine the consistency of causal estimates across different variants, the horizontal pleiotropy analysis to examine whether certain variants affect AS risk through pathways other than blood metabolites, and a leave-one-out cross-validation analysis to observe how the overall estimate changes when one variant is removed at a time. Furthermore, several other analyses, including replication, meta-analysis, Linkage disequilibrium score regression, reverse MR analysis, metabolic pathway analysis, and colocalization analysis were performed. Results: Out of 486 blood metabolites were investigated using MR and GWAS data from the FinnGen consortium, ten known metabolites exhibited significant causal associations with AS. Moreover, metabolites identified as risk factors for AS included 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (CMPF), butyrylcarnitine, tryptophan, N-acetylornithine, gamma-tocopherol, glycylvaline, taurodeoxycholate, hydroxyisovaleroyl carnitine, bilirubin, and aspartylphenylalanine. Conversely, taurodeoxycholate, hydroxyisovaleroyl carnitine, bilirubin, and aspartylphenylalanine were identified as protective factors for AS. Furthermore, Metabolic pathway analysis found that arginine biosynthesis, porphyrin and chlorophyll metabolism, tryptophan metabolism, and aminoacyl-tRNA biosynthesis were significantly linked to the development of AS. Conclusions: This study confirmed a causal relationship between blood metabolites and AS, and identified several metabolites of potential clinical and biological significance. These findings offer new insights for advancing diagnostic approaches and improving treatment options
The Expression of NXPH4 in Hepatocellular Carcinoma Tissues and Its Impact on the Biological Functions of Hepatic Cancer Cells
No full textBackground: There is an urgent need for reliable biomarkers for early detection and effective therapeutic targeting of hepatocellular carcinoma (HCC). This investigation aimed to elucidate neurexophilin 4 (NXPH4) expression in HCC and explore its impact on the biological functions of HCC cells. Methods: This research employed publicly available transcriptomic data from The Cancer Genome Atlas (TCGA) database to evaluate the expression profiles of the NXPH family members in HCC. We collected 70 HCC tissue samples and assessed NXPH4 expression levels using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot analysis, and immunohistochemistry. Furthermore, HCC cell lines with overexpressed and knocked down NXPH4 were established. Methyl thiazolyl tetrazolium (MTT) assay and colony formation analysis were conducted to assess the proliferation and colony formation capabilities of the HCC cells. Additionally, scratch assays and transwell experiments were employed to elucidate the migration and invasion capabilities of these cells. Results: NXPH4 showed significant differential expression in HCC compared to adjacent non-cancerous tissues (p < 0.001), which was associated with overall patient survival (p < 0.001). Moreover, the knockdown of NXPH4 (shNXPH4-1 and shNXPH4-2) significantly suppressed the proliferation and clonogenicity of HepG2 HCC cells (p < 0.01). Conversely, transfection with an overexpression vector (Ov-NXPH4) substantially elevated the proliferation and clonogenicity of Hub-7 cells (p < 0.01). Furthermore, knockdown of NXPH4 (shNXPH4-1 and shNXPH4-2) significantly suppressed the migration and invasion of HepG2 cells (p < 0.01), while its overexpression vector (Ov-NXPH4) significantly promoted these capabilities (p < 0.01). Conclusion: Elevated expression of NXPH4 in HCC regulates crucial biological processes, such as proliferation, migration, and invasion, suggesting its potential role as a biomarker for the progression of hepatocellular carcinoma
Heparanase Inhibitor OGT2115 Alleviates Myocardial Hypoxia/Reoxygenation Injury in H9c2 Cells through the MAPK/ERK Pathway
No full textBackground: Heparanase (HPSE) is an endo-beta-D-glucuronidase, and its upregulation is associated with many inflammatory diseases, such as atherosclerosis, fibrosis, and cancer. However, the role of HPSE in myocardial infarction remains unclear. Methods: Cell Counting Kit-8 (CCK8) assay was used to detect the development of the oxygen-glucose deprivation/reoxygenation (OGD/R) model and the effect of HPSE inhibitor OGT2115. Real-time quantitative PCR (RT-qPCR) and western blotting were used to detect HSPE expression in OGD/R model, the effect of HPSE inhibitor OGT2115, and the expression of cell proliferation-related protein, apoptosis-related protein, and fibrosis-related protein. The immunofluorescence assay was used to detect the expression of 5-Ethynyl-2′-Deoxyuridine (EdU) and TdT-mediated dUTP nick-end labeling (TUNEL). Enzyme-linked immunosorbent assay (ELISA) assay was used to detect the secretion of cytokines associated with the OGD/R model. Results: HPSE was highly expressed in the OGD/R model, and its inhibitor OGT2115 significantly promoted the proliferation and inhibited the apoptosis and fibrosis of H9C2 cells in the OGD/R model. Simultaneously, OGT2115 inhibited the secretion of inflammatory factors—tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-8—in the OGD/R model. Further mechanistic studies demonstrated that OGT2115 could inhibit the phosphorylation of mitogen-activated protein kinases (MAPK) and extracellular signal-regulated kinase (ERK). Conclusion: HPSE inhibitor OGT2115 alleviates myocardial IR injury in H9c2 cells and inhibits the MAPK/ERK pathway, indicating that HPSE is crucial in regulating myocardial infarction, and its inhibitor OGT2115 may be a potential drug for the treatment of myocardial infarction
Piperine Exhibits Anticancer Potential by Inhibiting the Cell Cycle Progression in Vitro
No full textBackground: Leukaemia is a blood-related cancer that is the leading cause of death worldwide. Piperine, a spicy alkaloid found in the spice Piper nigrum, has multifunctional properties. However, its anticancer effects against human chronic myeloid leukemia (CML) cells are yet to be investigated. The present study aims to investigate the anticancer potential of piperine using the human CML cell line KCL22. Methods: The anticancer effects of piperine were investigated through 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, generation of reactive oxygen species (ROS), cellular apoptosis, and cell cycle arrest. Additionally, in silico molecular docking was utilized to predict cell death by detecting the binding interactions of piperine with surface proteins of leukemia cells, specifically death receptors 4 (DR4) and death receptors 5 (DR5). Results: Data in the MTT assay showed that piperine treatment inhibited the cellular proliferation of KCL22 cells in a dose-dependent manner. Furthermore, nuclear condensation and ROS generation significantly increased when KCL22 cells were treated with piperine. Further study on cell cycle progression showed that piperine treatment inhibited cell cycle progression at G2/M and S phases. In silico molecular docking study showed that piperine has a good binding interaction with both DR4 and DR5. No violation of Lipinskis rule of five was observed. Piperine exhibited drug-likeness properties without toxicity. Conclusions: These findings suggest that piperine may have potential as an anticancer drug against human chronic myeloid leukemia