Expression Of Marker Proteins Research Articles

Hypomyelinating Leukodystrophy 14 (HLD14)-Related UFC1 p.Arg23Gln Decreases Cell Morphogenesis: A Phenotype Reversable with Hesperetin

Introduction: In the central nervous system (CNS), proper interaction between neuronal and glial cells is crucial for the development of mature nervous tissue. Hypomyelinating leukodystrophies (HLDs) are a group of genetic CNS disorders characterized by hypomyelination and/or demyelination. In these conditions, genetic mutations disrupt the biological functions of oligodendroglial cells, which are responsible for wrapping neuronal axons with myelin sheaths. Among these, an amino acid mutation of the ubiquitin-fold modifier conjugating enzyme 1 (UFC1) is associated with HLD14-related disease, characterized by hypomyelination and delayed myelination in the brain. UFC1 is a critical component of the UFMylation system, functioning similarly to E2-conjugating enzymes in the ubiquitin-dependent protein degradation system. Methodology: We describe how a missense mutation in UFC1 (p.Arg23Gln) leads to the aggregation of UFC1 primarily in lysosomes in FBD-102b cells, which are undergoing oligodendroglial cell differentiation. Results: Cells with mutated UFC1 exhibit reduced Akt kinase phosphorylation and reduced expression of differentiation and myelination marker proteins. Consistently, these cells exhibit impaired morphological differentiation with a reduced ability to extend widespread membranes. Interestingly, hesperetin, a citrus flavonoid with known neuroprotective properties, was found to restore differentiation abilities in cells with the UFC1 mutation. Conclusions: These findings indicate that the HLD14-related mutation in UFC1 causes its lysosomal aggregation, impairing its morphological differentiation. Furthermore, the study highlights potential therapeutic insights into the pathological molecular and cellular mechanisms underlying HLD14 and suggests hesperetin as a promising candidate for treatment.

Mechanism Study on the Regulation of Th1/Th2 in the Treatment of Idiopathic Membranous Nephropathy by Shengyang Yiwei Decoction.

Shengyang Yiwei Decoction showed efficacy in idiopathic membranous nephropathy treatment, and this study aimed to assess the underlying molecular mechanisms. Rats with passive Heymann nephritis were divided into the model group, the Shengyang Yiwei Decoction group, the JAK2 inhibitor group, and the STAT3 inhibitor group. Healthy rats served as the normal control. 24-hour urinary protein excretion, IgG deposition, renal histopathology, the expression levels of synaptopodin, nephrin, podocalyxin, interferon-γ, interleukin- 4, T-box expressed in T cells, GATA binding protein-3, and relevant pathway proteins were measured. Within the model group, a notable elevation in the 24-h urinary protein level was observed, accompanied by evident IgG deposition, increased glomerular volume, eosinophilic deposits, diminished expression of podocyte marker proteins, and a discernible imbalance in Th1/Th2 cellular immunity. Conversely, in Shengyang Yiwei Decoction and both inhibitor groups, enhancements were observed across the aforementioned indexes. Shengyang Yiwei Decoction may reduce glomerular podocyte injury through the suppression of the JAK2/STAT3 signaling pathway and modulation of the Th1/Th2 immune balance.

Fisetin Alleviates d-Galactose-Induced Senescence in C2C12 Myoblasts: Metabolic and Gene Regulatory Mechanisms.

Skeletal muscle aging poses a major threat to the health and quality of life of elderly individuals. Fisetin, a natural polyphenolic compound, exhibits various biological activities; however, its role in preventing skeletal muscle cell aging is still unclear. This study aimed to elucidate the effects of fisetin on skeletal muscle aging using a d-galactose-induced C2C12 myoblast senescence model. Fisetin treatment effectively ameliorated d-galactose-induced aging damage and restored cellular functionality by improving cell viability, reducing the accumulation of the senescence marker enzyme SA-β-gal, and decreasing the expression of key aging marker proteins, p16 and p53. NMR-based metabolomics and RNA-seq transcriptomics analyses revealed that fisetin regulates several critical metabolic pathways, including glutathione metabolism, glycine, serine and threonine metabolism, as well as taurine and hypotaurine metabolism. This regulation led to the restoration of amino acid metabolism, stabilization of cellular energy homeostasis, and the preservation of membrane integrity. In addition, fisetin inhibited calcium signaling and JAK-STAT pathways, reduced cellular stress responses and reversed senescence-induced cell cycle arrest. Together, these findings highlight the potential of fisetin as a therapeutic agent to combat skeletal muscle aging and restore cellular homeostasis, offering a promising avenue for the development of antiaging treatments for skeletal muscle degeneration.

Short-Chain Chlorinated Paraffins May Induce Ovarian Damage in Mice via AIM2- and NLRP12-PANoptosome.

Humans may intake 0.02 mg/kg/day of short-chain chlorinated paraffins (SCCPs), and no study is available on mammalian ovarian damage caused by low-level SCCPs. In this study, four groups of 5-week-old female Institute of Cancer Research (ICR) mice were orally administered 0, 0.01, 0.1, and 1.0 mg/kg/day SCCPs for 21 consecutive days, and serum and ovaries were collected 20 h after the last SCCPs-administration. SCCPs at ≥0.1 mg/kg/day were found to reduce follicle counts at each stage, induce dose-dependent oxidative stress in mice, and lower serum E2 and ovarian anti-Müllerian hormone levels. The data indicated that cellular PANoptosis increased in the ovaries of all SCCP-treated mice. Furthermore, AIM2- and NLRP12-PANoptosome gene and protein levels were considerably elevated. Female germline stem cells (FGSCs) in the cortical portion of the ovary exhibited substantial damage in all SCCP groups, additionally, the expression of FGSC marker genes and major marker proteins was diminished in the ovaries. Oral administration of SCCPs with 0.01, 0.1, and 1.0 mg/kg/day to mice resulted in PANoptosis of the ovaries. Therefore, it was suggested that the oral administration of ≥0.1 mg/kg/day of SCCPs suppressed ovarian function, which may be attributed to the fact that SCCPs induced the generation of AIM2- and NLRP12-PANoptosome in ovary cells.

CAVIN2 attenuates ventilator-induced lung injury in rats by MAPK/ERK1/2 signaling pathway.

Mechanical ventilation is an important treatment in medical treatment, but it may cause or aggravate lung injury, which is called ventilator-induced lung injury (VILI). Studies have shown that CAVIN2 plays an important role in regulating inflammatory responses and cell death. However, its functional mechanism in VILI remains unclear. This study explores the potential role and mechanisms of CAVIN2 in the pathogenesis of VILI. We constructed rat and cell models of VILI. Real-time quantitative polymerase chain reactions (qRT-PCR), Western blot (WB), immunohistochemistry (IHC) and immunofluorescence (IF) were used to detect CAVIN2, ERK1/2, p-ERK1/2, autophagy-associated marker proteins LC3II/I, Beclin1 and P62, and the expression level of pro-inflammatory factors IL-1β and IL-6. Hematoxylin and eosin (H&E) staining were used to evaluate the degree of pathological injury of lung tissue, and the lung permeability was evaluated by measuring the wet-dry ratio of lung tissue and the total protein content in bronchoalveolar lavage fluid (BALF). Molecular docking, co-immunoprecipitation and immunofluorescence were used to verify the binding of CAVIN2 and ERK1/2, and the regulatory mechanisms of both were investigated by rescue experiments. CAVIN2 expression was downregulated in VILI rat lung tissues and ATII cells, whereas p-ERK1/2 expression was up-regulated. Overexpressing CAVIN2 alleviated pathological damage in VILI rat lung tissues and reduced the expression of pro-inflammatory factors and autophagy-related marker proteins in both lung tissues and ATII cells. Conversely, knockdown of CAVIN2 led to increased expression of pro-inflammatory factors and autophagy-related marker proteins in ATII cells. Further mechanism studies showed that CAVIN2 binds to ERK1/2 and inhibits ERK1/2 phosphorylation. Conversely when treated with the p-ERK1/2 agonist Ro67-7476, the protective, anti-inflammatory, and anti-autophagic effects of overexpressing CAVIN2 in VILI rats and ATII cells were reversed. CAVIN2 can bind to ERK1/2 and inhibit the activation of MAPK/ERK1/2 signaling pathway to reduce inflammatory response and autophagy in VILI, thereby reducing lung injury. Therefore, CAVIN2 may be a potential intervention target to provide a new strategy for the treatment of VILI.

Quinine inhibits myogenic differentiation by disrupting AKT signaling pathway.

Sarcopenia is a disease characterized by decreased muscle fibers and mass. Although it mainly affects the older adults, it can also occur in various age groups as a secondary effect of medications used for treating certain diseases, such as cancer and diabetes. With population aging, sarcopenia has drawn significant attention owing to its increasing prevalence. However, its pathogenesis remains unclear, and no specific treatment is available. Natural products containing bioactive compounds have long been used as therapeutic agents and are crucial sources for drug development. However, the use of drugs derived from natural extracts is limited because of their ambiguous mechanisms of action and potential side effects. Therefore, a systematic analysis of the potential effects of using natural products is required. In this study, we investigated the effects of the antimalarial drug quinine on myogenic differentiation. Our findings revealed that quinine significantly inhibited the expression of marker genes and proteins associated with myogenic differentiation and markedly impaired muscle regeneration following injury. Furthermore, this reduction occurred when quinine selectively decreased the AKT signaling activity. Quinine reduced muscle protein and gene expression by modulating AKT signaling and inhibiting myogenic differentiation and muscle regeneration. Therefore, quinine may cause sarcopenia, and this risk should be considered when using quinine for treatment.

Apical-out intestinal organoids as an alternative model for evaluating deoxynivalenol toxicity and Lactobacillus detoxification in bovine

Small intestinal organoids are similar to actual small intestines in structure and function and can be used in various fields, such as nutrition, disease, and toxicity research. However, the basal-out type is difficult to homogenize because of the diversity of cell sizes and types, and the Matrigel-based culture conditions. Contrastingly, the apical-out form of small intestinal organoids is relatively uniform and easy to manipulate without Matrigel. Therefore, we sought to investigate the possibility of replacing animal testing with bovine apical-out small intestinal organoids (Apo-IOs) by confirming the toxicity of mycotoxins and effectiveness of L. plantarum as mycotoxin-reducing agents. The characteristics and functions of Apo-IOs were first confirmed. The gene and protein expression of stem cell, proliferation, mucous, and adherence markers were detected, and the absorption capacity of amino and fatty acids was also confirmed. FITC-4 kDa dextran, a marker of intestinal barrier function, did not penetrate the Apo-IOs, confirming the role of the organoids as a barrier. However, when co-treated with deoxynivalenol (DON), FITC-4 kDa dextran was detected deep within the organoids. Moreover, qPCR and immunofluorescence staining confirmed a decrease in the expression of key markers, such as LGR5, Ki67, Mucin2, Villin2, and E-cadherin. In addition, when Apo-IOs were treated with Lactobacillus plantarum ATCC14917 culture supernatant (LCS) and DON together, cell death was reduced compared to when treated with DON alone, and FITC-4 kDa dextran was confirmed to flow only to the peripheral part of the organoid. The qPCR and immunofluorescence staining results of LCS and DON co-treatment group showed that LGR5, Ki67, Mucin2, Villin2, and E-cadherin were expressed at significant higher levels than those in the DON treatment group alone. In this study, we found that the characteristics and functions of bovine Apo-IOs were similar to those of the intestinal structure in vivo. Additionally, the effects of mycotoxins and effectiveness of L. plantarum as mycotoxin-reducing agents were confirmed using bovine Apo-IOs. Therefore, bovine Apo-IOs could be applied in toxicity studies of mycotoxins and could also be used as in vitro models to replace animal testing and improve animal welfare.

PDGFR-α shRNA-polyplex for uveal melanoma treatment via EMT mediated vasculogenic mimicry interfering

Up to 50% of individuals with uveal melanoma (UM), a frequent cancer of the eye, pass away from metastases. One of the major challenges in treating UM is the role of receptor tyrosine kinases (RTKs), which mediate the epithelial-mesenchymal transition (EMT) of tumors. RTKs are involved in binding multiple growth factors, leading to angiogenesis and vasculogenic mimicry (VM) phenomena. Currently, most anti-angiogenic drugs have shown a tendency to increase the VM of tumors in clinical trials, resulting in limited efficacy. The existing gap in UM treatment lies in the lack of effective strategies to target RTK-mediated EMT and VM. While some approaches have been attempted, there is still a need for novel therapeutic interventions that can specifically interfere with these processes. This research employed the gene vector PEI-g-PEG to interfere with the platelet derived growth factor-alpha receptor (PDGFR-α)-mediated EMT process, thereby retarding the growth of UM. The cell experiments demonstrated that the gene polyplex exhibited favorable cell uptake and lysosome escape properties, effectively suppressing the expression of PDGFR-α protein and EMT marker proteins and the occurrence of VM phenomenon. In vivo animal studies also inhibited the growth of UM, and PAS assays showed that the treatment reduced the generation of VM in tumor tissue. This study broadens the application of PEI-g-PEG while interfering with the RTK-mediated tumor EMT process with the help of RNAi technology, providing a new idea for tumor reduction research.

15-Deoxy-Δ-12,14-Prostaglandin J2 Represses Immune Escape of Lung Adenocarcinoma by Polarizing Macrophages Through Epidermal Growth Factor Receptor/Ras/Raf Pathway.

Lung adenocarcinoma (LUAD) is a widespread and deadly form of cancer. Prostaglandin 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) possesses antioxidant, anti-inflammatory, and anticancer properties. However, it is unclear whether this effect on LUAD progression stems from its ability to influence macrophage polarization. By utilizing 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, colony formation, transwell assays, and enzyme linked immunosorbent assay (ELISA), we investigated how 15d-PGJ2 affects A549 cell viability, proliferation, apoptosis, and invasion, as well as levels of interleukin (IL)-4, IL-13, and IL-17. Human monocytic cell line THP-1 was induced into M2 macrophages using phorbol 12-myristate 13-acetate and IL-4/IL-13, followed by treatment with 15d-PGJ2. The study employed flow cytometry to observe the polarization of macrophages, quantitative reverse transcription polymerase chain reaction (qRT-PCR) to identify epidermal growth factor receptor (EGFR) expression, western blot for identifying expression of macrophage marker proteins, and examining EGFR/rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf) activation. In a coculture setup, CD8+ T cells were shown to have a proliferation capacity by carboxifluorescein diacetate succinimidyl ester (CFSE), a killing ability by lactate dehydrogenase, and an analysis of their interferon gamma and tumor necrosis factor alpha levels by ELISA. 15d-PGJ2 reduced invasion capacity and expression of inflammatory cytokines, lowered A549 cell viability in a dose-dependent way, and promoted apoptosis. 15d-PGJ2 facilitated the transition of M2 macrophages to the M1 type, inhibited Ras/Raf pathway activation, reduced EGFR expression in macrophages, and stimulated CD8+ T cells to enhance anti-tumor immunity. 15d-PGJ2 repressed M2 macrophage polarization and LUAD immune evasion by targeting the EGFR/Ras/Raf pathway in macrophages.

Coleus vettiveroides Root Extract Protects Against Thioacetamide-Induced Chronic Liver Injury by Inhibiting NF-κB Signaling Pathway.

The roots of Coleus vettiveroides (CV) have been traditionally used in Indian medicinal systems such as Ayurveda and Siddha for its antioxidant, anti-inflammatory, and antidiabetic effects. This study examines the antifibrotic potential of CV ethanolic root extract (CVERE) against thioacetamide (TAA)-induced liver fibrosis in Wistar rats. TAA was administered via i.p., thrice weekly for 11 weeks to induce liver fibrosis in rats. In separate groups, rats were administered with TAA and were concurrently treated with CVERE 125 mg/kg, CVERE 250 mg/kg, and silymarin (SIL) 100 mg/kg. Liver marker enzymes of hepatotoxicity, oxidative stress markers, proinflammatory marker gene expression (TNF-α, NF-κB, COX, and ILs), fibrotic marker gene expression (collagen I and III), immune histochemical expression of fibrosis marker proteins, and histopathologic changes were analyzed. TAA administration led to a significant (p < 0.001) increase in the serum level of hepatotoxic marker enzymes. The TAA-treated group showed higher levels (p < 0.001) of MDA and reduced activities of SOD and CAT in the liver. TAA administration increased CYP2E1 expression, proinflammatory, and fibrotic marker gene expressions in rat liver. The histopathology of the liver confirms TAA-induced architectural distortion and fibrotic changes. CVERE and SIL simultaneous treatments significantly protected against TAA-induced oxidative stress, inflammation, and liver fibrosis. In conclusion, CVERE inhibited TAA-induced liver fibrosis through downregulation of TAA metabolic activation, redox imbalance, and inflammation through repression of the NF-κB pathway.

Skin Barrier-Improving and Skin-Soothing Effects of Autophagy-Activating Peptide on Sensitive Skin

Among the complex and diverse triggering and aggravating factors for sensitive skin syndrome, potential defects in skin barrier function are considered one of the most important ones. Previously, we have reported improvements in skin barrier function thanks to autophagy-activating peptide derivatives. Further investigation revealed that the activation of autophagy signaling in skin cells also attenuated inflammatory responses induced by UV irradiation or exposure to pollution. In this study, in vitro and ex vivo human skin explant models were used to evaluate the potential benefits of the autophagy-activating peptide, pentasodium tetracarboxymethyl palmitoyl didpeptide-12 (PTPD-12), on sensitive skin-related parameters. Clinical efficacy testing was also performed to confirm the skin barrier-improving and skin-soothing activities of the autophagy-activating peptide. As a result, significant reductions in inflammatory cytokine (IL-8 and TNF-α) and enzyme activity (PDE4) were observed in the in vitro system. Increased expression of barrier marker proteins by PTPD-12 in UV-irradiated human skin tissue was observed ex vivo. In a clinical study, delayed response to topical capsaicin-induced vascular activation, which represents enhanced epidermal permeability barrier function, was observed after 4 weeks of application of PTPD-12 in healthy volunteers. In another clinical study with sensitive skin carriers identified via a lactic acid stinging test, a significant reduction in trans-epidermal water loss (TEWL) and skin erythema index was observed after 4 weeks of PTPD-12 usage. These results suggest that the activation of autophagy can be a potential treatment regimen for sensitive skin syndrome, specifically in terms of skin barrier function enhancement and skin soothing.

Inducing Neural Fate: The Impact of Phenylacetate and Calcium on Human Adipose-Derived Mesenchymal Stem Cells Differentiation.

Human adipose-derived stem cells (hADSCs) are considered a promising source for cell replacement therapy in degenerative and traumatic conditions. This study explores the effects of phenylacetate and calcium on the neural differentiation of hADSCs for regenerative medicine. We assessed cell viability and cytotoxicity using the MTT assay, revealing that treatment with 1μM phenylacetate significantly enhanced cell viability compared to control groups over five days, while higher concentrations resulted in cytotoxic effects. Additionally, qualitative analysis through Acridine orange/ethidium bromide (AO/EB) staining indicated normal cellular characteristics at lower phenylacetate concentrations, whereas higher doses led to observable cell death. A subsequent evaluation of intracellular calcium levels demonstrated a significant increase when hADSCs were treated with both phenylacetate and calcium. The neural differentiation potential was further assessed through the relative quantification of neuronal-specific genes, showing marked upregulation of NSE, Oligo-2, β-tubulin III, and MAP-2 in all treatment groups compared to controls. Immunohistochemistry confirmed elevated protein expression of neural markers in cultures supplemented with phenylacetate and calcium. These findings suggest that phenylacetate, particularly in conjunction with calcium, enhances the neural differentiation of hADSCs, highlighting its potential utility in regenerative medicine strategies targeting neurodegenerative conditions.

Plakin Expression in Serous Epithelial Ovarian Cancer Has the Potential to Impede Metastatic Spread and Epithelial-Mesenchymal Transition: A Comparative Expression Analysis of Immunohistochemical and In Silico Datasets.

Epithelial ovarian cancer is aggressive and causes high mortality among women worldwide. Members of the plakin family are essential to maintain cytoskeletal integrity and key cellular processes. In this study we characterised the expression of plakins, particularly plectin (PLEC), periplakin (PPL), envoplakin (EVPL), and EMT-related proteins by immunohistochemistry in n = 48 patients' samples to evaluate a potential correlation of plakin expression with EMT as EOC progresses. These tissue plakin and EMT expression analyses were further evaluated by in vitro cell line expression and correlated with the expression of these molecules using publicly available datasets such as Cancer Genome Atlas (TCGA) and Clinical Proteome Tumour Analysis Consortium (CPTAC) datasets. We demonstrate that the expression of PPL and PLEC plakins is decreased in high-grade compared to low-grade EOCs with mixed EMT marker protein expression. This is supported by the correlation of high PPL and PLEC expression with an epithelial rather than mesenchymal phenotype. Our data suggest a partial loss of plakin expression as EOC tumours progress. This may impact the connections of plakins with membrane-bound receptors, which impede the downstream signalling required for the initiation of EMT as the tumours progress.

Gα12 and Gα13 proteins are required for transforming growth factor-β-induced myofibroblast differentiation.

Myofibroblast differentiation, characterized by accumulation of cytoskeletal and extracellular matrix proteins by fibroblasts, is a key process in wound healing and pathogenesis of tissue fibrosis. Transforming growth factor-β (TGF-β) is the most powerful known driver of myofibroblast differentiation. TGF-β signals through transmembrane receptor serine/threonine kinases that phosphorylate Smad transcription factors (Smad2/3) leading to activation of transcription of target genes. Heterotrimeric G proteins mediate distinct signaling from seven-transmembrane G protein coupled receptors, which are not known to be linked to Smad activation. We tested whether G protein signaling plays any role in TGF-β-induced myofibroblast differentiation, using primary cultured human lung fibroblasts. Activation of Gαs by cholera toxin blocked TGF-β-induced myofibroblast differentiation without affecting Smad2/3 phosphorylation. Neither inhibition of Gαi by pertussis toxin nor siRNA-mediated combined knockdown of Gαq and Gα11 had a significant effect on TGF-β-induced myofibroblast differentiation. In contrast, combined knockdown of Gα12 and Gα13 significantly inhibited TGF-β-stimulated expression of myofibroblast marker proteins (collagen-1, fibronectin, smooth-muscle α-actin), with siGα12 being significantly more potent than siGα13. Mechanistically, combined knockdown of Gα12 and Gα13 resulted in substantially reduced phosphorylation of Smad2 and Smad3 in response to TGF-β, which was accompanied by a significant decrease in the expression of TGF-β receptors (TGFBR1, TGFBR2) and of Smad3. Thus, our study uncovers a novel role of Gα12/13 proteins in the control of TGF-β signaling and myofibroblast differentiation.

Wild-Type p53 Regulates Apoptosis of Human Breast Cancer Cells.

The tumor suppressor wild-type p53 is known for its role in inducing apoptosis in tumor cells. This study investigated the relationship between wild-type p53 and protein phosphatase 1 (PP1) and caspase in promoting apoptosis of breast cancer cells. Human breast cancer cell lines MCF-7 and MDA-MB-231 obtained from the American Type Culture Collection were used in this study. Small interference RNAs (Si-RNA) and plasmids were used to regulate wild-type p53 expression in these two tumor cell lines through liposome-mediated transfection. GSK-2830371 (PP1 inhibitor) and zVAD (Caspase inhibitor) were employed to further verify the PP1 activating function of wild-type p53 in Caspase-dependent MCF-7 and MDA-MB-231 apoptosis. PP1 activity was quantitatively detected by phosphorus colorimetric assay. Co-immunoprecipitation (Co-IP), flow cytometry assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Western blot, the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), and immunofluorescence staining were used to analyze cell apoptosis degree and marker protein expression. The expression level of PP1 in the breast cancer cells was successfully regulated by cell transfection. The phosphatase activity was increased, and obvious apoptotic cytological characteristics were observed in p53-overexpressed breast cancer cells. p53 knockdown/overexpression increased/decreased the level of B cell lymphoma 2 (Bcl-2), and decreased/increased levels of Caspase-3, cleaved Caspase-3, cleaved Caspase-8, Cytochrome C (Cyt-C), Truncated BID (tBid), Bcl-2-associated X (Bax), and cell apoptosis (p < 0.01). The promotion of proteins and apoptosis induced by p53 overexpression was reversed by GSK-2830371 or zVAD. Wild-type p53 might promote Caspase-dependent apoptosis of human breast cancer cells through PP1 activation.

In vitro and ex vivo screening of microRNA combinations with enhanced cell penetrating peptides to stimulate intervertebral disc regeneration.

Low back pain (LBP) is predominantly caused by degeneration of the intervertebral disc (IVD) and central nucleus pulposus (NP) region. Conservative treatments fail to restore disc function, motivating the exploration of nucleic acid therapies, such as the use of microRNAs (miRNAs). miRNAs have the potential to modulate expression of discogenic factors, while silencing the catabolic cascade associated with degeneration. To deliver these miRNAs, nonviral cell penetrating peptides (CPPs) are gaining favor given their low immunogenicity and strong targeting ability. Single miRNA therapies have been investigated for IVD repair, however dual miRNA delivery strategies have not been commonly examined and may augment regeneration. Transfection of four pro-discogenic miRNAs (miRNA mimics:140-5p; 149-5p and inhibitors: 141-3p; 221-3p) and dual delivery of six miRNA pairings was performed using two CPPs, RALA and GET peptide (FLR), in primary rat NP monolayer culture, and in an ex vivo organ culture model of rat caudal discs. Protein expression of discogenic (aggrecan, collagen type II, and SOX9) and catabolic markers (ADAMTS5 and MMP13) were assessed. Monolayer investigations signified enhanced discogenic marker expression following dual miRNA delivery, signifying a synergistic effect when compared to single miRNA transfection. Utilization of an appropriate model was emphasized in our ex vivo organ culture experiment, revealing the establishment of a regenerative microenvironment characterized by reduced catabolic enzyme activity and enhanced matrix deposition, particularly following concurrent delivery of FLR-miRNA-149-5p mimic and miRNA-221-3p inhibitor. Bioinformatics analysis of miRNA-149-5p mimic and miRNA-221-3p inhibitor identified distinct targets, pathways, and interactions, suggesting a mode of action for this amplified response. Our findings suggest the potential of FLR-miRNA-149-5p + miRNA-221-3p inhibitor to create an anti-catabolic niche within the disc to foster regeneration in moderate cases of disc degeneration, which could be utilized in further studies with the overarching aim of developing treatments for LBP.

Macrophage P2Y12 regulates iron transport and its inhibition protects against atherosclerosis.

Iron retention is commonly observed in atherosclerotic plaques and is believed to be detrimental to atherosclerosis. Platelet P2Y12 is a target of antiplatelet therapy in preventing thrombotic complications of atherosclerosis. The protective effect of P2Y12 on hematopoiesis reported by our previous work implies the involvement of P2Y12 in iron metabolism. This study further investigated the role of P2Y12 in the iron metabolism of macrophages, the key player in systemic iron homeostasis and atherosclerosis. The association between serum iron and the use of P2Y12 inhibitors was evaluated by a case-control study in human. Secondary iron overload and atherosclerosis animal models were established in P2Y12-deficient zebrafish to explore the role of P2Y12 in macrophage iron metabolism in vivo. Both iron-overloaded murine primary peritoneal macrophages (PMs) and ox-LDL-treated PMs with P2Y12 knockdown were used for in vitro studies. RNA sequencing and pharmacological approaches were performed to investigate the downstream mechanisms. Increased serum iron level was positively associated with P2Y12 inhibitor usage [odds ratio (OR) = 10.333 (1.281-83.370)]. Elevated serum iron level and transferrin saturation, reduced hepatic and splenic iron content, and decreased iron staining in macrophages were observed in secondary iron overload P2Y12-deficient zebrafish. Deficiency of P2Y12 in ApoEb-/- zebrafish fed a high-fat diet reduced atherosclerosis progression and intraplaque iron retention. Furthermore, reduced ferritin, restored cell viability and expression of ferroptosis marker proteins, and decreased ROS formation and inflammatory cytokines were observed in both iron-overloaded and ox-LDL-treated PMs with P2Y12 knockdown in vitro, while reversed phenotypes were observed after agonist-induced P2Y12 activation. Mechanistically, P2Y12 inhibition in iron-overloaded or ox-LDL-treated PMs suppressed NF-κB p65 phosphorylation and hepcidin expression, both of which were reversed by P2Y12 activation. P2Y12 inhibition decreased hepcidin autocrine through repressing NF-κB p65 phosphorylation in macrophages, preventing intracellular iron retention and atherosclerosis.

Good Manufacturing Practice-grade fibronectin for hollow-fiber bioreactor cell manufacture: a mesenchymal stromal cell case study.

The need for large-scale production of mesenchymal stromal cell (MSC)-based cellular therapeutics continues to grow around the globe. Manual cell expansion processes can be highly variable between operators, require significant hands-on time from skilled staff and, because of the large number of open manipulation steps required to produce cells in dose-relevant quantities, be prone to greater risk of contamination relative to automated processes. All of these can increase overall production costs and risks to the patient. In order to meet the needs of this growing industry, viable options for large-scale automation coupled with consistent and compliant ancillary materials needed to drive cell expansion are needed. In the work described herein, the automated and functionally closed hollow-fiber bioreactor system Quantum Flex (Terumo Blood and Cell Technologies, Inc., Lakewood, CO, USA) was used in conjunction with Good Manufacturing Practice (GMP)-compliant, virus-inactivated human fibronectin (FN) from Akron Bio (Boca Raton, FL, USA) to expand MSCs to clinically relevant numbers. In order to assess the performance of Akron Bio's GMP-grade FN, use of this product in the production of MSCs was referenced against use of a research-use-only (RUO)-grade FN product used extensively for MSC expansion in Quantum. Because many MSC-based processes require passaging of cells to attain the appropriate number of cells needed, a two-passage process was employed comparing the transfer of MSCs expanded on RUO FN to RUO FN, GMP FN to GMP FN and RUO FN to GMP FN to assess the impacts of transitioning from one grade of FN to another, as a product might be required to do as it moves from pre-clinical to clinical stages and beyond. No statistically significant differences were noted when MSCs were transferred from RUO FN to RUO FN, GMP FN to GMP FN or RUO FN to GMP FN in terms of harvest yield, population doubling time, seeding efficiency estimates or fold expansion. All MSCs harvested from all groups met International Society for Cell & Gene Therapy standards for MSCs in terms of protein marker expression measured by flow cytometry, adherence to plastic, downstream cell morphology and trilineage differentiation. The combination of Quantum Flex as an expansion platform and Akron Bio's GMP FN is seen as an attractive option for larger-scale manufacture of GMP-grade MSC products.

CALML3-AS1 enhances malignancies and stemness of small cell lung cancer cells through interacting with DAXX protein and promoting GLUT4-mediated aerobic glycolysis

The lncRNA CALML3 antisense RNA 1 (CALML3-AS1) is a biomarker for various cancers, including non-small cell lung cancer (NSCLC). However, the role of CALM3-AS1 in small cell lung cancer (SCLC) is still unclear. Here, we found that the CALML3-AS1 was upregulated in SCLC tissues and cells. SCLC cells (NCI-H69 and NCI-H466 cells) were transfected with small interfering RNA of CALML-AS1 (si-CALML3-AS1) and Death domain-associated protein (DAXX) (si-DAXX) or an overexpression vector of CALML-AS1 (dCas9-CALML3-AS1) and DAXX (dCas9-DAXX). The results showed that silencing CALML3-AS1 inhibited SCLC cell proliferation, colony formation, migration, invasion, and spheroid formation, and reduced the expression of stemness marker proteins (Nanog. Oct4, and Lin28). Moreover, silencing CALML3-AS1 reduced glycolysis rate, glucose utilization, and lactate production, and decreased the levels of key glycolytic regulatory proteins (GLUT1, GLUT4, HK2, and PKM2) in SCLC cells, while overexpression of CALML3-AS1 promoted malignant growth and stemness and enhanced glucose transporters type 4 (GLUT4)-mediated aerobic glycolysis by interacting with DAXX in NCI-H69 and NCI-H466 cells. Silencing DAXX or GLUT4, or treatment with 2-Deoxy-d-glucose (2-DG, a glycolysis inhibitor) reversed the effects of CALML3-AS1 overexpression on aerobic glycolysis, malignant growth, and stemness of SCLC cells. Finally, NCI-H69 cells transfected with CALML3-AS1, sh-CALML3-AS1, and sh-DAXX lentiviral vectors were subcutaneously injected into nude mice to construct xenograft models. Knockdown of CALML3-AS1 or DAXX inhibited tumor growth in SCLC in vivo. In conclusion, CALML3-AS1, an oncogene, promotes the malignancy and stemness of SCLC cells by interacting with DAXX to enhance GLUT4-mediated aerobic glycolysis, thereby promoting SCLC progression.

Pericytes require physiological oxygen tension to maintain phenotypic fidelity

Pericytes function to maintain tissue homeostasis by regulating capillary blood flow and maintaining endothelial barrier function. Pericyte dysfunction is associated with various pathologies and has recently been found to aid cancer progression. Despite having critical functions in health and disease, pericytes remain an understudied population due to a lack of model systems which accurately reflect in vivo biology. In this study we developed a protocol to isolate and culture murine lung, brain, bone, and liver pericytes, that maintains their known phenotypes and functions. We demonstrate that pericytes, being inherently plastic, benefit from controlled oxygen tension culture conditions, aiding their expansion ex vivo. Primary pericytes grown in physiologically relevant oxygen tensions (10% O2 for lung; 5% O2 for brain, bone, and liver) also better retain pericyte phenotypes indicated by stable expression of characteristic transcriptional and protein markers. In functional tube formation assays, pericytes were observed to significantly associate with endothelial junctions. Importantly, we identified growth conditions that limit expression of the plasticity factor Klf4 to prevent spontaneous phenotypic switching in vitro. Additionally, we were able to induce pathological pericyte phenotypic switching in response to metastatic stimuli to accurately recapitulate in vivo biology. Here, we present a robust method for studying pericyte biology in both physiology and disease.