Changes in Parameters after High Tibial Osteotomy: Comparison of EOS System and Computed Tomographic Analysis.

Unintended rotation of the distal tibia occurs during medial open-wedge high tibial osteotomy (MOWHTO). Computed tomography (CT) is the standard method of measuring lower limb alignment; however, the new low-dose EOS system allows three-dimensional limb modeling with automated measurements of lower limb alignment. This study investigated the differences between the changes in lower limb alignment profiles obtained using the EOS system and CT in patients who underwent MOWHTO. We investigated whether any factors contributed to the degree of deformation. Thirty patients were prospectively enrolled between October 2019 and February 2023. Changes in femoral and tibial torsion, femorotibial rotation, and posterior tibial slope were measured using pre- and post-MOWHTO CT and EOS images. We found no significant difference in pre- and postoperative tibial torsion or posterior tibial slope between CT and EOS. No variables showed a significant correlation with changes in the tibial torsion or posterior tibial slope. This study confirmed the possibility that the EOS system could replace CT in measuring changes in several parameters pre- and postoperatively. Furthermore, we confirmed that the distal tibia tended to be internally rotated after MOWHTO; however, we found no significantly related parameters related to deformation caused by MOWHTO.

Common and distinct functions of mouse Dot1l in the regulation of endothelial transcriptome.

Epigenetic mechanisms are mandatory for endothelial called lymphangioblasts during cardiovascular development. Dot1l-mediated gene transcription in mice is essential for the development and function of lymphatic ECs (LECs). The role of Dot1l in the development and function of blood ECs blood endothelial cells is unclear. RNA-seq datasets from Dot1l-depleted or -overexpressing BECs and LECs were used to comprehensively analyze regulatory networks of gene transcription and pathways. Dot1l depletion in BECs changed the expression of genes involved in cell-to-cell adhesion and immunity-related biological processes. Dot1l overexpression modified the expression of genes involved in different types of cell-to-cell adhesion and angiogenesis-related biological processes. Genes involved in specific tissue development-related biological pathways were altered in Dot1l-depleted BECs and LECs. Dot1l overexpression altered ion transportation-related genes in BECs and immune response regulation-related genes in LECs. Importantly, Dot1l overexpression in BECs led to the expression of genes related to the angiogenesis and increased expression of MAPK signaling pathways related was found in both Dot1l-overexpressing BECs and LECs. Therefore, our integrated analyses of transcriptomics in Dot1l-depleted and Dot1l-overexpressed ECs demonstrate the unique transcriptomic program of ECs and the differential functions of Dot1l in the regulation of gene transcription in BECs and LECs.

Epigenetic regulator Cfp1 safeguards male meiotic progression by regulating meiotic gene expression.

Meiosis occurs specifically in germ cells to produce sperm and oocytes that are competent for sexual reproduction. Multiple factors are required for successful meiotic entry, progression, and termination. Among them, trimethylation of histone H3 on lysine 4 (H3K4me3), a mark of active transcription, has been implicated in spermatogenesis by forming double-strand breaks (DSBs). However, the role of H3K4me in transcriptional regulation during meiosis remains poorly understood. Here, we reveal that mouse CXXC finger protein 1 (Cfp1), a component of the H3K4 methyltransferase Setd1a/b, is dynamically expressed in differentiating male germ cells and safeguards meiosis by controlling gene expression. Genetic ablation of mouse CFP1 in male germ cells caused complete infertility with failure in prophase I of the 1st meiosis. Mechanistically, CFP1 binds to genes essential for spermatogenesis, and its loss leads to a reduction in H3K4me3 levels and gene expression. Importantly, CFP1 is highly enriched within the promoter/TSS of target genes to elevate H3K4me3 levels and gene expression at the pachytene stage of meiotic prophase I. The most enriched genes were associated with meiosis and homologous recombination during the differentiation of spermatocytes to round spermatids. Therefore, our study establishes a mechanistic link between CFP1-mediated transcriptional control and meiotic progression and might provide an unprecedented genetic basis for understanding human sterility.

Role of Transcriptional and Epigenetic Regulation in Lymphatic Endothelial Cell Development.

The lymphatic system is critical for maintaining the homeostasis of lipids and interstitial fluid and regulating the immune cell development and functions. Developmental anomaly-induced lymphatic dysfunction is associated with various pathological conditions, including lymphedema, inflammation, and cancer. Most lymphatic endothelial cells (LECs) are derived from a subset of endothelial cells in the cardinal vein. However, recent studies have reported that the developmental origin of LECs is heterogeneous. Multiple regulatory mechanisms, including those mediated by signaling pathways, transcription factors, and epigenetic pathways, are involved in lymphatic development and functions. Recent studies have demonstrated that the epigenetic regulation of transcription is critical for embryonic LEC development and functions. In addition to the chromatin structures, epigenetic modifications may modulate transcriptional signatures during the development or differentiation of LECs. Therefore, the understanding of the epigenetic mechanisms involved in the development and function of the lymphatic system can aid in the management of various congenital or acquired lymphatic disorders. Future studies must determine the role of other epigenetic factors and changes in mammalian lymphatic development and function. Here, the recent findings on key factors involved in the development of the lymphatic system and their epigenetic regulation, LEC origins from different organs, and lymphatic diseases are reviewed.

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo.

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

Comparison of the Predicting Performance for Fate of Medial Meniscus Posterior Root Tear Based on Treatment Strategies: A Comparison between Logistic Regression, Gradient Boosting, and CNN Algorithms.

This study aimed to validate the accuracy and prediction performance of machine learning (ML), deep learning (DL), and logistic regression methods in the treatment of medial meniscus posterior root tears (MMPRT). From July 2003 to May 2018, 640 patients diagnosed with MMPRT were included. First, the affecting factors for the surgery were evaluated using statistical analysis. Second, AI technology was introduced using X-ray and MRI. Finally, the accuracy and prediction performance were compared between ML&DL and logistic regression methods. Affecting factors of the logistic regression method corresponded well with the feature importance of the six top-ranked factors in the ML&DL method. There was no significant difference when comparing the accuracy, F1-score, and error rate between ML&DL and logistic regression methods (accuracy = 0.89 and 0.91, F1 score = 0.89 and 0.90, error rate = 0.11 and 0.09; p = 0.114, 0.422, and 0.119, respectively). The area under the curve (AUC) values showed excellent test quality for both ML&DL and logistic regression methods (AUC = 0.97 and 0.94, respectively) in the evaluation of prediction performance (p = 0.289). The affecting factors of the logistic regression method and the influence of the ML&DL method were not significantly different. The accuracy and performance of the ML&DL method in predicting the fate of MMPRT were comparable to those of the logistic regression method. Therefore, this ML&DL algorithm could potentially predict the outcome of the MMRPT in various fields and situations. Furthermore, our method could be efficiently implemented in current clinical practice.

Can Deep Learning Using Weight Bearing Knee Anterio-Posterior Radiograph Alone Replace a Whole-Leg Radiograph in the Interpretation of Weight Bearing Line Ratio?

Weight bearing whole-leg radiograph (WLR) is essential to assess lower limb alignment such as weight bearing line (WBL) ratio. The purpose of this study was to develop a deep learning (DL) model that predicts the WBL ratio using knee standing AP alone. Total of 3997 knee AP & WLRs were used. WBL ratio was used for labeling and analysis of prediction accuracy. The WBL ratio was divided into seven categories (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6). After training, performance of the DL model was evaluated. Final performance was evaluated using 386 subjects as a test set. Cumulative score (CS) within error range 0.1 was set with showing maximum CS in the validation set (95% CI, 0.924-0.970). In the test set, mean absolute error was 0.054 (95% CI, 0.048-0.061) and CS was 0.951 (95% CI, 0.924-0.970). Developed DL algorithm could predict the WBL ratio on knee standing AP alone with comparable accuracy as the degree primary physician can assess the alignment. It can be the basis for developing an automated lower limb alignment assessment tool that can be used easily and cost-effectively in primary clinics.

Insights from the Applications of Single-Cell Transcriptomic Analysis in Germ Cell Development and Reproductive Medicine.

Mechanistic understanding of germ cell formation at a genome-scale level can aid in developing novel therapeutic strategies for infertility. Germ cell formation is a complex process that is regulated by various mechanisms, including epigenetic regulation, germ cell-specific gene transcription, and meiosis. Gonads contain a limited number of germ cells at various stages of differentiation. Hence, genome-scale analysis of germ cells at the single-cell level is challenging. Conventional genome-scale approaches cannot delineate the landscape of genomic, transcriptomic, and epigenomic diversity or heterogeneity in the differentiating germ cells of gonads. Recent advances in single-cell genomic techniques along with single-cell isolation methods, such as microfluidics and fluorescence-activated cell sorting, have helped elucidate the mechanisms underlying germ cell development and reproductive disorders in humans. In this review, the history of single-cell transcriptomic analysis and their technical advantages over the conventional methods have been discussed. Additionally, recent applications of single-cell transcriptomic analysis for analyzing germ cells have been summarized.

Conjugation of vascular endothelial growth factor to poly lactic-co-glycolic acid nanospheres enhances differentiation of embryonic stem cells to lymphatic endothelial cells.

OBJECTIVE: Pluripotent stem cell-derived lymphatic endothelial cells (LECs) show great promise in their therapeutic application in the field of regenerative medicine related to lymphatic vessels. We tested the approach of forced differentiation of mouse embryonal stem cells into LECs using biodegradable poly lactic-co-glycolic acid (PLGA) nanospheres in conjugation with growth factors (vascular endothelial growth factors [VEGF-A and VEGF-C]). METHODS: We evaluated the practical use of heparin-conjugated PLGA nanoparticles (molecular weight ~15,000) in conjugation with VEGF-A/C, embryoid body (EB) formation, and LEC differentiation using immunofluorescence staining followed by quantification and quantitative real-time polymerase chain reaction analysis. RESULTS: We showed that formation and differentiation of EB with VEGF-A/C-conjugated PLGA nanospheres, compared to direct supplementation of VEGF-A/C to the EB differentiation media, greatly improved yield of LYVE1(+) LECs. Our analyses revealed that the enhanced potential of LEC differentiation using VEGF-A/C-conjugated PLGA nanospheres was mediated by elevation of expression of the genes that are important for lymphatic vessel formation. CONCLUSION: Together, we not only established an improved protocol for LEC differentiation using PLGA nanospheres but also provided a platform technology for the mechanistic study of LEC development in mammals.

ATP-Dependent Chromatin Remodeler CHD9 Controls the Proliferation of Embryonic Stem Cells in a Cell Culture Condition-Dependent Manner.

Emerging evidence suggests that chromodomain-helicase-DNA-binding (CHD) proteins are involved in stem cell maintenance and differentiation via the coordination of chromatin structure and gene expression. However, the molecular function of some CHD proteins in stem cell regulation is still poorly understood. Herein, we show that Chd9 knockdown (KD) in mouse embryonic stem cells (ESCs) cultured in normal serum media, not in 2i-leukemia inhibitory factor (LIF) media, causes rapid cell proliferation. This is caused by transcriptional regulation related to the cell cycle and the response to growth factors. Our analysis showed that, unlike the serum cultured-Chd9 KD ESCs, the 2i-LIF-cultured-Chd9 KO ESCs displayed elevated levels of critical G1 phase regulators such as p21 and p27. Consistently, the DNA binding sites of CHD9 overlap with some transcription factor DNA motifs that are associated with genes regulating the cell cycle and growth pathways. These transcription factors include the cycle gene homology region (CHR), Arid5a, and LIN54. Collectively, our results provide new insights into CHD9-mediated gene transcription for controlling the cell cycle of ESCs.

Ascorbic Acid Promotes Functional Restoration after Spinal Cord Injury Partly by Epigenetic Modulation.

Axonal regeneration after spinal cord injury (SCI) is difficult to achieve, and no fundamental treatment can be applied in clinical settings. DNA methylation has been suggested to play a role in regeneration capacity and neuronal growth after SCI by controlling the expression of regeneration-associated genes (RAGs). The aim of this study was to examine changes in neuronal DNA methylation status after SCI and to determine whether modulation of DNA methylation with ascorbic acid can enhance neuronal regeneration or functional restoration after SCI. Changes in epigenetic marks (5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC)); the expression of Ten-eleven translocation (Tet) family genes; and the expression of genes related to inflammation, regeneration, and degeneration in the brain motor cortex were determined following SCI. The 5hmC level within the brain was increased after SCI, especially in the acute and subacute stages, and the mRNA levels of Tet gene family members (Tet1, Tet2, and Tet3) were also increased. Administration of ascorbic acid (100 mg/kg) to SCI rats enhanced 5hmC levels; increased the expression of the Tet1, Tet2, and Tet3 genes within the brain motor cortex; promoted axonal sprouting within the lesion cavity of the spinal cord; and enhanced recovery of locomotor function until 12 weeks. In conclusion, we found that epigenetic status in the brain motor cortex is changed after SCI and that epigenetic modulation using ascorbic acid may contribute to functional recovery after SCI.

TMEM100 is a key factor for specification of lymphatic endothelial progenitors.

BACKGROUND: TMEM100 is identified as a downstream gene of bone morphogenetic protein 9 (BMP9) signaling via activin receptor-like kinase 1 (ALK1), which is known to participate in lymphangiogenesis as well as angiogenesis. TMEM100 has been shown to be important for blood vessel formation and maintenance, but its role in the development of lymphatic vasculature remains unknown. The objective is to investigate the role of TMEM100 in development of the lymphatic system. METHODS AND RESULTS: Global Tmem100 gene deletion was induced by tamoxifen on 10.5 days post-coitus. Tmem100-inducible knockout (iKO) embryos in embryonic days (E)14.5-16.5 exhibited edema and blood-filled enlarged lymphatics with misconnections between veins and lymphatic vessels. For a reciprocal approach, we have generated a novel mouse line in which TMEM100 overexpression (OE) can be induced in endothelial cells by intercrossing with Tie2-Cre driver. TMEM100-OE embryos at E12.5-14.5 exhibited edema with small size and number of lymphatic vessels, the exact opposite phenotypes of Tmem100-iKOs. In Tmem100-iKO embryos, the number of progenitors of lymphatic endothelial cells (LECs) in the cardinal vein was increased, while it was decreased in TMEM100-OE embryos. The activity of NOTCH signaling, which limits the number of progenitors of LECs in the cardinal vein, was decreased in Tmem100-iKO embryos, whereas it was increased in TMEM100-OE embryos. CONCLUSION: TMEM100 plays an important role in the specification of LECs in the cardinal veins, at least in part, by regulating the NOTCH signaling.

Anisotropic Platinum Nanoparticle-Induced Cytotoxicity, Apoptosis, Inflammatory Response, and Transcriptomic and Molecular Pathways in Human Acute Monocytic Leukemia Cells.

The thermoplasmonic properties of platinum nanoparticles (PtNPs) render them desirable for use in diagnosis, detection, therapy, and surgery. However, their toxicological effects and impact at the molecular level remain obscure. Nanotoxicology is mainly focused on the interactions of nanostructures with biological systems, particularly with an emphasis on elucidating the relationship between the physical and chemical properties such as size and shape. Therefore, we hypothesized whether these unique anisotropic nanoparticles could induce cytotoxicity similar to that of spherical nanoparticles and the mechanism involved. Thus, we synthesized unique and distinct anisotropic PtNPs using lycopene as a biological template and investigated their biological activities in model human acute monocytic leukemia (THP-1) macrophages. Exposure to PtNPs for 24 h dose-dependently decreased cell viability and proliferation. Levels of the cytotoxic markers lactate dehydrogenase and intracellular protease significantly and dose-dependently increased with PtNP concentration. Furthermore, cells incubated with PtNPs dose-dependently produced oxidative stress markers including reactive oxygen species (ROS), malondialdehyde, nitric oxide, and carbonylated protein. An imbalance in pro-oxidants and antioxidants was confirmed by significant decreases in reduced glutathione, thioredoxin, superoxide dismutase, and catalase levels against oxidative stress. The cell death mechanism was confirmed by mitochondrial dysfunction and decreased ATP levels, mitochondrial copy numbers, and PGC-1α expression. To further substantiate the mechanism of cell death mediated by endoplasmic reticulum stress (ERS), we determined the expression of the inositol-requiring enzyme (IRE1), (PKR-like ER kinase) PERK, activating transcription factor 6 (ATF6), and activating transcription factor 4 ATF4, the apoptotic markers p53, Bax, and caspase 3, and the anti-apoptotic marker Bcl-2. PtNPs could activate ERS and apoptosis mediated by mitochondria. A proinflammatory response to PtNPs was confirmed by significant upregulation of interleukin-1-beta (IL-1ß), interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL-6). Transcriptomic and molecular pathway analyses of THP-1 cells incubated with the half maximal inhibitory concentration (IC50) of PtNPs revealed the altered expression of genes involved in protein misfolding, mitochondrial function, protein synthesis, inflammatory responses, and transcription regulation. We applied transcriptomic analyses to investigate anisotropic PtNP-induced toxicity for further mechanistic studies. Isotropic nanoparticles are specifically used to inhibit non-specific cellular uptake, leading to enhanced in vivo bio-distribution and increased targeting capabilities due to the higher radius of curvature. These characteristics of anisotropic nanoparticles could enable the technology as an attractive platform for nanomedicine in biomedical applications.

Epigenetic priming by Dot1l in lymphatic endothelial progenitors ensures normal lymphatic development and function.

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

Nonylphenol Induces Apoptosis through ROS/JNK Signaling in a Spermatogonia Cell Line.

Nonylphenol (NP) is an endocrine-disruptor chemical that negatively affects reproductive health. Testes exposure to NP results in testicular structure disruption and a reduction in testicular size and testosterone levels. However, the effects of NP on spermatogonia in testes have not been fully elucidated. In this study, the molecular mechanisms of NP in GC-1 spermatogonia (spg) cells were investigated. We found that cell viability significantly decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to NP. Furthermore, the expression levels of the pro-apoptotic proteins increased, whereas anti-apoptosis markers decreased in NP-exposed GC-1 spg cells. We also found that NP increased reactive oxygen species (ROS) generation, suggesting that ROS-induced activation of the MAPK signaling pathway is the molecular mechanism of NP-induced apoptosis in GC-1 spg cells. Thus, NP could induce c-Jun phosphorylation; dose-dependent expression of JNK, MKK4, p53, and p38; and the subsequent inhibition of ERK1/2 and MEK1/2 phosphorylation. The genes involved in apoptosis and JNK signaling were also upregulated in GC-1 spg cells treated with NP compared to those in the controls. Our findings suggest that NP induces apoptosis through ROS/JNK signaling in GC-1 spg cells.

Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells.

Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.

Activation of peroxisome proliferator-activated receptor delta suppresses BACE1 expression by up-regulating SOCS1 in a JAK2/STAT1-dependent manner.

Neuronal expression of beta-secretase 1 (BACE1) has been implicated in the progression of Alzheimer's disease. However, the mechanisms that regulate BACE1 expression are unclear. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) decreases BACE1 expression by up-regulating suppressor of cytokine signaling 1 (SOCS1) in SH-SY5Y neuroblastoma cells. The activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited expression of BACE1. This effect was abrogated by shRNA-mediated knockdown of PPARδ and by treatment with the PPARδ antagonist GSK0660, indicating that PPARδ is involved in GW501516-mediated suppression of BACE1 expression. On the other hand, GW501516-activated PPARδ induced expression of SOCS1, which is a negative regulator of cytokine signal transduction, at the transcriptional level by binding to a PPAR response element in its promoter. This GW501516-mediated induction of SOCS1 expression led to down-regulation of BACE1 expression via inactivation of signal transducer and activator of transcription 1. GW501516-activated PPARδ suppressed the generation of neurotoxic amyloid beta (Aß) in accordance with the decrease in BACE1 expression. Taken together, these results indicate that PPARδ attenuates BACE1 expression via SOCS1-mediated inhibition of signal transducer and activator of transcription 1 signaling, thereby suppressing BACE1-associated generation of neurotoxic Aß.

Cytotoxicity and Transcriptomic Analyses of Biogenic Palladium Nanoparticles in Human Ovarian Cancer Cells (SKOV3).

Ovarian cancer incidence continues to increase at an alarming rate. Although various therapeutic approaches exist for ovarian cancer, they have limitations, including undesired side effects. Therefore, nanoparticle (NP)-mediated therapy may be a viable, biocompatible, and suitable alternative. To the best of our knowledge, no comprehensive analysis has been undertaken on the cytotoxicity and cellular pathways involved in ovarian cancer cells, particularly SKOV3 cells. Here, we investigated the effect of palladium NPs (PdNPs) and the molecular mechanisms and cellular pathways involved in ovarian cancer. We assayed cell viability, proliferation, cytotoxicity, oxidative stress, DNA damage, and apoptosis and performed an RNA-Seq analysis. The results showed that PdNPs elicited concentration-dependent decreases in cell viability and proliferation and induced increasing cytotoxicity at increasing concentrations, as determined by leakage of lactate dehydrogenase, increased levels of reactive oxygen species and malondialdehyde, and decreased levels of antioxidants like glutathione and superoxide dismutase. Furthermore, our study revealed that PdNPs induce mitochondrial dysfunction by altering mitochondrial membrane potential, reducing adenosine triphosphate levels, inducing DNA damage, and activating caspase 3, all of which significantly induced apoptosis in SKOV3 cells following PdNPs treatment. Gene ontology (GO) term analysis of PdNPs-exposed SKOV3 cells showed various dysregulated pathways, particularly nucleosome assembly, telomere organization, and rDNA chromatin silencing. When genes downregulated by PdNPs were applied to GO term enrichment analysis, nucleosome assembly was the top-ranked biological pathway. We also provide evidence for an association between PdNPs exposure and multiple layers of epigenetic transcriptional control and establish a molecular basis for NP-mediated apoptosis. These findings provide a foundation, potential targets, and novel insights into the mechanism underlying toxicity and pathways in SKOV3 cells, and open new avenues to identify novel targets for ovarian cancer treatment.

Cytotoxicity and Transcriptomic Analysis of Silver Nanoparticles in Mouse Embryonic Fibroblast Cells.

The rapid development of nanotechnology has led to the use of silver nanoparticles (AgNPs) in biomedical applications, including antibacterial, antiviral, anti-inflammatory, and anticancer therapies. The molecular mechanism of AgNPs-induced cytotoxicity has not been studied thoroughly using a combination of cellular assays and RNA sequencing (RNA-Seq) analysis. In this study, we prepared AgNPs using myricetin, an anti-oxidant polyphenol, and studied their effects on NIH3T3 mouse embryonic fibroblasts as an in vitro model system to explore the potential biomedical applications of AgNPs. AgNPs induced loss of cell viability and cell proliferation in a dose-dependent manner, as evident by increased leakage of lactate dehydrogenase (LDH) from cells. Reactive oxygen species (ROS) were a potential source of cytotoxicity. AgNPs also incrementally increased oxidative stress and the level of malondialdehyde, depleted glutathione and superoxide dismutase, reduced mitochondrial membrane potential and adenosine triphosphate (ATP), and caused DNA damage by increasing the level of 8-hydroxy-2'-deoxyguanosine and the expressions of the p53 and p21 genes in NIH3T3 cells. Thus, activation of oxidative stress may be crucial for NIH3T3 cytotoxicity. Interestingly, gene ontology (GO) term analysis revealed alterations in epigenetics-related biological processes including nucleosome assembly and DNA methylation due to AgNPs exposure. This study is the first demonstration that AgNPs can alter bulk histone gene expression. Therefore, our genome-scale study suggests that the apoptosis observed in NIH3T3 cells treated with AgNPs is mediated by the repression of genes required for cell survival and the aberrant enhancement of nucleosome assembly components to induce apoptosis.

Cytotoxic Potential and Molecular Pathway Analysis of Silver Nanoparticles in Human Colon Cancer Cells HCT116.

Silver nanoparticles (AgNPs) have gained attention for use in cancer therapy. In this study, AgNPs were biosynthesized using naringenin. We investigated the anti-colon cancer activities of biogenic AgNPs through transcriptome analysis using RNA sequencing, and the mechanisms of AgNPs in regulating colon cancer cell growth. The synthesized AgNPs were characterized using UV⁻visible spectroscopy (UV⁻vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The AgNPs were spherical with sizes of 2⁻10 nm. Cytotoxicity assays indicated that the AgNPs in HCT116 colorectal cancer cells were very effective at low concentrations. The viability and proliferation of colon cancer cells treated with 5 µg/mL biogenic AgNPs were reduced by 50%. Increased lactate dehydrogenase leakage (LDH), reactive oxygen species (ROS) generation, malondialdehyde (MDA), and decreased dead-cell protease activity and ATP generation were observed. This impaired mitochondrial function and DNA damage led to cell death. The AgNPs upregulated and downregulated the most highly ranked biological processes of oxidation⁻reduction and cell-cycle regulation, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that AgNPs upregulated GADD45G in the p53 pathway. Thus, the AgNP tumor suppressive effects were mediated by cell apoptosis following DNA damage, as well as by mitochondrial dysfunction and cell-cycle arrest following aberrant regulation of p53 effector proteins. It is of interest to mention that, to the best of our knowledge, this study is the first report demonstrating cellular responses and molecular pathways analysis of AgNPs in HCT116 colorectal cancer cells.