Proliferation Gene Research Articles

MAF Translocations Are Enriched in NDMM Patients with Elevated Levels of Circulating Tumor Cells Suggesting a Genetic Basis for Aggressive Disease Course

Introduction A subset of Multiple Myeloma (MM) patients responds poorly to treatment, highlighting the need to understand the pathobiology of high-risk MM. The correlation between aggressive disease and elevated levels of circulating tumor cells (CTCs) has been recognized. We recently identified a subset of high-risk newly diagnosed MM (NDMM) patients based on a transcriptomic classifier derived from patients with high levels of CTC, indicating that CTC levels are a relevant reflection of tumor cell biology. Here, we hypothesized that comparing bone marrow plasma cells (BM PCs) of MM patients with high and low CTC levels will provide an opportunity to identify mechanistic drivers of aggressive disease. Results We first set out to exclude the presence of a circulating aggressive subclone of MM cells in patients with high levels of CTCs. Paired single cell transcriptomes from BM and peripheral blood MM cells of 7 NDMM patients were generated. Analysis of 55,230 CTCs and BM PCs revealed patient specific clustering, but with large transcriptional overlap between individual paired CTCs and BM PCs. Since only 11 genes were significantly upregulated in the BM PCs (P < 0.05) compared to paired CTCs, we excluded the presence of circulating aggressive subclones in patients with high CTC levels. Next, we aimed to identify differences in MM cell pathobiology between patients with high and low levels of CTCs, we generated single cell transcriptomes from 89,092 BM MM cells from 8 NDMM patients with high (≥0.5%) and 14 patients with low (0.004-0.006%) percentages of CTCs. Single cell data integration across all patients led to the identification of 8 distinct PC clusters. Gene set enrichment analysis between both patient groups revealed upregulation of cell cycle pathways in MM cells of patients with high CTC levels. Based on cell cycle analyses an increase in cycling PCs (53% vs 32%, p=0.007) was associated with high CTC levels (Figure 1). Ki67 labeling of BM biopsies validated this finding. Interestingly, proliferation was increased across all transcriptomically-defined plasma cell clusters, suggesting a common cell-intrinsic driver of increased proliferation. To analyze whether the prevalence of genetic lesions previously associated with proliferation was increased in patients with high CTC levels, WGS was performed on the same 22 patients. This revealed an elevated mutation burden and a predilection for MAF translocations, as 50% (4/8) of the patients with high CTC levels carried MAF translocations versus none in the patients with low CTC levels (0/14). We also observed increased transcription of cyclin D2 (CCND2), CCR1 and IntegrinB7 (ITGB7) in patients with high CTC levels. These proliferation or tumor survival genes have been shown to be increased in patients with t(14;16), a translocation also resulting in overexpression of MAF. To validate these findings in a larger patient cohort, clinical, genomic and CTC data were retrieved from the MMRF-CoMMpass database (n=725). Strikingly, 21% of patients with CTC levels above 0.3% showed MAF translocations vs only 3% in patients with CTC levels below 0.3% (p<0.001), solidifying the correlation between MAF translocations and high levels of CTCs in NDMM patients with a high-risk disease profile. Conclusion Single cell RNA sequencing and WGS of MM cells from patients with high and low CTC levels identified a correlation between CTC levels and overrepresentation of MAF translocations in NDMM patients. This finding suggests that MAF target genes and downstream proliferation could be drivers of aggressive disease and might be used for future patient stratification. Figure 1. Based on cell cycle analyses in single cell transcriptomics an increase in cycling PCs was associated with high CTC levels. a) UMAP of cell cycle analysis of 89,902 BM MM cells from 22 patients with high (n=8) and low (n=14) CTC levels. Colors represent cell cycle phase. b) Bar graph representing percentage of cycling cells. Data points are individual patients (high CTC levels, n=8; low CTC levels, n=14). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting N-Myristoylation for Adult Acute Myeloid Leukemia Therapy

Protein N-myristoylation is the covalent attachment of myristic acid (C14:0) by an amide linkage to the NH2-terminal glycine residue of numerous eukaryotic and viral proteins, thereafter regulating their membrane targeting or enzymatic activity. A single myristoyl-CoA: protein N-myristoyltransferase (NMT) isoform catalyses the acylation reaction in yeast, insects, plants, and unicellular parasites. It is two separate isoforms however; NMT1 and NMT2, with predominantly overlapping substrates that perform this task in mammals. Myristoylated proteins that fail to undergo the modification are recognised by a dedicated glycine N-degron system and rapidly degraded. We recently identified a remarkable reduction of NMT2 expression in a wide variety of cancers, especially hematological cancers. As a consequence, we analysed if NMT1/NMT2 could be associated with AML progression. Data analysis from the TCGA transcriptome database showed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. One oncogenic myristoylated target identified initially in B-cell lymphoma is the family of Src kinases (SFKs). Here, we demonstrate that PCLX-001 significantly reduces total and phosphorylated SFKs, which are essential for pro-survival signalling downstream frequently mutated c-Kit and FLT3 receptors in multiple AML cell lines. Moreover, inhibiting myristoylation led to ER stress induction and apoptosis in cell lines and patient blast samples at concentrations sparing most peripheral blood lymphocytes and monocytes from healthy individuals. We further validated the therapeutic potential of our lead myristoylation inhibitor ongoing clinical trial evaluation against lymphoma and solid tumours (PCLX-001) in a variety of AML xenograft models. PCLX-001 monotherapy had a dose-dependent tumoricidal effect in an ectopic sub-cutaneous MV-4-11 xenograft model, in two tail vein injected patient derived xenografts (PDXs) as well as in 3 out of 5 intra-femoral PDXs. Of note, five days of PCLX-001 treatment at the highest concentration was sufficient to obtain complete tumour eradication in our CDX model. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow with minimal toxicity or long-term impact on body weight (Fig.1). Further analyses are required to characterize the cytogenetics, common mutations or NMTs expression profile that could explain the sensitivity of certain patient models compared to the others. An ongoing challenge in treatment of leukemia resides in leukemic stem cells (LSCs), a low-frequency sub-population functioning as progenitor cells. Without eradication of LSCs no patient can be cured. Relapse rates are high in AML and are often featuring additional mutations and drug resistance. Here, we demonstrate that LSC subpopulations (CD34+/CD38- and CD117+/ CD15-) in the OCI-AML-22 patient derived model are exquisitely sensitive to PCLX-001 treatment in vitro and began shrinking after 48 hours of treatment. In addition, the LSC subpopulation showed lower IC50 compared to the general cell population at 72 hours. Interestingly, we identified that functionally validated LSC+ patient subpopulations were expressing lower amount of NMT2 compared to LSC-. This observation could partially explain the sensitivity of the LSC subpopulation. In aggregate, our preclinical observations suggest that PCLX-001 is highly potent against AML cells in vitro and in vivo (both circulating and in the bone marrow), and, that LSCs are also more sensitive to PCLX-001 than non-leukemic stem cells. This further supports the initiation of PCLX-001evaluation in AML clinical trials scheduled to start in late 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting CNS Lymphoma with Intravenous Axicabtagene Ciloleucel: Evidence for Transcriptional Evolution Towards a Prominent Interferon Signature in CAR-T Cells Trafficking to the Tumor Site

Background: Chemorefractory primary and secondary central nervous system lymphomas (CNSL) have a dismal prognosis, and represent a major unmet need in oncology. To address this, we conducted a pilot trial of axi-cel in patients with relapsed/refractory CNSL. All patients had Ommaya reservoirs, such that after CAR-T infusion, paired CSF and blood could be collected and analyzed daily, allowing the interrogation of single-cell CAR-T transcriptional profiles at an unprecedented level of detail. This analysis enabled us to address key mechanistic questions relevant to CAR-T therapy for CNS lymphoma, including determining the impact of tumor site (brain vs lymphoid tissue) on CAR-T efficacy and toxicity, and discovering the drivers of CAR-T cell transcriptional evolution as these cells move between their sites of action. Methods: Adult patients with either primary or secondary relapsed/refractory CNSL were enrolled on the 'Axi-cel In CNS Lymphoma' Trial (Clinicaltrials.gov# NCT04608487). Patients received fludarabine/cyclophosphamide lymphodepletion followed by axi-cel infusion. Blood and CSF were sampled daily between D0-14 after infusion, with data from CAR-T maximum expansion (Days 5-9) reported here. 5' 10x Single-cell RNA-Seq (scRNA-Seq) and TCR-Seq were performed on peripheral blood mononuclear cells (PBMC), enriched T cells, and CSF-derived cells. scRNA-Seq data was analyzed with the Python packages 'scanpy' and 'scVI' to identify and characterize high-quality CD4+ and CD8+ T cells, and to conduct differential expression tests. Gene set enrichment analysis (GSEA) was used to compare CSF and blood CD4+ and CD8+ CAR-T cells using the 'clusterProfiler' package in R. Results: Detailed clinical results from this trial are reported in an accompanying abstract. In brief, data from the first 7 patients on-study demonstrated no treatment-limiting toxicities and an ORR of 86%, with 6 of 6 responding patients achieving a CR by 3 months. 2 of the responding patients have progressed, 1 at 6 months and 1 at 15 months. To interrogate the mechanisms driving axi-cel efficacy in inducing CNS responses, we analyzed 129,088 CAR+ and non-CAR T cells from the peripheral blood (64,337 cells), the CSF (37,070 cells), and the axi-cel product (27,681 cells) from 5 enrolled patients, with blood/CSF collected around the time of maximum CAR-T expansion (Days 5-9 post-infusion). Enumeration of CAR-T cells in the blood vs CSF revealed a mean 13% CAR-Ts of total blood T cells (range 7-24%), and a mean 18% (range 13-28%) in the CSF. CD4+ CAR-T cells predominated in the infused product (mean 79% CAR-T cells were CD4+, range 68-95%), with the CD4:CD8 balance maintained at peak expansion (with 77% (range 60-83%) CAR-T cells in the blood being CD4+, and 95% (range 91-97%) CAR-T cells in the CSF being CD4+). Sc-TCR sequencing enabled an evaluation of the T cell repertoire, revealing a similarly broad CAR-T repertoire in both the blood (mean 1,375 clones (range 313-2,563) and CSF (mean 1,090 clones, range 268-1,965). scRNA-Seq gene expression and clustering analysis revealed a striking distinction between blood and CSF CAR-Ts: While blood CAR-Ts exhibited a prominent proliferation gene expression signature (analyzed by single cell GSEA), CSF CAR-Ts, obtained on the same days as those from the blood, exhibited strong enrichment for interferon-pathway associated genes (Panel 1). Although the majority of CAR-T clones identified were unique to either the blood or CSF, we were able to identify 54 shared clones between these compartments. Analysis of these clones further substantiated the transcriptional evolution of CAR-Ts between the blood and CSF. Thus, individual shared clones tracked in blood and CSF from multiple patients exhibited an evolution from a more diverse proliferation/IFN pathway profile in the blood, to predominantly IFN pathways enriched in CSF CAR-Ts (Panel 2). Conclusions: Daily examination of the blood and CSF by scRNA-Seq and scTCR-Seq has identified a major distinction between blood and CSF CAR-Ts at the level of individual CAR-T clones, revealing the evolution of a prominent interferon pathway transcriptomic signature of CAR-Ts in the CSF. These results suggest that IFN pathway enrichment may play a major role in CAR-T efficacy for the eradication of CNS disease in patients with CNSL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CTIM-25. NEOADJUVANT ANTI-PD1 IMMUNOTHERAPY FOR SURGICALLY ACCESSIBLE RECURRENT GLIOBLASTOMA: CLINICAL AND MOLECULAR OUTCOMES OF A STAGE 2 SINGLE-ARM EXPANSION COHORT

Abstract Glioblastoma is immunologically “cold”, with little clinical response to single-agent immune-checkpoint inhibitors (ICI). Our previous randomized study of neoadjuvant and adjuvant pembrolizumab versus adjuvant pembrolizumab in recurrent glioblastoma identified a molecular signature for ICI and suggested that neoadjuvant pembrolizumab improved survival (PMID30742122). We extended these original findings with a single-arm expansion cohort of glioblastoma patients at first or second relapse undergoing surgical resection for recurrent disease. Eligibility was identical to the original study. Patients receiving &amp;gt; 4mg dexamethasone, or with prior immunotherapy or anti-angiogenic therapy were excluded. The primary objective was to determine the effect of neoadjuvant pembrolizumab on cell cycle/cancer proliferation genes. Secondary objectives were PFS-6 and OS. Twenty-five patients received neoadjuvant pembrolizumab followed by tumor resection and adjuvant pembrolizumab until progression or unacceptable toxicity. The median age of participants was 60 years and median KPS was 90. Ninety-six percent of tumors were IDH wild-type and 48% were MGMT promoter methylated. Neoadjuvant pembrolizumab continued to induce alterations in the tumor microenvironment (T-cell/IFN upregulation, cell cycle downregulation; molecular responders (MR)), but the proportion of MR in this trial was 36% compared with 70% in the original treatment cohort. Median OS was 6.8 months. There were no CTCAE grade 4-5 AEs attributable to pembrolizumab. Further analysis of prognostic differences between stage 1 and 2 subjects is underway, as are additional immune and genotypic correlates to further define molecular changes induced by pembrolizumab. Anti-PD1 monotherapy triggers an intra-tumoral genetic signature associated with interferon-γ response and suppression of cell cycle/cancer proliferation in recurrent glioblastoma, though does not extend OS. This surgical window-of-opportunity paradigm allows for attribution of molecular effects to single agents. Future work will evaluate the differences in patient population, tumor characteristics, and address additional immune checkpoints and/or signaling pathways that can be targeted to augment the effectiveness of anti-PD1 in glioblastoma.

BIOM-15. GLIOBLASTOMA CELLS SHOW ELEVATED CHITINASE 3-LIKE PROTEIN 1 GENE EXPRESSION IN RESPONSE TO HUMAN CEREBROSPINAL FLUID WITH INCREASED MIGRATION AND EPITHELIAL-MESENCHYMAL TRANSITION RELATED ACTIVITY

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. GBM tumors proximal to the lateral ventricles have a worse prognosis with multifocality, recurrence, and lower overall survival. Studies suggest this increased aggressiveness is attributed to cerebrospinal fluid (CSF), as we have previously observed transcriptomic changes in GBM cells upon CSF exposure. GlioVis database reveals chitinase 3-like protein 1 (CHI3L1), a glycoprotein secreted by immune and cancer cells, is associated with poor prognosis and co-expressed with pro-inflammatory and tissue remodeling genes. We suggest exposure to CSF elevates CHI3L1 expression, consequently impacting malignancy-associated gene expression in patient-derived GBM lines. We first evaluated if patient CSF influences the expression of CHI3L1. GBM cells were treated with artificial CSF, non-cancer CSF, or cancer CSF for 72 hours. qPCR revealed significant increases in CHI3L1 (p&amp;lt; 0.0001), SPP1, and CD44 expression in cancer CSF groups compared to artificial CSF. CHI3L1 expression at the protein level is also increased by cancer CSF when profiled using Western Blot. Additionally, ELISA results indicated significantly higher levels of CHI3L1 in CSF samples obtained from patients with high-grade tumors compared to non-cancer samples. Next, we assessed if exogenous CHI3L1 stimulates GBM malignancy at the cellular level. An effective concentration of recombinant human CHI3L1 was determined through Alamar Blue assay, showing 500 ng/ml CHI3L1 increases viability in primary patient cell lines compared to non-treated media. Recombinant CHI3L1 also increases cell migration in Transwell assay. qPCR revealed Vimentin (p&amp;lt; 0.001) and Twist (p&amp;lt; 0.0001) expression significantly increases while E-cadherin (p&amp;lt; 0.001) significantly decreases in CHI3L1-treated groups, consistent with EMT and migratory activity. Ultimately, this study shows CHI3L1 is a CSF-induced factor in GBM and could be a therapeutic target as it enhances tumor malignancy by increasing proliferation, migration, and malignancy-promoting gene expression.

Clinicogenomic characteristics and synthetic lethal implications of germline homologous recombination-deficient hepatocellular carcinoma.

We performed an in-depth examination of pathogenic germline variants (PGVs) and somatic variants in DNA damage response (DDR) genes in hepatocellular carcinoma (HCC) to explore their clinical and genomic impacts. We used a merged whole-exome or RNA sequencing data set derived from in-house ( n = 230) and The Cancer Genome Atlas ( n = 362) databases of multiethnic HCC samples. We also evaluated synthetic lethal approaches targeting mutations in homologous recombination (HR) genes using HCC cells selected from five genomic databases of cancer cell lines. A total of 110 PGVs in DDR pathways in 96 patients were selected. Of the PGV carriers, 44 were HR-altered and found to be independently associated with poorer disease-free survival after hepatectomy. The most frequently altered HR gene in both germline and somatic tissues was POLQ , and this variant was detected in 22.7% (10/44) and 23.8% (5/21) of all the corresponding carriers, respectively. PGVs in HR were significantly associated with upregulation of proliferation and replication-related genes and familial risk of HCC. Samples harboring PGVs in HR with loss of heterozygosity were most strongly correlated with the genomic footprints of deficient HR, such as mutation burden and denovoSig2 (analogous to Catalogue of Somatic Mutations in Cancer [COSMIC] 3), and poor outcome. Pharmacologic experiments with HCC cells defective in BRCA2 or POLQ suggested that tumors with this phenotype are synthetic lethal with poly(ADP-ribose) polymerase inhibitors. Our findings suggest that germline HR defects in HCC tend to confer a poor prognosis and result in distinctive genomic scarring. Tests of the clinical benefits of HR-directed treatments in the affected patients are needed.

Molecular mechanism of di-n-butyl phthalate promotion of bladder cancer development

PurposeTo determine whether di-n-butyl phthalate (DBP) promotes the occurrence of bladder cancer (BCa) and explore the action of DBP acts on BCa cells at the cellular and molecular levels. MethodsMTT and Transwell assays were used to investigate the tumorigenic actions of DBP on BCa cells. Second-generation sequencing was used to identify differences in gene expression before and after DBP treatment. Differential gene expression was verified by q-PCR and analyzed using bioinformatics. Cells were transfected to overexpress genes of interest and proliferation and migration were measured using MTT and Transwell assays, respectively. ResultsDBP treatment stimulated both proliferation and invasion in BCa cells. Second-generation sequencing identified differences in the expression of FOSB, JUND, ATP6V1C2, and RHOQ before and after DBP treatment. FOSB expression was confirmed by q-PCR and bioinformatic analyses. FOSB overexpression increased both proliferation and invasion in BCa cells. ConclusionDBP promoted BCa tumorigenesis by inducing changes in gene expression.

Electrical stimulation enhances the neuronal differentiation of neural stem cells in three-dimensional conductive scaffolds through the voltage-gated calcium ion channel

Electrical stimulation (ES) or electroconductive scaffold has been proved to have the positive effects on the behavior of neural stem cells (NSCs). We previously developed a novel three-dimensional conductive composite scaffold of poly (3, 4-ethylenedioxythiophen)/chitosan/gelatin (PEDOT/Cs/Gel) for neural tissue engineering. In the present study, we further studied the effect of three-dimensional conductive scaffolds combined with ES on the neuronal differentiation of NSCs. The sandwiched ES device was designed to apply single-phase pulse voltage on NSCs cultured in conductive scaffold for 7 days (4 h/day). Proliferation and differentiation related proteins and genes were analyzed by immunofluorescence staining and RT-qPCR. The role of voltage-gated ion channels (VGICs) in regulating NSCs’ neuronal differentiation by ES was investigated in presence of ion channels blockers. The results of protein and gene expression indicated that ES not only promoted the proliferation of NSCs cultured in the conductive scaffold, but also enhanced the differentiation of NSCs into neurons. Especially, the voltage-gated calcium channel (Cav2+) played an important role in the neuronal differentiation of NSCs under ES. Our findings demonstrated that ES combined with three-dimensional conductive scaffolds would be a promising strategy to regulate the neuronal differentiation of NSCs for neural regeneration.

Parasympathetic-macrophages-ductal epithelial cells axis promotes female rat submandibular gland regeneration after excretory duct ligation/deligation

ObjectiveSubmandibular gland (SMG) regeneration depends on parasympathetic signals that modulate this process primarily by promoting ductal epithelial cell proliferation. The purpose of this study was to elucidate the mechanism underlying of parasympathetic regulation on salivary gland regeneration. Materials and methodsWe performed duct ligation/deligation with or without chorda lingual innervation in female Sprague-Dawley rats. Clodronate liposomes were administered retroductally and intravenously into the SMG to deplete macrophages. The proliferative ability and cell cycle-related genes of SMG were assessed using western blotting, quantitative real-time PCR, and immunohistochemical staining. ResultsParasympathetic stimulation boosted Interleukin-6 (IL-6) release by macrophage via muscarinic signalling. This process was suppressed by denervation, muscarinic blockade, or SMG macrophage depletion. SMG IL-6 release led to signal transducer and activator of transcription 3 (STAT3) phosphorylation in ductal epithelial cells to promote proliferation and regeneration by upregulating the expression of cell cycle-related genes. SMG parasympathetic denervation and macrophage depletion suppressed cell cycle-related genes upregulation and ductal cells proliferation. ConclusionsThese data indicated that the parasympathetic-macrophage-ductal epithelial cells axis promote SMG regeneration after duct ligation and deligation.

Impacts of PFOAC8, GenXC6, and their mixtures on zebrafish developmental toxicity and gene expression provide insight about tumor-related disease

Concerns about per- and polyfluoroalkyl substances (PFASs) have grown in importance in the fields of ecotoxicology and public health. This study aims to compare the potential effects of long-chain (carbon atoms ≥ 7) and short-chain derivatives and their mixtures' exposure according to PFASs-exposed (1, 2, 5, 10, and 20 mg/L) zebrafish's (Danio rerio) toxic effects and their differential gene expression. Here, PFOAC8, GenXC6, and their mixtures (v/v, 1:1) could reduce embryo hatchability and increase teratogenicity and mortality. The toxicity of PFOAC8 was higher than that of GenXC6, and the toxicity of their mixtures was irregular. Their exposure (2 mg/L) caused zebrafish ventricular edema, malformation of the spine, blood accumulation, or developmental delay. In addition, all of them had significant differences in gene expression. PFOAC8 exposure causes overall genetic changes, and the pathways of this transformation were autophagy and apoptosis. More importantly, in order to protect cells from PFOAC8, GenXC6, and their mixtures' influences, zebrafish inhibited the expression of ATPase and Ca2+ transport gene (atp1b2b), mitochondrial function-related regulatory genes (mt-co2, mt-co3, and mt-cyb), and tumor or carcinogenic cell proliferation genes (laptm4b and ctsbb). Overall, PFOAC8, GenXC6, and their mixtures' exposures will affect the gene expression effects of zebrafish embryos, indicating that PFASs may pose a potential threat to aquatic biological safety. These results showed that the relevant genes in zebrafish that were inhibited by PFASs exposure were related to tumorigenesis. Therefore, the effect of PFASs on zebrafish can be further used to study the pathogenesis of tumors.

Citrullus colocynthis-Mediated Green Synthesis of Silver Nanoparticles and Their Antiproliferative Action against Breast Cancer Cells and Bactericidal Roles against Human Pathogens.

The present study investigated the biomedical potential of eco-friendly Citrullus colocynthis-mediated silver nanoparticles (Cc-AgNPs). The antibacterial efficacy of Cc-AgNPs was evaluated against two multidrug-resistant pathogenic bacterial strains, Escherichia coli and Pseudomonas aeruginosa. The antiproliferative and antilipidemic performance of the prepared particles was determined against the MCF7 cell line, a breast cancer cell line. The in vitro antibacterial assay revealed that Cc-AgNPs induced dose-dependent bactericidal activity, as a considerable increase in the zone of inhibition (ZOI) was noted at higher concentrations. Reduced proliferation, migration, spheroid size, and colony formation exhibited the substantial antiproliferative potential of Cc-AgNPs against MCF7 cells. Significant alterations in the expression of cell surface markers, apoptosis, and cell proliferation genes further confirmed the antiproliferative impact of Cc-AgNPs. Moreover, Cc-AgNPs exhibited antilipidemic activity by reducing cellular cholesterol and triglyceride levels and regulating key genes involved in lipogenesis. In conclusion, these results propose that Cc-AgNPs can be employed as a potent tool for future antibacterial and anticancer applications.

In vitro application of drug-loaded hydrogel combined with 3D-printed porous scaffolds

Titanium mesh and three-dimensional titanium alloy scaffolds play a key role in addressing oral and maxillofacial bone defects, which can provide a specific environment and structure for bone growth and development. The two main causes of implant surgery failure are aseptic loosening and bacterial-induced implant-associated infections. To make bone defect implants effective for a long time, the ideal scaffold should take into account the two functions of osseointegration and anti-infection. Therefore, on the basis of the low-elastic-modulus Ti-10Ta-2Nb-2Zr (TTNZ) alloys developed by the research group in the early stage, this study intends to combine the vancomycin-loaded hydrogel with the 3D-printed through-hole porous titanium alloy scaffold to endow 3D-printed TTNZ scaffolds with antibacterial properties. The antibacterial properties of the complex were investigated by the zone of inhibition test and the adhesion/free antibacterial test. The effects of the composite system on osseointegration were investigated from the aspects of cell adhesion, cell proliferation and osteogenesis-related gene expression. The results showed that loading 2.5 wt.% and 5 wt.% vancomycin did not affect the structure of chitosan–hyaluronic acid hydrogel. The properties of the hydrogels were examined by scanning electron microscopy, Fourier-transform infrared, degradation experiment in vitro and vancomycin release experiment in vitro. When combined with porous scaffolds, the drug-loaded hydrogels exhibited slower drug release rates and longer release times. In addition, in vitro studies found that the TTNZ scaffolds loaded with 5 wt.% vancomycin had a certain effect on the expression of osteogenesis-related genes in cells, but the antibacterial effect was the best. The porous scaffolds loaded with 2.5 wt.% vancomycin hydrogel TTNZ scaffolds did not inhibit cell proliferation, adhesion, alkaline phosphatase activity, and osteogenesis-related gene ex-pression, but the antibacterial effect on free bacteria was not as good as that of TTNZ scaffolds loaded with 5 wt.% vancomycin. This study, complementing the advantages of the two and controlling the local release rate of vancomycin, provides a new idea for future 3D printing of titanium alloy stents for anti-infection.

MicroRNA-499-5p promotes vascular smooth muscle cell proliferation and migration via inhibiting SOX6.

Atherosclerosis (AS) is the primary etiology of cardiovascular disease, which is considered the leading cause of death all over the world. MicroRNA miR-499-5p was involved in the functional regulation of myocardial and skeletal muscle, whereas its role in atherosclerosis, especially in vascular smooth muscle cells (VSMCs), remains unclear. Our study aims to investigate the effects of miR-499-5p in the proliferation and migration of VSMCs and potential mechanisms. We used mouse aortic vascular smooth muscle cells (MOVAS) and ApoE-/- mice to establish the models of AS in vitro and in vivo, respectively. RT-PCR was performed to detect the expression level of miR-499-5p. Subsequently, Cell Counting Kit-8 (CCK-8) assays, Transwell assays, and wound-healing assays were used to evaluate cell proliferation and migration. Dual-luciferase reporter assay was performed to validate the interaction between miR-499-5p and SOX6. miR-499-5p significantly increased in aorta tissues of mice in AS tissues and vascular smooth muscle cells treated with ox-LDL. miR-499-5p overexpression could promote the proliferation and migration of MOVAS. Bioinformatics analysis predicted and further experiments verified that miR-499-5p could directly bind to the 3'-untranslated region (UTR) region of SOX6. Further, miR-499-5p induced an increased expression of smooth muscle proliferation and migration-related genes, PCNA, cyclin D1, and matrix metalloproteinase (MMP2), as well as the decreased expression of proliferation inhibiting factor p21, which was significantly reversed by SOX6 overexpression. miR-499-5p boosts the proliferation and migration of smooth muscle cells by binding and inhibiting SOX6 expression. The miR-499-5p/SOX6 axis may present a promising therapeutic implication for the prevention and treatment of cardiovascular diseases.

Effects of sodium hypochlorite and ethylenediaminetetraacetic acid on proliferation, osteogenic/odontogenic differentiation, and mechanosensitive gene expression of human dental pulp stem cells

Sodium Hypochlorite (NaOCl) and Ethylene Diamine Tetraacetic Acid (EDTA) can change the biochemical and biophysical properties of dentin. However, the response of human dental pulp stem cells (hDPSCs) to NaOCl and EDTA-treated dentin remains unknown. This study was conducted to investigate the effect of NaOCl and EDTA on cell proliferation, osteogenic/odontogenic differentiation, and the response to mechanosensitive gene expression in hDPSCs. Dentin slices were treated with 5.25% NaOCl, 17% EDTA, and saline (0.9% NaCl) separately. The cell viability and osteogenic/odontogenic differentiation of hDPSCs were analyzed using scanning electron microscopy, cell counting assay, alkaline phosphatase (ALP) staining, and quantitative polymerase chain reaction (qPCR). Besides, the hardness was measured by a Vickers microhardness tester. The expression of mechanosensitive genes was detected by the qPCR assay. All the irrigant-treated dentin allowed cell attachment. The EDTA-treated dentin significantly boosted the ALP and osteogenic/odontogenic differentiation, followed by NaCl and NaOCl groups. Remarkably, these trends were similar to the expression of mechanosensitive genes but were different from the trends of hardness values. The effect of irrigant-treated dentin on regulating hDPSCs differentiation might correlate with mechanosensitive signals. Whereas, the hardness changes between groups might not produce significant roles in regulating osteogenic/odontogenic differentiation of stem cells on dentin surfaces.

Conserved roles for Hnf4 family transcription factors in zebrafish development and intestinal function.

Transcription factors play important roles in the development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte nuclear factor 4 family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have 3 hepatocyte nuclear factor 4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other hepatocyte nuclear factor 4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a; hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a; hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a; hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a; hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine.

MiR-4638-3p regulates transforming growth factor-β1-induced activating transcription factor-3 and cell proliferation, invasion, and apoptosis in human breast cancer cells

TGF-β1 (transforming growth factor-beta1), a secreted polypeptide cytokine, stimulates ATF-3 (activating transcription factor-3) expression in a sustained and prolonged manner in human breast cancer cells (MDA-MB231), but not in normal human mammary epithelial cells (MCF-10A). Cyclin A (cell proliferation gene), Runx2 (metastasis gene), and MMP-13 (matrix metalloproteinase-13; invasive gene) were identified as ATF-3 target genes in these cells. Because ATF-3 has very few druggable sites, its direct targeting is difficult. Recent evidence has indicated that microRNAs (miRNAs) are key players in the post-transcriptional modulation of gene expression under several conditions. Bioinformatic analysis suggested a list of putative miRNAs that target ATF-3. Therefore, we hypothesized that TGF-β1 downregulates the miRNAs that target ATF-3, resulting in the activation of genes that participate in breast cancer progression and skeletal metastasis. Our findings indicate that TGF-β1 downregulated the expression of miR-4638-3p in MDA-MB231 cells. At the molecular level, forced expression of miR-4638-3p reduced the expression of ATF-3 and its downstream targets, Runx2 and MMP-13, in these cells. At the cellular level, overexpression of miR-4638-3p reduced proliferation, invasion, and migration, and induced G0/G1 cell cycle arrest and apoptosis in MDA-MB231 cells. Overall, this study highlights the possibility of utilizing miR-4638-3p as a therapeutic molecule to curb skeletal metastasis of breast cancer cells.

Clinical and molecular characterization of 1q43q44 deletion and corpus callosum malformations: 2 new cases and literature review

BackgroundCorpus callosum malformations (CCM) represent one of the most common congenital cerebral malformations with a prevalence of around one for 4000 births. There have been at least 230 reports in the literature concerning 1q43q44 deletions of varying sizes discovered using chromosomal microarrays. This disorder is distinguished by global developmental delay, seizures, hypotonia, corpus callosum defects, and significant craniofacial dysmorphism. In this study, we present a molecular cytogenetic analysis of 2 Tunisian patients with corpus callosum malformations. Patient 1 was a boy of 3 years old who presented psychomotor retardation, microcephaly, behavioral problems, interventricular septal defect, moderate pulmonary stenosis, hypospadias, and total CCA associated with delayed encephalic myelination. Patient 2 was a boy of 9 months. He presented a facial dysmorphia, a psychomotor retardation, an axial hypotonia, a quadri pyramidal syndrome, a micropenis, and HCC associated with decreased volume of the periventricular white matter. Both the array comparative genomic hybridization and fluorescence in situ hybridization techniques were used.ResultsArray CGH analysis reveals that patient 1 had the greater deletion size (11,7 Mb) at 1q43. The same region harbors a 2,7 Mb deletion in patient 2. Here, we notice that the larger the deletion, the more genes are likely to be involved, and the more severe the phenotype is likely to be. In both patients, the commonly deleted region includes six genes: PLD5, AKT3, ZNF238, HNRNPU, SDCCAG8 and CEP170. Based on the role of the ZNF238 gene in neuronal proliferation, migration, and cortex development, we hypothesized that the common deletion of ZNF238 in both patients seems to be the most responsible for corpus callosum malformations. Its absence may directly cause CCM. In addition, due to their high expression in the brain, PLD5 and FMN2 could modulate in the CCM phenotype.ConclusionOur findings support and improve the complex genotype–phenotype correlations previously reported in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of several genes related to this pathology.

Immunohistochemical expression of proliferation markers in canine osteosarcoma

Canine osteosarcoma is an extremely malignant bone tumor that often arises in the bones of the limbs. It is a highly metastatic disease distinguished by proliferative bone lesions and a tendency for pulmonary metastasis. Overexpression of proliferative proteins are associated with bad prognosis in human osteosarcoma. Here, we tested the expression of the different proliferative proteins (p53, p16, vimentin, and mdm2) in nine archival samples with canine osteosarcoma. Paraffin-embedded tissue sections were confirmed by histopathology and stained by immunohistochemistry for p53, p16, vimentin and mdm2. Positive expression of these proteins was evaluated as the ratio of positive cancer cells and the intensity staining was assessed in several areas. Histopathologically, 95% of samples were grade II and III. All high-grade osteosarcomas were particularly cellular. The cancer cells were generally large spindle-shaped and large nucleus with distribution of osteoid between the cancer cells. Immunohistochemical detection of p53, p16, vimentin and mdm2 was 89%, 56%, 78%, and 89% of samples respectively. The staining intensity for p53, p16, vimentin and mdm2 was particularly nuclear in 81%, 66%, 78%, and 79% of the cancer cells respectively. Our present work suggests that p53, p16, vimentin, and mdm2 were detected in grade III canine osteosarcomas samples. In addition, these proliferative markers are the significant biomarkers in canine osteosarcomas and can be used as a predictor for diagnostic and prognostic value and allowing cancer differentiation. This primary data supports that both canine and human osteosarcomas share same molecular characters which are approved by expression of proliferative genes.

Growth Factor Cocktail to Facilitate Epithelial Differentiation of Exfoliated Deciduous Teeth Stem Cells

Epithelial cells are important in the regeneration of oral mucosal tissue. The cell source is commonly derived from tissue biopsy, which is obtained through surgery. Stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to differentiate into multiple cell types. As it can be readily available from exfoliated deciduous teeth, it can be induced and become a potential source of epithelial cell for oral tissue study. This study aims to examine a mixture of growth factors in the differentiation of epithelial-like cells obtained from human exfoliated deciduous teeth (SHED) stem cells. This growth factor cocktail constitutes hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), insulin-like growth factor 2 (IGF-II), and epidermal growth factor (EGF). After introducing the cocktail, the treated SHED were assessed for epithelial characteristics and markers using cell proliferation test, morphological transformation, protein and gene expression by immunofluorescence staining and cytometry assessment, respectively. The proliferation rate was analysed statistically using Analysis of Variance (ANOVA) with Repeated Measures (p&lt;0.05). SHED cells demonstrated morphological changes on the 7th day after using the cocktail. These transformations are in alignment with the identification of genes associated with epithelial cells and positive stain outcomes for pan-cytokeratin, E-cadherin, and p63. The cell proliferation test indicated that proliferation of cells and growth factor introduction were significantly correlated. The growth factor cocktail used for this research facilitated SHED differentiation for epithelial-like cells. The outcomes validate the production of epithelial cells using SHED; tissue production studies focus on these aspects immensely.

Identification of Prognostic Markers and Potential Therapeutic Targets in Gastric Adenocarcinoma by Machine Learning Based on mRNAsi Index.

Background Cancer stem cells (CSCs), characterized by self-renewal and therapeutic resistance, play important roles in stomach adenocarcinoma (STAD). However, the molecular mechanism of STAD stem cells is still unclear. In this study, our purpose is to explore the expression of stem cell-related genes in STAD. Methods The stemness index based on mRNA expression (mRNAsi) was used to analyze STAD cases in The Cancer Genome Atlas (TCGA). Firstly, mRNAsi was used and analyzed by differential expression, survival analysis, clinical stage, and gender in STAD. Then, weighted gene coexpression network analysis (WGCNA) was used to discover the fascinating modules and key genes. Enrichment analysis was carried out to annotate the functions and pathways of key genes. The gene expression comprehensive database (GEO) in STAD was used to verify the expression levels of key genes in all cancers. Protein-protein interaction networks is used to determine the relationships between key genes. Results The mRNAsi was obviously upregulated in tumor cases. With the increase of tumor stage and T stage, the mRNAsi score decreased, and the overall survival rate of high score group patients was better. According to the degree of association with mRNAsi, different modules and key genes were screened out. A total of 6,740 differential genes were found, of which 1,147 genes were downregulated and 5,593 genes were upregulated. 19 key genes (BUB1, BUB1B, KIF14, NCAPH, RACGAP, KIF15, CENPF, TPX2, RAD54L, KIF18B, KIF4A, TTK, SGO2, PLK4, ARHGAP11A, XRCC2, Clorf112, NCAPG, and ORC6) were screened due to significant upregulation in STAD. And they had been proven that enriched from the cell cycle Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, relating to cell proliferation Gene Ontology (GO) terms, as well. Among them, 9 genes have been extensively associated to OS, and 3 genes had been associated to receive chemotherapy resistance. PPI protein network suggests that there is a sturdy correlation between these key genes. Conclusion A total of 19 key genes were found to play an essential position in retaining the traits of STAD stem cells. These genes can be used to evaluate the prognosis of STAD patients or become specific therapeutic targets.