Repair Mechanisms Research Articles

RNA-Sequencing Identification of Genes Supporting HepG2 as a Model Cell Line for Hepatocellular Carcinoma or Hepatocytes

Background/Objectives: Cell lines do not faithfully replicate the authentic transcriptomic condition of the disease under study. The HepG2 cell line is widely used for studying hepatocellular carcinoma (HCC), but not all biological processes and genes exhibit congruent expression patterns between cell lines and the actual disease. The objective of this study is to perform a comparative transcriptomic analysis of the HepG2 cell line, HCC, and primary hepatocytes (PH) in order to identify genes suitable for research in HepG2 as a model for PH or HCC research. Methods: We conducted a differential expression analysis between publicly available data from HCC patients, PH, and HepG2. We examined specific overlaps of differentially expressed genes (DEGs) in a pairwise manner between groups in order to obtain a valuable gene list for studying HCC or PH using different parameter filtering. We looked into the function and druggability of these genes. Conclusions: In total, we identified 397 genes for HepG2 as a valuable HCC model and 421 genes for HepG2 as a valuable PH model, and with more stringent criteria, we derived a smaller list of 40 and 21 genes, respectively. The majority of genes identified as a valuable set for the HCC model are involved in DNA repair and protein degradation mechanisms. This research aims to provide detailed guidance on gene selection for studying diseases like hepatocellular carcinoma, primary hepatocytes, or others using cell lines.

Intergenerational crosstalk of brain-gut axis in parental Nd2O3 exposure-induced offspring neurotoxicity and cognitive dysfunction: a mechanistic study

IntroductionRare earth elements (REEs) are widely used in plenty of fields. REEs have significant neurotoxicity and it may adversely affect the development of cognitive. For example, neodymium will causing neurological damage through penetrate the blood–brain barrier (BBB). However, whether it disrupts the balance of brain-gut axis (BGA) crosstalk and affects the intestinal microecology disorder of host is still unclear. This study investigated the neural damage on children caused by maternal exposure to Neodymium oxide (Nd2O3) during pregnancy, and its involved mechanism of BGA injury.MethodsWe used rat model to investigated the mechanisms of the offspring’s neural damage that Nd2O3 exposure in pregnancy. To verify the neural damage of offspring rats, we examed BBB-related factors, such like glutamate and ROS levels in brain tissue, behavioral tests, hippocampal and cortical damage, as well as changes in gut microbiota, intestinal mucosal barrier, and SCFAs in the intestine. Also, we observed some specific indicators of intestinal immune barrier function and gut nerve-related indicators.ResultsMaternal Nd2O3 exposure reduced the content of offspring tight junction proteins, increased BBB permeability, leading to Nd accumulation and brain tissue inflammation, affecting offspring’s neural development and weakening their spatial learning ability. Nd2O3 also disrupted BBB integrity by regulating SCFAs and BGA. Probiotic intervention in the offspring rats exposed to 2% Nd2O3 showed significant recovery of inflammation in both brain and colon tissues, and reduced BBB permeability.ConclusionMaternal exposure to Nd2O3 affects the offspring’s BGA, targeting brain and colon tissues, increasing BBB permeability, affecting neural development, causing damage to the intestinal mucosa, and impacting children’s gut development. Probiotics can alleviate these effects. These findings provide valuable insights into understanding the neurodevelopmental and intestinal developmental toxicity of Nd2O3 and its prevention and treatment. It also calls for a comprehensive assessment of the health risks of susceptible populations to Nd2O3, such as pregnant women. It may providing theoretical basis for preventing and controlling neodymium-induced harm in children by examing the repair mechanism of the damage through probiotic intervention.

Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair

Abstract The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems.

DNAR-15. GROUP 3 MEDULLOBLASTOMAS EXPLOIT A TRANSIENT MECHANISM OF TELOMERE REPAIR MEDIATED BY ZSCAN4 WHICH IS TARGETABLE FOR THERAPEUTIC BENEFIT WITH BRAIN-PENETRANT DRUGS

Abstract All cancers need some mechanism of telomere repair. However, it is unknown how Group 3 (G3) medulloblastomas (MB) repair their telomeres, even though MB is the most common malignant brain tumor in kids and G3 is its most aggressive subtype. With rare exceptions, G3 MBs lack telomerase gain-of-function alterations or evidence of constitutive alternative-lengthening-of-telomeres pathway activation. We profiled tumors from N=38 MB patients from UCSF via single-nucleus RNA sequencing; 13 cases were also subjected to single-cell assay for transposase-accessible chromatin by sequencing, and 4 were subjected to single-cell cleavage under targets and tagmentation for H3K27ac. These data identified a network of gene enhancers that were specifically active in rhombic-lip progenitor-like MB cells, the putative G3 MB cell of origin. This program was driven by zinc finger and SCAN domain containing 4 (ZSCAN4), a gene reported to drive transient telomere repair during zygotic genome activation in early embryos. CRISPR inhibition of ZSCAN4 led to significant telomere shortening, telomeric DNA damage, telomere fusions, decreased proliferation, and increased survival of orthotopic xenografts. Conversely, ZSCAN4 over-expression induced significant telomere elongation, increased proliferation, and decreased survival in vivo. Analysis of endogenous-reporter cells indicated that ZSCAN4 is transiently expressed in response to telomere shortening and increases global histone acetylation by inducing chromatin-remodeling factors. Rebastinib targeted ZSCAN4, histone acetylation, and telomere repair, was brain penetrant, and significantly enhanced orthotopic-xenograft survival with no toxicity. Acetyltransferase inhibitor CPI-1612 was brain penetrant and extended xenograft survival with no toxicity. Our data support the hypothesis that G3 MBs exploit a transient mechanism of telomere repair mediated by ZSCAN4. Targeting ZSCAN4 or downstream histone acetylation is therapeutically viable. Pan-cancer whole-genome sequencing data showed that MBs have lower telomeric content than most other cancers. Thus, MBs may be particularly vulnerable to therapeutic strategies that inhibit telomere repair.

EXTH-65. EXPLORING ANTIHISTAMINES TO TREAT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and lethal primary malignant brain tumor with a 5-year survival of 6.8%. Interestingly, people with allergies, atopy, or asthma are less likely to develop GBM. A recent study recognized that high histamine receptor 1 (HRH1) expression is inversely related to overall and progression-free survival. METHODS We used a bioinformatics tool, iLINCS, which, after analyzing 99 GBM and 38 healthy samples, identified 36 drugs that can reverse GBM signatures. Based on the concordance score and blood-brain barrier permeability, we selected brompheniramine (Brom), a first-generation HRH1 antagonist, as a potential candidate. HRH1 expression analysis was done on human and mouse GBM cell lines by immunoblotting. The effect of Brom alone or in combination with temozolomide (TMZ) was investigated on proliferation, DNA damage and apoptosis in human (U118 and U251) and three syngeneic (EGFRvIII, p16-/- & GFAP Cre, PTEN+/-, p53R172H+/-, EGFRvIII & GFAP Cre, PTEN-/-, p53R172H-/- & GFAP Cre) cell lines. GBM orthograft mouse models were used to test the efficacy of combination therapy. RESULTS HRH1 is highly expressed in human and syngeneic GBM cells. This expression is further increased in TMZ-resistant cells. Interestingly, the addition of histamine, induced TMZ resistance in GBM cells. TMZ with Brom decreases proliferation in mouse and human GBM cells, and Brom is non-toxic to non-transformed cells. Combination treatment increases DNA damage and decreases the DNA repair mechanism, possibly by downregulating the expression of O6methylguanine-DNA methyltransferase (MGMT). In the GBM orthograft mouse model, the combination therapy reduced tumor volume and significantly increased survival compared to single-agent treatment. CONCLUSIONS Brom is non-toxic; Brom, in combination with TMZ, enhanced the anti-tumor efficacy of TMZ both in in vitro and in vivo GBM models. Further studies in spontaneous mouse models are planned.

DDDR-30. ENHANCED CHEMO AND RADIO-SENSITIZATION OF GLIOMAS BY HSP90 INHIBITION WITH JIN-001, A NOVEL BLOOD-BRAIN BARRIER PENETRANT HSP90 INHIBITOR

Abstract BACKGROUND HSP90, an abundant molecular chaperone, plays a pivotal role as a key regulator in the growth and survival of malignant cells and has been a therapeutic target in various cancers. We have previously reported a strong role for HSP90 inhibitors to disable DNA damage repair mechanisms and induce cell death. Here, we determined the effects of JIN-001 mediated HSP90 inhibition on alkylator and radiation induced DNA damage. Method: Using a panel of patient-derived glioma stem cells (GSCs) we determined the synergy scores for cell viability of JIN-001 (0.1µM to 0.5µM) in combination with radiation (RT) and Temozolomide (TMZ). We also examined cell cycle dynamics and cell death of the combination using flow cytometry (PI and PI/Annexin method) and cell viability assays. Additionally, organotypic human glioma slice cultures treated with JIN-001 and chemoradiation were analyzed for changes in gene and protein expression as well as cell viability in situ. Ongoing in-vivo studies are being conducted to further elucidate drug combination efficacy in mouse models. RESULTS JIN-001 sensitized the cells to chemoradiation leading to cell cycle arrest and cell death which was higher in the combination compared to single agent. Significant changes in expression of downstream molecules of homologous recombination pathway was noted (ATM, pATM, CHK1, pCHK1, ATR, pATR) in the cell lines and confirmed in human glioma slice culture. Based on the synergy score from four cell lines, an effective dose for combination of JIN-001+RT+TMZ was identified; Results of ongoing ex-vivo human glioma gene expression changes and in-vivo mouse studies will be presented. CONCLUSION Through a comprehensive analysis, we demonstrated that HSP90 inhibitor JIN-001 sensitizes glioma cells to radio-chemotherapy including in human glioma tissue. Our study underscores the potential of combining JIN-001 with standard therapies in glioma treatment in future clinical trials for patients with glioblastoma.

DNAR-12. STEAROYL-COA DESATURASE REGULATES DNA DAMAGE REPAIR IN GBM BY MODULATING PARP1 ACTIVITY

Abstract Glioblastoma (GBM) is the most prevalent malignant brain cancer in adults, notorious for its aggressiveness and resistance to therapy. GBM patients commonly receive radiation therapy combined with temozolomide (TMZ), which engages tumor cells to promote lethal DNA damage and subsequent cytotoxicity. Active DNA repair mechanisms, particularly in a subset population of GBM stem-like cells (GSCs), drive therapeutic resistance, thus largely contributing to the bleak prognosis of GBM. Poly (ADP-ribose) polymerases (PARPs) are essential in various cellular processes, including DNA repair. We have previously reported that fatty acid desaturation mediated by Stearoyl-CoA desaturase 1 (SCD1) plays a cytoprotective role in GSCs. Inhibiting SCD prevents tumor initiation and inhibits DNA damage repair, hence sensitizing to radiation and TMZ. Yet, the mechanistic role of SCD in DNA damage regulation remained unknown. Here, we found that the expression of both human isoforms of SCD, SCD1, and SCD5, directly correlates with PARP protein levels and subcellular localization. Downregulating SCD1/5 in GSCs resulted in significantly lower levels of PARP1, coupled with increased DNA damage, as well as reduced DNA damage repair and chromosome assembly. Conversely, cells overexpressing SCD1/5 exhibited higher PARP1 expression and lower levels of basal DNA damage compared to control GSCs. Notably, SCD1/5 overexpression also led to increased resistance to TMZ. We show that SCD1/5 regulates PARP activity by controlling PARP1 subcellular localization. In addition to decreased total levels of PARP following SCD1/5 downregulation, we also found increased PARP1 self-PARylation. Using confocal microscopy, we show that when SCD1/5 is repressed, PARylated PARP translocates from the nucleus to the cytosol, rendering it unable to bind DNA and recruit DNA-repairing machinery. In conclusion, our study provides a direct link between fatty acid metabolism and PARP1-mediated DNA damage repair.

Molecular Insights into Radiation Effects and Protective Mechanisms: A Focus on Cellular Damage and Radioprotectors

Ionizing radiation has been a critical tool in various fields, such as medicine, agriculture, and energy production, since its discovery in 1895. While its applications—particularly in cancer treatment and diagnostics—offer significant benefits, ionizing radiation also poses risks due to its potential to cause molecular and cellular damage. This damage can occur through the direct ionization of biological macromolecules, such as deoxyribonucleic acid (DNA), or indirectly through the radiolysis of water, which generates reactive oxygen species (ROS) that further damage cellular components. Radioprotectors, compounds that protect against radiation-induced damage, have been extensively researched since World War II. These agents work by enhancing DNA repair, scavenging free radicals, and boosting antioxidant defenses, thereby protecting healthy tissues. Furthermore, some radioprotective agents also stimulate DNA repair mechanisms even after radiation exposure, aiding in recovery from radiation-induced damage. This article explores the molecular mechanisms of radiation-induced damage, focusing on both direct and indirect effects on DNA, and discusses the role of radioprotectors, their mechanisms of action, and recent advancements in the field. The findings underscore the importance of developing effective radioprotective strategies, particularly in medical and industrial settings, where radiation exposure is prevalent.

Bioactive Peptide Hydrogel Scaffold with High Fluidity, Thermosensitivity, and Neurotropism in 3D Spatial Structure for Promoted Repair of Spinal Cord Injury.

Spinal cord injury (SCI) has been considered a clinically challenging disease that is characterized by local disturbance of the microenvironment, which inhibits post-injury neural regeneration. The simulation of a microenvironment conducive to the regeneration of spinal cord is beneficial for SCI repair. In this study, bioactive composite hydrogels are developed that mimic the regenerative microenvironment of spinal cord for enhanced SCI repair. The fabricated composite hydrogels (CRP) based on chitosan (CS), RADA16 nanofibers, and nerve-promoted peptide (PPFLMLLKGSTR) exhibit excellent injectability, superior biodegradability and biocompatibility. In addition, the CRP hydrogels can formquickly (a few minutes) by mixing three components at human body temperature, showing high potential as a biomimetic matrix for in situ repair of SCI. The in vitro studies demonstrate that the CRP hydrogels can not only promote the proliferation and migration of bone marrow mesenchymal stem cells but also induce the proliferation and differentiation of neural stem cells (NSCs) into neurons. Meanwhile, the hydrogels reveal the efficiency of protecting neurons and promoting axonal growth. Furthermore, the in vivo tests prove that the CRP hydrogels can reduce post-SCI inflammatory responses, inhibit reactive astrocyte over-proliferation, and promote the migration, proliferation, and differentiation of endogenous NSCs, which agree well with the in vitro results. The pre-clinical test demonstrates that the CRP hydrogels restore the motor function in completely transected spinal cord rats, and the SCI repair mechanism may involve the activation of the PI3K/AKT/mTOR pathway. It is believed that the strategies shown in this work will be valuable for the design and synthesis of novel hydrogels for biomedical and tissue engineering applications.

Modelling the Repair of Carbon-Centered Protein Radicals by Phenolic Antioxidants

Oxidative stress is a biological process that has been linked to many diseases, hence understanding how to prevent and repair it is essential to medicine. The thermodynamics and kinetics of the repair reactions of radically damaged leucine (a lateral chain in a simplified protein environment) by twenty phenolic antioxidants are studied at the M06-2X(SMD)/6-31++G(d,p) level of theory in water and pentyl ethanoate. The two repair mechanisms modelled are formal-hydrogen atom transfer (f-HAT) and single electron transfer (SET). Although all f-HAT reactions are thermodynamically favourable, only one of the phenols produced rate constants in the diffusion limit, exhibiting biological relevance. SET is not suspected to be an important repair pathway for the phenols studied. We show that the Bell–Evans–Polanyi principle, which relates thermodynamics and kinetics properties for a reaction, breaks down when comparing between the solvents, protein repair sites, and the phenolic antioxidants. While thermodynamic data can be used as valuable screening tools, the kinetic calculation of rate constants in solution is crucial for enhancing the biological relevance of theoretical studies.

TET2 germline variants promote kidney disease by impairing DNA repair and activating cytosolic nucleotide sensors

Genome-wide association studies (GWAS) have identified over 800 loci associated with kidney function, yet the specific genes, variants, and pathways involved remain elusive. By integrating kidney function GWAS with human kidney expression and methylation quantitative trait analyses, we identified Ten-Eleven Translocation (TET) DNA demethylase 2 (TET2) as a novel kidney disease risk gene. Utilizing single-cell chromatin accessibility and CRISPR-based genome editing, we highlight GWAS variants that influence TET2 expression in kidney proximal tubule cells. Experiments using kidney/tubule-specific Tet2 knockout mice indicated its protective role in cisplatin-induced acute kidney injury, as well as in chronic kidney disease and fibrosis induced by unilateral ureteral obstruction or adenine diet. Single-cell gene profiling of kidneys from Tet2 knockout mice and TET2-knockdown tubule cells revealed the altered expression of DNA damage repair and chromosome segregation genes, notably including INO80, another kidney function GWAS target gene itself. Remarkably, both TET2-null and INO80-null cells exhibited an increased accumulation of micronuclei after injury, leading to the activation of cytosolic nucleotide sensor cGAS-STING. Genetic deletion of cGAS or STING in kidney tubules, or pharmacological inhibition of STING, protected TET2-null mice from disease development. In conclusion, our findings highlight TET2 and INO80 as key genes in the pathogenesis of kidney diseases, indicating the importance of DNA damage repair mechanisms.

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Mechanisms of Alternative Lengthening of Telomeres.

In recent years, significant advances have been made in understanding the intricate details of the mechanisms underlying alternative lengthening of telomeres (ALT). Studies of a specialized DNA strand break repair mechanism, known as break-induced replication, and the advent of telomere-specific DNA damaging strategies and proteomic methodologies to profile the ribonucleoprotein composition of telomeres enabled the discovery of networks of proteins that coordinate the stepwise homology-directed DNA repair and DNA synthesis processes of ALT. These networks couple mediators of homologous recombination, DNA template-switching, long-range template-directed DNA synthesis, and DNA strand resolution with SUMO-dependent liquid condensate formation to create discrete nuclear bodies where telomere extension occurs. This review will discuss the recent findings of how these networks may cooperate to mediate telomere extension by the ALT mechanism and their impact on telomere function and integrity in ALT cancer cells.

Regulation of ferroptosis in osteoarthritis and osteoarthritic chondrocytes by typical MicroRNAs in chondrocytes

Osteoarthritis (OA) is a progressive degenerative disorder impacting bones and joints, worsened by chronic inflammation, immune dysregulation, mechanical stress, metabolic disturbances, and various other contributing factors. The complex interplay of cartilage damage, loss, and impaired repair mechanisms remains a critical and formidable aspect of OA pathogenesis. At the genetic level, multiple genes have been implicated in the modulation of chondrocyte metabolism, displaying both promotive and inhibitory roles. Recent research has increasingly focused on the influence of non-coding RNAs in the regulation of distinct cell types within bone tissue in OA. In particular, an expanding body of evidence highlights the regulatory roles of microRNAs in OA chondrocytes. This review aims to consolidate the most relevant microRNAs associated with OA chondrocytes, as identified in recent studies, and to elucidate their involvement in chondrocyte metabolic processes and ferroptosis. Furthermore, this study explores the complex regulatory interactions between long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in OA, with an emphasis on microRNA-mediated mechanisms. Finally, critical gaps in the current research are identified, offering strategic insights to advance the understanding of OA pathophysiology and guide therapeutic developments in this field.

Protective Effect of Biochanin A on Gamma Radiation-Induced Oxidative Stress, Antioxidant Status, Apoptotic, and DNA Repairing Molecules in Swiss Albino Mice.

Radiation therapy is indispensable in medical practice but often causes adverse effects on healthy tissues, necessitating the search for natural radioprotectors. This study investigates the protective effect of Biochanin A (BCA) against gamma radiation-induced oxidative stress and DNA damage in Swiss albino mice. Gamma radiation, a potent ionizing source, generates reactive oxygen species (ROS) that damage cellular biomolecules, including DNA. Antioxidants play a crucial role in neutralizing ROS and preventing oxidative damage. Swiss albino mice were divided into control, BCA control (10 mg/kg body weight), radiation alone (7 Gy), and radiation+ BCA pretreatment groups. BCA, a natural isoflavone with known antioxidant and cytoprotective properties, was administered intraperitoneally before radiation exposure. After irradiation, lipid peroxidation levels, antioxidant enzyme activities/level (superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), expression levels of DNA repair genes (P53, P21, GADD45α), apoptotic markers (Bax, Bcl-2, Caspase-3, -9 and Cytochrome-C), and inflammatory marker (NF-κB) were analyzed in small intestine tissue. Our findings indicate that gamma radiation significantly elevated lipid peroxidation levels and altered antioxidant enzyme activities, indicating oxidative stress. However, BCA pretreatment mitigated these effects by bolstering antioxidant defences, reducing radiation-induced oxidative damage. Additionally, BCA altered apoptotic markers, NF-κB expression, promoting cell survival mechanisms. At the molecular level, BCA pretreatment upregulated key DNA repair genes (P53, P21, GADD45α), crucial for repairing radiation-induced DNA damage and maintaining genomic stability. These results underscore BCA potential as a radioprotector, suggesting its efficacy in mitigating radiation-induced oxidative stress and preserving cellular integrity. In conclusion, BCA demonstrates promising radioprotective properties by attenuating oxidative stress, enhancing antioxidant defences, modulating apoptotic pathways, and promoting DNA repair mechanisms following gamma radiation exposure. Further research is necessary to elucidate its precise mechanisms of action and explore its potential therapeutic applications in radiation oncology and environmental radioprotection.

Nuclear poly-glutamine aggregates rupture the nuclear envelope and hinder its repair.

Huntington's disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington's disease and other polyglutamine diseases.

Exosomes derived from platelet-rich plasma present a novel potential in repairing knee articular cartilage defect combined with cyclic peptide-modified β-TCP scaffold

BackgroundThe aim of this study was to investigate the therapeutic effects and mechanisms of PRP-exos combined with cyclic peptide-modified β-TCP scaffold in the treatment of rabbit knee cartilage defect.MethodsPRP-exos were extracted and characterized by TEM, NTA and WB. The therapeutic effects were evaluated by ICRS score, HE staining, Immunohistochemistry, qRT-PCR and ELISA. The repair mechanism of PRP-exos was estimated and predicted by miRNA sequencing analysis and protein–protein interaction network analysis.ResultsThe results showed that PRP-exos had a reasonable size distribution and exhibited typical exosome morphology. The combination of PRP-exos and cyclic peptide-modified β-TCP scaffold improved ICRS score and the expression level of COL-2, RUNX2, and SOX9. Moreover, this combination therapy reduced the level of MMP-3, TNF-α, IL-1β, and IL-6, while increasing the level of TIMP-1. In PRP-exos miRNA sequencing analysis, the total number of known miRNAs aligned across all samples was 252, and a total of 91 differentially expressed miRNAs were detected. The results of KEGG enrichment analysis and the protein–protein interaction network analysis indicated that the PI3K/AKT signaling pathway could impact the function of chondrocytes by regulating key transcription factors to repair cartilage defect.ConclusionPRP-exos combined with cyclic peptide-modified β-TCP scaffold effectively promoted cartilage repair and improved chondrocyte function in rabbit knee cartilage defect. Based on the analysis and prediction of PRP-exos miRNAs sequencing, PI3K/AKT signaling pathway may contribute to the therapeutic effect. These findings provide experimental evidence for the application of PRP-exos in the treatment of cartilage defect.

Screening and design of PARP12 inhibitors from traditional Chinese medicine small molecules using computational modeling and simulation

The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a pivotal role in orchestrating a multitude of cellular processes, including DNA repair mechanisms, transcriptional regulation, and modulation of immune responses. Within this family, PARP12 emerges as a noteworthy candidate for targeted cancer therapeutics. Consequently, this investigation endeavors to screen and design potential PARP12 inhibitors derived from traditional Chinese medicinal compounds by employing sophisticated molecular modeling and computational medicinal chemistry approaches. The compound RBN2397 is utilized as a benchmark, and the binding efficacies of the newly identified small molecules are assessed against a spectrum of criteria, encompassing molecular interactions, binding free energy, and extensive post-simulation analyses. The outcomes demonstrated that the identified small molecules, specifically tcm8650 and its derivative XC-1, possess remarkable binding affinities and exhibit reduced binding free energies compared to RBN2397. The molecular docking and interaction profiles of these compounds were also comprehensively scrutinized. Moreover, ADMET profiling meticulously evaluated the pharmacokinetic profiles and physicochemical characteristics of these promising molecules and their projected human physiological impact. These computational studies indicated their potential therapeutic applicability and predicted acceptable safety profile, advocating their further exploration as viable candidates in cancer treatment.

Mid-late gestation leptin infusion induces placental mitochondrial and endoplasmic reticulum unfolded protein responses in a mouse model of preeclampsia

IntroductionPreeclamptic patients, both lean and obese, present with elevated leptin levels which are associated with the development of maternal endothelial dysfunction and adverse fetal outcomes, such as growth restriction, leading to low birth weight. Recent studies in pregnant mice demonstrate that mid-late gestation leptin infusion induces clinical characteristics of preeclampsia, including elevated maternal blood pressure, maternal endothelial dysfunction and fetal growth restriction. However, whether leptin triggers placental stress responses that contribute to adverse fetal outcomes as in preeclampsia is unknown. MethodsIn the current study we measured the expression of proteins involved in the endoplasmic reticulum (UPRer) and mitochondrial (UPRmt) unfolded protein responses in placentas of wild-type sham normal pregnant and leptin-infused preeclamptic mice. ResultsThe data show that mid-late gestation leptin infusion induced activation of indices of placental UPRer and UPRmt, while reducing placental repair mechanisms to UPRmt in preeclamptic mice. Mid-late gestation infusion with leptin upregulated markers of placental oxidative stress, reduced the placental expression levels of mitochondrial electron transport chain complexes I and II and increased the expression of placental endothelin-1 (ET-1) in preeclamptic mice. The leptin-induced activation of several placental UPRmt markers as well as ET-1 levels correlated with fetal growth restriction and impaired maternal endothelial function in preeclamptic mice. DiscussionCollectively, these data indicate that elevated levels of leptin in mid-late pregnancy in mice promote placental stress responses, akin to those in pregnant women with preeclampsia.

Severe ischemia and reperfusion injury induces epigenetic inactivation of LHX1 in kidney progenitor cells after kidney transplantation

Severe ischemia-reperfusion injury (IRI) causes acute and chronic kidney allograft damage. As therapeutic interventions to reduce damage are limited yet, research on how to promote kidney repair has gained significant interest. To address this question, we performed genome-wide transcriptome and epigenome profiling in progenitor cells isolated from urine of deceased (severe IRI) and living (mild IRI) donor human kidney transplants and identified LIM homebox-1 (LHX1) as an epigenetically regulated gene whose expression depends on the IRI severity. Using a mice model of IRI, we observed a relationship between IRI severity, LHX1 promoter hypermethylation and LHX1 gene expression. By functional studies we confirmed that LHX1 expression is involved in proliferation of epithelial tubular cells and podocyte differentiation from kidney progenitor cells. Our results provide evidence that severe IRI may reduce intrinsic mechanisms of kidney repair through epigenetic signaling.