Cell Biology Experiments Research Articles

Amyloid Nanofilm-Induced surface mineralization of 3D-Printed Polyetheretherketone scaffolds for in situ orbital bone regeneration and repair

Orbital bone defect repair is both challenging and crucial and requires the comprehensive consideration of anatomical complexity, functional preservation, aesthetic outcomes, postoperative risks, and long-term effects. Polyetheretherketone (PEEK) is a promising orthopedic substitute material due to its cortical bone-like elastic modulus, biocompatibility, chemical stability, and natural radiolucency. However, PEEK is bioinert and lacks interfacial bioactivity, which limits its ability to promote bone growth and osseointegration. In this study, we fabricated porous PEEK scaffolds using Fused Deposition Modeling (FDM) 3D printing technology. We employed a phase-transitioned lysozyme (PTL) nanofilm as the organic matrix template to construct a robust hydroxyapatite (HAp) coating both inside and outside the porous PEEK scaffold, generating HAp@PTL@PO-PEEK. The PTL nanofilm acted as a strong glue, enhancing the interfacial bonding strength between the HAp coating and PEEK. In vitro cell biology experiments revealed that HAp@PTL@PO-PEEK promoted the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Furthermore, the modified scaffolds exhibited excellent osteoconductivity and osteoinductivity in the in vivo repair of rabbit orbital bone defects, promoting new bone formation and guiding new bone growth into the scaffold. Therefore, HAp@PTL@PO-PEEK scaffolds hold potential for clinical craniomaxillofacial bone regeneration and repair.

Pyrotinib and trastuzumab combination treatment synergistically overcomes HER2 dependency in HER2-positive breast cancer: insights from the PHILA trial

The PHILA study suggests that pyrotinib, trastuzumab, and docetaxel significantly improved progression-free survival (PFS) compared with placebo, trastuzumab, and docetaxel in patients with untreated HER2-positive metastatic breast cancer. In this study, we aimed to investigate the synergistic mechanisms of pyrotinib plus trastuzumab and provide further insights for the PHILA trial. The invitro activity of combination treatments was assessed through cell biological and biochemical experiments. The invivo efficacy was evaluated in cell-derived xenografts, a TUBO tumour model, and one clinical case. Next-generation sequencing was performed on circulating tumour DNA (ctDNA) from patients in the PHILA trial. The combination of pyrotinib and trastuzumab more effectively inhibited cell growth than pyrotinib or trastuzumab alone in models of HER2-dependent breast cancer. It potentiated membrane HER2 ubiquitination and downregulation, which resulted in a comprehensive blockade of the HER2 signalling pathway. The pyrotinib-altered membrane HER2 levels had no significant effect on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). We further validated the synergistic mechanisms in TUBO tumours and one clinical case, rather than models of HCC1954cells harbouring the PIK3CA H1047R mutation. Similarly, in our centre cohort of the PHILA study, patients with genetic alterations in the HER2 signalling cascade had significantly shorter median PFS than individuals with the wild-type pathway. Our findings underscore the robust synergy between pyrotinib and trastuzumab in overcoming HER2 dependency and provide a rationale for pyrotinib, trastuzumab, and docetaxel as one of the optimal choices for patients with untreated HER2-positive metastatic breast cancer, who are dependent on the HER2 signalling cascade. This work was supported by the National Key Research and Development Program of China (2021YFF1201300), the National Natural Science Foundation of China (82172875), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2022-I2M-2-001), and the Joint Innovative Fund of Beijing Natural Science Foundation and Changping District (L234004).

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.

Likelihood-based inference, identifiability, and prediction using count data from lattice-based random walk models.

In vitro cell biology experiments are routinely used to characterize cell migration properties under various experimental conditions. These experiments can be interpreted using lattice-based random walk models to provide insight into underlying biological mechanisms, and continuum limit partial differential equation(PDE) descriptions of the stochastic models can be used to efficiently explore model properties instead of relying on repeated stochastic simulations. Working with efficient PDE models is of high interest for parameter estimation algorithms that typically require a large number of forward model simulations. Quantitative data from cell biology experiments usually involve non-negative cell counts in different regions of the experimental images, and it is not obvious how to relate finite, noisy count data to the solutions of continuous PDE models that correspond to noise-free density profiles. In this work, we illustrate how to develop and implement likelihood-based methods for parameter estimation, parameter identifiability, and model prediction for lattice-based models describing collective migration with an arbitrary number of interacting subpopulations. We implement a standard additive Gaussian measurement error model as well as a new physically motivated multinomial measurement error model that relates noisy count data with the solution of continuous PDE models. Both measurement error models lead to similar outcomes for parameter estimation and parameter identifiability, whereas the standard additive Gaussian measurement error model leads to nonphysical prediction outcomes. In contrast, the new multinomial measurement error model involves a lower computational overhead for parameter estimation and identifiability analysis, as well as leading to physically meaningful model predictions.

Integrative Human Genetic and Cellular Analysis of the Pathophysiological Roles of AnxA2 in Alzheimer's Disease.

Alzheimer's disease (AD) is an intractable and progressive neurodegenerative disease. Amyloid beta (Aβ) aggregation is the hallmark of AD. Aβ induces neurotoxicity through a variety of mechanisms, including interacting with membrane receptors to alter downstream signaling, damaging cellular or organelle membranes, interfering with protein degradation and synthesis, and inducing an excessive immune-inflammatory response, all of which lead to neuronal death and other pathological changes associated with AD. In this study, we extracted gene expression profiles from the GSE5281 and GSE97760 microarray datasets in the GEO (Gene Expression Omnibus) database, as well as from the Human Gene Database. We identified differentially expressed genes in the brain tissues of AD patients and healthy persons. Through GO, KEGG, and ROC analyses, annexin A2 (AnxA2) was identified as a putative target gene. Notably, accumulating evidence suggests that intracellular AnxA2 is a key regulator in various biological processes, including endocytosis, transmembrane transport, neuroinflammation, and apoptosis. Thus, we conducted a series of cell biology experiments to explore the biological function of AnxA2 in AD. The results indicate that AnxA2 gene knockdown primarily affects oxidative phosphorylation, cell cycle, AD, protein processing in the endoplasmic reticulum, SNARE interactions in vesicular transport, and autophagy. In SH-SY5Y cells secreting Aβ42, AnxA2 gene knockdown exacerbated Aβ42-induced cytotoxicity, including cell death, intracellular ROS levels, and neuronal senescence, altered cell cycle, and reduced ATP levels, suggesting its critical role in mitochondrial function maintenance. AnxA2 gene knockdown also exacerbated the inhibitory effect of Aβ42 on cell migration. AnxA2 overexpression reduced the inflammatory response induced by Aβ42, while its absence increased pro-inflammatory and decreased anti-inflammatory responses. Furthermore, AnxA2 gene knockdown facilitated apoptosis and decreased autophagy. These results indicated potential pathophysiological roles of AnxA2 in AD.

A Comparative Study of the Chemical Composition and Skincare Activities of Red and Yellow Ginseng Berries.

This study was conducted to investigate the differences in chemical composition between red (RGBs) and yellow ginseng berries (YGBs) and their whitening and anti-aging skincare effects. The differences in the chemical composition between RGB and YGB were analyzed by ultra-high-performance liquid chromatography tandem quadrupole electrostatic field orbit trap mass spectrometry (UHPLC-Q-Exactive-MS/MS) combined with multivariate statistics. An aging model was established using UVB radiation induction, and the whitening and anti-aging effects of the two ginseng berries were verified in vitro and in vivo using cell biology (HaCaT and B16-F10 cells) and zebrafish model organisms. A total of 31 differential compounds, including saponins, flavonoids, phenolic acids, and other chemical constituents, were identified between the two groups. Superoxide dismutase (SOD) activity was more significantly increased (p < 0.05) and malondialdehyde (MDA) content was more significantly decreased (p < 0.01) in RGB more than YGB induced by UVB ultraviolet radiation. In terms of whitening effects, YGB was more effective in inhibiting the melanin content of B16-F10 cells (p < 0.01). The results of zebrafish experiments were consistent with those of in vitro experiments and cell biology experiments. The DCFH fluorescence staining results revealed that both ginseng berries were able to significantly reduce the level of reactive oxygen species (ROS) in zebrafish (p < 0.01). Comparison of chemical composition and skin care activities based on RGB and YGB can provide a theoretical basis for the deep development and utilization of ginseng berry resources.

Toll-Like Receptor 4 (TLR4) Promotes DRG Regeneration and Repair after Sciatic Nerve Injury via the ERK-NF-kB Pathway.

Previously, we found that the expression of Toll-like receptor 4 (TLR4) is altered after sciatic nerve injury, and its differential expression plays a key role in recovery. However, the mechanisms by which TLR4 affects neuronal function in the dorsal root ganglion (DRG) have not been completely evaluated. The objective is to determine TLR4 expression in DRG tissues after sciatic neural injury and exploring the effects of TLR4 knockdown and overexpression in the DRG on neuronal function and nerve regeneration in rats in vivo and in vitro. We established a model of nerve injury and utilized molecular biology and cell biology experiments to explore the molecular mechanisms by which TLR4 in the DRG affects sciatic nerve restoration and regeneration after injury. Verified the localization of TLR4 in DRG neurons. Investigated pathways that related to apoptosis or nerve regeneration by which TLR4 regulates the function of DRG neurons. TLR4 expression was upregulated in the DRG tissues of rats after sciatic nerve injury. TLR4 overexpression promoted axon regeneration and inhibited apoptosis in DRG neurons. TLR4 promoted the regeneration of axons and the recovery of motor and sensory functions in the sciatic nerve after injury in vivo, and the data showed that TLR4 may regulate the function of DRG neurons and promote nerve repair and regeneration through the ERK and NF-κB signaling pathways in vivo and ex vivo. The study suggests that TLR4 may regulate the function of DRG neurons and promote nerve regeneration by affecting the ERK and NF-κB signaling pathways.

Forecasting and Predicting Stochastic Agent-Based Model Data with Biologically-Informed Neural Networks.

Collective migration is an important component of many biological processes, including wound healing, tumorigenesis, and embryo development. Spatial agent-based models (ABMs) are often used to model collective migration, but it is challenging to thoroughly predict these models' behavior throughout parameter space due to their random and computationally intensive nature. Modelers often coarse-grain ABM rules into mean-field differential equation (DE) models. While these DE models are fast to simulate, they suffer from poor (or even ill-posed) ABM predictions in some regions of parameter space. In this work, we describe how biologically-informed neural networks (BINNs) can be trained to learn interpretable BINN-guided DE models capable of accurately predicting ABM behavior. In particular, we show that BINN-guided partial DE (PDE) simulations can (1) forecast future spatial ABM data not seen during model training, and (2) predict ABM data at previously-unexplored parameter values. This latter task is achieved by combining BINN-guided PDE simulations with multivariate interpolation. We demonstrate our approach using three case study ABMs of collective migration that imitate cell biology experiments and find that BINN-guided PDEs accurately forecast and predict ABM data with a one-compartment PDE when the mean-field PDE is ill-posed or requires two compartments. This work suggests that BINN-guided PDEs allow modelers to efficiently explore parameter space, which may enable data-driven tasks for ABMs, such as estimating parameters from experimental data. All code and data from our study is available at https://github.com/johnnardini/Forecasting_predicting_ABMs .

Long non-coding RNA PVT1 regulates TGF-β and promotes the proliferation, migration and invasion of hypopharyngeal carcinoma FaDu cells

Hypopharyngeal carcinoma is one of the malignant tumors of the head and neck with a particularly poor prognosis. Recurrence and metastasis are important reasons for poor prognosis of hypopharyngeal cancer patients, and malignant proliferation, migration, and invasion of tumor cells are important factors for recurrence and metastasis of hypopharyngeal cancer. Therefore, elucidating hypopharyngeal cancer cells’ proliferation, migration, and invasion mechanism is essential for improving diagnosis, treatment, and prognosis. Plasmacytoma Variant Translocation 1 (PVT1) is considered a potential diagnostic marker and therapeutic target for tumors. However, it remains unclear whether PVT1 is related to the occurrence and development of hypopharyngeal cancer and its specific mechanism. In this study, the promoting effect of PVT1 on the proliferation, migration, and invasion of hypopharyngeal carcinoma FaDu cells was verified by cell biology experiments and animal studies, and it was found that PVT1 inhibited the expression of TGF-β, suggesting that PVT1 may regulate the occurrence and development of hypopharyngeal carcinoma FaDu cells through TGF-β.

Polarly localized Bro1 domain proteins regulate PIN-FORMED abundance and root gravitropic growth in Arabidopsis

The developmental plasticity of the root system plays an essential role in the adaptation of plants to the environment. Among many other signals, auxin and its directional, intercellular transport are critical in regulating root growth and development. In particular, the PIN-FORMED2 (PIN2) auxin exporter acts as a key regulator of root gravitropic growth. Multiple regulators have been reported to be involved in PIN2-mediated root growth; however, our information remains incomplete. Here, we identified ROWY Bro1-domain proteins as important regulators of PIN2 sorting control. Genetic analysis revealed that Arabidopsis rowy1 single mutants and higher-order rowy1 rowy2 rowy3 triple mutants presented a wavy root growth phenotype. Cell biological experiments revealed that ROWY1 and PIN2 colocalized to the apical side of the plasma membrane in the root epidermis and that ROWYs are required for correct PM targeting of PIN2. In addition, ROWYs also affected PIN3 protein abundance in the stele, suggesting the potential involvement of additional PIN transporters as well as other proteins. A global transcriptome analysis revealed that ROWY genes are involved in the Fe2+ availability perception pathway. This work establishes ROWYs as important novel regulators of root gravitropic growth by connecting micronutrient availability to the proper subcellular targeting of PIN auxin transporters.

3-Aminobenzamide-linked multifunctional nanoparticles enhances anticancer activity of low-dose cisplatin chemotherapy in lung adenocarcinoma

Chemotherapy is one of the main treatment methods for cancer patients, but its effectiveness is limited by drug resistance. Combining a chemotherapeutic agent with targeted molecular therapy may improve the curative effect of the chemotherapeutic agent. In this study, we investigate the efficacy of combining a 3-Aminobenzamide (3AB)-linked multifunctional platform with low-dose cisplatin chemotherapy aiming to modulate poly [ADP-ribose] polymerase 1 (PARP1) function in DNA repair to increase cytotoxic activity of the platinum-based cisplatin. The structure of the synthesized nanoplatforms was characterized by several physicochemical techniques, including dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and an in vitro pH-dependent release study. Cellular uptake experiments demonstrated preferentially targeted delivery of nanoparticles in lung carcinoma A549 cells, whereas the cellular uptake capacity was minimal in normal lung BEAS-2B cells. On the other hand, cytotoxicity experiments showed a reduction of cancer cell viability compared to free formulations. Furthermore, the combination treatment was examined by detecting the loss of mitochondrial membrane potential and the apoptotic cell population, confirming the treatment's functional involvement in apoptosis. Soft agar colony formation and cell invasion tests were also performed to detect the cancer cell's tumorigenic potential, confirming the synergistic effect of this combination in the reduction of tumorigenicity. Moreover, we analyzed the expression profiles of three candidate genes, which play important roles in cancer initiation, promotion and progression. Cell biology experiments indicated that this novel combination treatment possesses significant synergy between 3AB and low-dose cisplatin and is promising for development as an antitumor treatment for lung cancer.

Aquaporin-3 is down-regulated by LMP1 in nasopharyngeal carcinoma cells to regulate cell migration and affect EBV latent infection.

Epstein-Barr virus (EBV) infection has a strong correlation with the development of nasopharyngeal carcinoma (NPC). Aquaporin 3 (AQP3), a member of the aquaporin family, plays an important role in tumor development, especially in epithelial-mesenchymal transition. In this study, the expression of AQP3 in EBV-positive NPC cells was significantly lower than that in EBV-negative NPC cells. Western blot and qRT-PCR analysis showed that LMP1 down-regulated the expression of AQP3 by activating the ERK pathway. Cell biology experiments have confirmed that AQP3 affects the development of tumor by promoting cell migration and proliferation in NPC cells. In addition, AQP3 can promote the lysis of EBV in EBV-positive NPC cells. The inhibition of AQP3 expression by EBV through LMP1 may be one of the mechanisms by which EBV maintains latent infection-induced tumor progression.

M6A modification of VEGFA mRNA by RBM15/YTHDF2/IGF2BP3 contributes to angiogenesis of hepatocellular carcinoma.

Vascular endothelial growth factor A (VEGFA) plays a critical role as a potent angiogenesis factor and is highly expressed in hepatocellular carcinoma (HCC). Although the expression of VEGFA has been strongly linked to the aggressive nature of HCC, the specific posttranscriptional modifications that might contribute to VEGFA expression and HCC angiogenesis are not yet well understood. In this study, we aimed to investigate the epitranscriptome regulation of VEGFA in HCC. A comprehensive analysis integrating MeRIP-seq, RNA-seq, and crosslinking-immunprecipitation-seq data revealed that VEGFA was hypermethylated in HCC and identified the potential m6A regulators of VEGFA including a m6A methyltransferase complex component RBM15 and the two readers, YTHDF2 and IGF2BP3. Through rigorous cell and molecular biology experiments, RBM15 was validated as a key component of methyltransferase complex responsible for m6A methylation of VEGFA, which was subsequently recognized and stabilized by IGF2BP3 and YTHDF2, leading to enhanced VEGFA expression and VEGFA-related functions such as human umbilical vascular endothelial cells (HUVEC) migration and tube formation. In the HCC xenograft model, knockdown of RBM15, IGF2BP3, or YTHDF2 resulted in reduced expression of VEGFA, accompanied by significant inhibition of tumor growth closely associated with VEGFA expression and angiogenesis. Furthermore, our analysis of HCC clinical samples identified positive correlations between the expression levels of VEGFA and the regulators RBM15, IGF2BP3, and YTHDF2. Collectively, these findings offer novel insights into the posttranscriptional modulation of VEGFA and provide potential avenues for alternative approaches to antiangiogenesis therapy targeting VEGFA.

CircHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

CircHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

Photothermal driven BMSCs osteogenesis and M2 macrophage polarization on polydopamine-coated Ti3C2 nanosheets/poly(vinylidene fluoride trifluoroethylene) nanocomposite coatings

Mild thermal stimulation plays an active role in bone tissue repair and regeneration. In this work, a bioactive polydopamine/Ti3C2/poly(vinylidene fluoride trifluoroethylene) (PDA/Ti3C2/P(VDF-TrFE)) nanocomposite coating with excellent near-infrared light (NIR)-triggered photothermal effect was designed to improve the osteogenic ability of implants. By incorporating dopamine (DA)-modified Ti3C2 nanosheets into the P(VDF-TrFE) matrix and combining them with alkali initiated in situ polymerization, the resulting PDA/Ti3C2/P(VDF-TrFE) nanocomposite coating gained high adhesion strength on Ti substrate, excellent tribological and corrosion resistance properties, which was quite important for clinical application of implant coatings. Cell biology experiments showed that NIR-triggered mild thermal stimulation on the coating surface promoted cell spreading and growth of BMSCs, and also greatly upregulated the osteogenic markers, including Runt-Related Transcription Factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN). Simultaneously, the synthesis of heat shock protein 47 (HSP47) was significantly promoted by the mild thermal stimulation, which strengthened the specific interaction between HSP47 and collagen Ⅰ (COL-Ⅰ), thereby activating the integrin-mediated MEK/ERK osteogenic differentiation signaling pathway. In addition, the results also showed that the mild thermal stimulation induced the polarization of macrophages towards M2 phenotype, which can attenuate the inflammatory response of injured bone tissue. Antibacterial results indicated that the coating exhibited an outstanding antibacterial ability against S. aureus and E. coli. Conceivably, the versatile implant bioactive coatings developed in this work will show great application potential for implant osseointegration.

The Low Tumorigenic Risk and Subtypes of Cardiomyocytes Derived from Human-induced Pluripotent Stem Cells

Background: Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a promising approach for the treatment of heart diseases. However, the tumorigenicity of hiPSC-CMs remains a concern for their clinical applications and the composition of the hiPSC-CM subtypes need to be clearly identified. Methods: In the present study, hiPSC-CMs were induced from hiPSCs via modulation of Wnt signaling followed by glucose deprivation purification. The structure, function, subpopulation composition, and tumorigenic risk of hiPSC-CMs were evaluated by single-cell RNA sequencing (scRNAseq), whole exome sequencing (WES), and integrated molecular biology, cell biology, electrophysiology, and/or animal experiments. Results: The high purity of hiPSC-CMs, determined by flow cytometry analysis, was generated. ScRNAseq analysis of differentiation day (D) 25 hiPSC-CMs did not identify the transcripts representative of undifferentiated hiPSCs. WES analysis showed a few newly acquired confidently identified mutations and no mutations in tumor susceptibility genes. Further, no tumor formation was observed after transplanting hiPSC-CMs into NOD-SCID mice for 3 months. Moreover, D25 hiPSC-CMs were composed of subtypes of ventricular-like cells (23.19%) and atrial-like cells (66.45%) in different cell cycle stages or mature levels, based on the scRNAseq analysis. Furthermore, a subpopulation of more mature ventricular cells (3.21%) was identified, which displayed significantly up-regulated signaling pathways related to myocardial contraction and action potentials. Additionally, a subpopulation of cardiomyocytes in an early differentiation stage (3.44%) experiencing nutrient stress-induced injury and heading toward apoptosis was observed. Conclusions: This study confirmed the biological safety of hiPSC-CMs and described the composition and expression profile of cardiac subtypes in hiPSC-CMs which provide standards for quality control and theoretical supports for the translational applications of hiPSC-CMs.

A comprehensive analysis of m6A/m7G/m5C/m1A-related gene expression and immune infiltration in liver ischemia–reperfusion injury by integrating bioinformatics and machine learning algorithms

BackgroundLiver ischemia–reperfusion injury (LIRI) is closely associated with immune infiltration, which commonly occurs after liver surgery, especially liver transplantation. Therefore, it is crucial to identify the genes responsible for LIRI and develop effective therapeutic strategies that target immune response. Methylation modifications in mRNA play various crucial roles in different diseases. This study aimed to identify potential methylation-related markers in patients with LIRI and evaluate the corresponding immune infiltration.MethodsTwo Gene Expression Omnibus datasets containing human liver transplantation data (GSE12720 and GSE151648) were downloaded for integrated analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to investigate the functional enrichment of differentially expressed genes (DEGs). Differentially expressed methylation-related genes (DEMRGs) were identified by overlapping DEG sets and 65 genes related to N6-methyladenosine (m6A), 7-methylguanine (m7G), 5-methylcytosine (m5C), and N1-methyladenosine (m1A). To evaluate the relationship between DEMRGs, a protein–protein interaction (PPI) network was utilized. The core DEMRGs were screened using three machine learning algorithms: least absolute shrinkage and selection operator, random forest, and support vector machine-recursive feature elimination. After verifying the diagnostic efficacy using the receiver operating characteristic curve, we validated the expression of the core DEMRGs in clinical samples and performed relative cell biology experiments. Additionally, the immune status of LIRI was comprehensively assessed using the single sample gene set enrichment analysis algorithm. The upstream microRNA and transcription factors of the core DEMRGs were also predicted.ResultsIn total, 2165 upregulated and 3191 downregulated DEGs were identified, mainly enriched in LIRI-related pathways. The intersection of DEGs and methylation-related genes yielded 28 DEMRGs, showing high interaction in the PPI network. Additionally, the core DEMRGs YTHDC1, METTL3, WTAP, and NUDT3 demonstrated satisfactory diagnostic efficacy and significant differential expression and corresponding function based on cell biology experiments. Furthermore, immune infiltration analyses indicated that several immune cells correlated with all core DEMRGs in the LIRI process to varying extents.ConclusionsWe identified core DEMRGs (YTHDC1, METTL3, WTAP, and NUDT3) associated with immune infiltration in LIRI through bioinformatics and validated them experimentally. This study may provide potential methylation-related gene targets for LIRI immunotherapy.

HIST1H2BK predicts neoadjuvant-chemotherapy response and mediates 5-fluorouracil resistance of gastric cancer cells

BackgroundNeoadjuvant chemotherapy (NACT) is routinely used to treat patients with advanced gastric cancer (AGC). However, the identification of reliable markers to determine which AGC patients would benefit from NACT remains challenging. MethodsA systematic screening of plasma proteins between NACT-sensitive and NACT-resistant AGC patients was performed by a mass spectrometer (n = 6). The effect of the most differential plasma protein was validated in two independent cohorts with AGC patients undergoing NACT (ELISA cohort: n = 155; Validated cohort: n = 203). The expression of this candidate was examined in a cohort of AGC tissues using immunohistochemistry (n = 34). The mechanism of this candidate on 5-Fluorouracil (5-FU) resistance was explored by cell-biology experiments in vitro and vivo. ResultsA series of differential plasma proteins between NACT-sensitive and NACT-resistant AGC patients was identified. Among them, plasma HIST1H2BK was validated as a significant biomarker for predicting NACT response and prognosis. Moreover, HIST1H2BK was over-expression in NACT-resistant tissues compared to NACT-sensitive tissues in AGC. Mechanistically, HIST1H2BK inhibited 5-FU-induced apoptosis by upregulating A2M transcription and then activating LRP/PI3K/Akt pathway, thereby promoting 5-FU resistance in GC cells. Intriguingly, HIST1H2BK-overexpressing 5-FU-resistant GC cells propagated resistance to 5-FU-sensitive GC cells through the secretion of HIST1H2BK. ConclusionThis study highlights significant differences in plasma protein profiles between NACT-resistant and NACT-sensitive AGC patients. Plasma HIST1H2BK emerged as an effective biomarker for achieving more accurate NACT in AGC. The mechanism of intracellular and secreted HIST1H2BK on 5-FU resistance provided a novel insight into chemoresistance in AGC.

Novel anti-Acanthamoeba effects elicited by a repurposed poly (ADP-ribose) polymerase inhibitor AZ9482.

Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood. In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses. Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment. Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.