3T3 Cells Research Articles

Unlocking the antiviral potential of rosmarinic acid against chikungunya virus via IL-17 signaling pathway

BackgroundThe Chikungunya virus is an Alphavirus that belongs to the Togaviridae family and is primarily transmitted by mosquitoes. It causes acute infection characterized by fever, headache, and arthralgia. Some patients also experience persistent chronic osteoarthritis-like symptoms. Dedicated antiviral treatments are currently unavailable for CHIKV. This study aims to explore the potential anti-CHIKV effect of rosmarinic acid using network pharmacology.MethodsThis study employed network pharmacology to predict and verify the molecular targets and pathways associated with ROSA in the context of CHIKV. The analysis outcomes were further validated using molecular docking and in vitro experiments.ResultsThe analysis of CHIKV targets using the Kyoto Encyclopedia of Genes and Genomes and MCODE identified IL-17 as an important pathogenic pathway in CHIKV infection. Among the 30 targets of ROSA against CHIKV, nearly half were found to be involved in the IL-17 signaling pathway. This suggests that ROSA may help the host in resisting CHIKV invasion by modulating this pathway. Molecular docking validation results showed that ROSA can stably bind to 10 core targets out of the 30 identified targets. In an in vitro CHIKV infection model developed using 293T cells, treatment with 60 μM ROSA significantly improved the survival rate of infected cells, inhibited 50% CHIKV proliferation after CHIKV infection, and reduced the expression of TNF-α in the IL-17 signaling pathway.ConclusionThis study provides the first confirmation of the efficacy of ROSA in suppressing CHIKV infection through the IL-17 signaling pathway. The findings warrant further investigation to facilitate the development of ROSA as a potential treatment for CHIKV infection.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Genotype-phenotype association and functional analysis of hnRNPA1 mutations in amyotrophic lateral sclerosis

Background Pathogenic variants in hnRNPA1 have been reported in amyotrophic lateral sclerosis (ALS) patients. However, studies on hnRNPA1 mutant spectrum and pathogenicity of variants were rare. Methods We performed whole exome sequencing of ALS-associated genes and subsequent verification of rare variants in hnRNPA1 in our ALS patients. The hnRNPA1 mutations reported in literature were reviewed and combined with our results to determine the genotype-phenotype relationship. Functional analysis of the novel variant p.G195A was performed in vitro by transfection of mutant hnRNPA1 into 293T cell. Results Among 207 ALS patients recruited, 3 rare hnRNPA1 variants were identified (mutant frequency 1.45%), including two recurrent mutations (p.P340S and p.G283R), and a novel rare variant p.G195A. In combination with previous reports, there are 27 ALS patients with 15 hnRNPA1 mutations identified. Disease onset age was 47.90 ± 1.52 years with predominant limb onset. The p.P340S mutation caused flail arm syndrome (FAS) in two independent families with extended life expectancy. The newly identified p.G195A mutation, lying at the start of the PrLD (“prion-like” domain)/LCD (low-complexity domain), causes local structural changes in 3D protein prediction. Upon sodium arsenite exposure, mutant hnRNPA1 retained in the nucleus but deficit of cytoplasmic G3BP1-positive stress granule clearance was observed. This is different from the p.P340S mutation which caused both cytoplasmic translocation and stress granule formation. No cytoplasmic TDP-43 translocation was observed. Conclusion Mutations in hnRNPA1 are overall minor in ALS patients. The p.P340S mutation is associated with manifestation of FAS. Mutations in LCD of hnRNPA1 cause stress granule misprocessing.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of β-AR-ROS signaling.

Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of β-adrenergic receptor (β-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and β-MHC), β-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of β-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-β-AR-ROS and NPR-A signaling.

Engineered microbubbles decorated with red emitting carbon nanoparticles for efficient delivery and imaging

Altering the route of uptake by the cells is an attractive strategy to overcome drug-receptor adaptation problems. Carbon nanoparticles (CNPs) with emission beyond tissue autofluorescence for imaging biological tissues were used to study the phenomenon of uptake by the cells. In this regard, red-emitting carbon nanoparticles (CNPs) were synthesized and incorporated onto lipid microbubbles (MBs). The CNPs showed red emissions in the range of 640 nm upon excitation with 480 nm wavelength of light. Atomic force microscopic and confocal microscopic images showed the successful loading of CNPs onto the MB. Carbon nanoparticle loaded microbubbles (CNP-MBs) were treated with NIH 3 T3 cells at different concentrations. Confocal microscopic imaging studies confirm the presence of CNPs inside the treated cells. Cytotoxicity studies revealed that the CNPs showed minimal toxicity towards cells after loading onto MBs. The CNPs are usually taken up by the cells through the clathrin-mediated (CME) pathway, but when loaded onto MBs, the mechanism of uptake of CNPs is altered, and the uptake by the cells was observed even in the presence of inhibitors for the CME pathway. Loading CNPs onto MBs resulted in the uptake of CNPs by the cell through micropinocytosis and sonophoresis in the presence of ultrasound. The in vivo uptake CNP-MBs were performed in Danio rerio (Zebrafish larvae). This study provides insights into altering the uptake pathway through reformulation by loading nanoparticles onto MBs.

F. Nucleatum enhances oral squamous cell carcinoma proliferation via E-cadherin/β-Catenin pathway

BackgroundFusobacterium nucleatum (F. nucleatum) is a microbial risk factor whose presence increases the risk of oral squamous cell carcinoma (OSCC) progression. However, whether it can promote the proliferation of OSCC cells remains unknown.MethodsIn this study, we investigated F. nucleatum effect on OSCC cell proliferation using in vitro and in vivo experiments.ResultsOur results showed that F. nucleatum promoted OSCC cell proliferation, doubling the cell count after 72 h (CCK-8 assay). Cell cycle analysis revealed G2/M phase arrest. F. nucleatum interaction with CDH1 triggered phosphorylation, upregulating downstream protein β-catenin and activating cyclinD1 and Myc. Notably, F. nucleatum did not affect noncancerous cells, unrelated to CDH1 expression levels in CAL27 cells. Overexpression of phosphorylated CDH1 in 293T cells did not upregulate β-catenin and cycle-related genes. In vivo BALB/c nude experiments showed increased tumor volume and Ki-67 proliferation index after F. nucleatum intervention.ConclusionOur study suggests that F. nucleatum promotes OSCC cell proliferation through the CDH1/β-catenin pathway, advancing our understanding of its role in OSCC progression and highlighting its potential as a therapeutic target.

Novel artesunate-metformin conjugate inhibits bladder cancer cell growth associated with Clusterin/SREBP1/FASN signaling pathway.

Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activity while high concentration of IC50 at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.

All-in-one hydrogel patches with sprayed bFGF-loaded GelMA microspheres for infected wound healing studies

The current wound healing process faces numerous challenges such as bacterial infection, inflammation and oxidative stress. However, wound dressings used to promote wound healing, are not well suited to meet the clinical needs. Hyaluronic acid (HA) not only has excellent water absorption and good biocompatibility but facilitates cell function and tissue regeneration. Dopamine, on the other hand, increases the overall viscosity of the hydrogel and possesses antioxidant property. Furthermore, chitosan exhibits outstanding performance in antimicrobial, anti-inflammatory and antioxidant activities. Basic fibroblast growth factor (bFGF) is conducive to cell proliferation and migration, vascular regeneration and wound healing. Hence, we designed an all-in-one hydrogel patch containing dopamine and chitosan framed by hyaluronic acid (HDC) with sprayed gelatin methacryloyl (GelMA) microspheres loaded with bFGF (HDC-bFGF). The hydrogel patch exhibits excellent adhesive, anti-inflammatory, antioxidant and antibacterial properties. In vitro experiments, the HDC-bFGF hydrogel patch not only showed significant inhibitory effect on RAW cell inflammation and Staphylococcus aureus (S. aureus) growth but also effectively scavenged free radicals, in addition to promoting the migration of 3 T3 cells. In the mice acute infected wound model, the HDC-bFGF hydrogel patch adhered to the wound surface greatly accelerated the healing process via its anti-inflammatory and antioxidant activities, bacterial inhibition and pro-vascularization effects. Therefore, the multifunctional HDC-bFGF hydrogel patch holds great promise for clinical application.

Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics

Overexpression of VSV-G elevates the secretion of EVs known as gectosomes which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (CNV-G) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison to VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity towards specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor (LDLR), which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Lamprey VDAC2: Suppressing Hydrogen Peroxide-Induced 293T Cell Apoptosis by downregulating BAK expression

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50% with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H2O2 (hydrogen peroxide)-induced 293T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans.

Colchicine modulates TIMP1, MMP1, and pMAP4 expression to enhance extracellular matrix degradation in 3T3 fibroblast-mediated myocardial infarction

Context: Cardiovascular disease is a leading cause of mortality worldwide. Following acute myocardial infarction (AMI), cardiac cells release many cytokines and proteolytic enzymes that regulate the extracellular matrix (ECM), including matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and phosphorylated microtubule-associated protein 4 (Phospho/pMAP4). Aims: To analyze the role of colchicine in ECM regulation post-ischemic in cell line culture towards MMPs, TIMPs, and pMAP4. Methods: This in vitro experimental study was conducted on the 3T3 fibroblast cell line subjected to hypoxic conditions (CoCl2 treatment) and compared with 3T3 cells experiencing hypoxia/ischemic and colchicine treatment. MMP-1, TIMP-1, and pMAP4 expression were measured using flow cytometry. Data analysis was performed using One-Way ANOVA with a 95% confidence level. Results: Treatment with colchicine in the 3T3 fibroblast cell culture regulated the extracellular matrix by increasing TIMP-1 expression and reducing MMP-1 and pMAP4 expression (p<0.0001) following ischemic conditions induced by CoCl2. Conclusions: Colchicine effectively mitigates ECM degradation by enhancing TIMP-1 expression and inhibiting MMP-1 activity and pMAP4 expression post-ischemic conditions.

A Comprehensive Study on Peppermint Oil and Cinnamon Oil as Nanoemulsion: Preparation, Stability, Cytotoxicity, Antimicrobial, Antifungal, and Antioxidant Activity.

Recent studies have shown that nanoemulsions prepared with essential oils have significant antimicrobial potential against multidrug-resistant pathogens due to increased chemical stability. Nanoemulsion also promotes controlled and sustained release, which increases their bioavailability and efficacy against multidrug-resistant bacteria. This study aimed to investigate the antimicrobial, antifungal, antioxidant, and cytotoxicity properties of cinnamon essential oil and peppermint essential oil as nanoemulsions compared to pure forms. For this purpose, analyses of the selected stable nanoemulsions were carried out. The droplet sizes and zeta potentials of peppermint essential oil nanoemulsions and cinnamon essential oil nanoemulsions were found to be 154.6±1.42 nm and -17.1±0.68 mV and 200.3±4.71 nm and -20.0±0.81 mV, respectively. Although the amount of essential oil used in nanoemulsions was 25% w/w, antioxidant and antimicrobial activities were found to be more effective compared to pure essential oils. In cytotoxicity studies on the 3T3 cell line, both essential oil nanoemulsions showed higher cell viability than pure essential oils. At the same time, cinnamon essential oil nanoemulsions exhibited a higher antioxidant property than peppermint essential oil nanoemulsions and showed superiority in the antimicrobial susceptibility test conducted against four bacteria and two fungi. Cell viability tests determined that cinnamon essential oil nanoemulsions showed considerably higher cell viability compared to pure cinnamon essential oil. These findings indicated that the prepared nanoemulsions in the current study might positively influence the dosing regimen and clinical outcomes of antibiotic therapy.

Tanshinone IIA alleviates pulmonary fibrosis by modulating glutamine metabolic reprogramming based on [U-13C5]-glutamine metabolic flux analysis

IntroductionGlutamine metabolic reprogramming, mediated by glutaminase (GLS), is an important signal during pulmonary fibrosis (PF) progression. Tanshinone IIA (Tan IIA) is a naturally lipophilic diterpene with antioxidant and antifibrotic properties. However, the potential mechanisms of Tan IIA for regulating glutamine metabolic reprogramming are not yet clear. ObjectivesThis study aimed was to evaluate the role of Tan IIA in intervening in glutamine metabolic reprogramming to exert anti-PF and to explore the potential new mechanisms of metabolic regulation. MethodsFibrotic characteristics was detected via immunofluorescence and western blotting analysis. Cell proliferation was examined with EdU Assay. Cell metabolites were labeled by using stable isotope [U-13C5]-glutamine. By utilizing 100% 13C glutamine tracers and employing network analysis to investigate the activation of metabolic pathways in fibroblasts, as well as evaluating the impact of Tan IIA on these pathways, we accurately quantified the absolute flux of glutaminolysis, proline synthesis, and the TCA cycle pathway using isotopomer network compartmental analysis (INCA), a user-friendly software tool for 13C metabolic flux analysis (13C-MFA). Molecular docking was used for identifying the binding of Tan IIA with target protein. ResultsTan IIA ameliorate TGF-β1-induced myofibroblast proliferation, reduce collagen I and III and α-SMA protein expression in MRC-5 and NIH-3 T3 cells. Furthermore, Tan IIA regulate mitochondrial energy metabolism by modulating TGF-β1-stimulated glutamine metabolic reprogramming in NIH-3 T3 cells and inhibiting GLS1 expression, which reduced the metabolic flux of glutamine into mitochondria in myofibroblasts, and also targeted inhibited the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), P5C reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), and reduced proline hydroxylation and blocked the collagen synthesis pathway. ConclusionTan IIA reverses glutamine metabolic reprogramming, reduces mitochondrial energy expenditure, and inhibits collagen matrix synthesis by modulating potential targets in glutamine metabolism. This novel perspective sheds light on the essential role of glutamine metabolic reprogramming in PF.

Biocompatibility assessment of organic semiconductor pigments epindolidione and quinacridone

Organic colorants epindolidione (EPI) and quinacridone (QUI) are commercially available hydrogen-bonded semiconductor pigments. Despite their suitable properties for bioelectronic applications, their biocompatibility has not been thoroughly examined yet. In this study, thin EPI and QUI layers were applied on well plates by vacuum deposition technique followed by short and long-term in vitro biocompatibility study. In vitro testing represents a relatively fast and cheap approach, especially suitable for screening testing and prioritization of materials for further research. LIVE/DEAD assay, cell cycle analysis, adhesion and morphology of NIH 3T3 mouse fibroblasts have been investigated up to 7 days using flow cytometry and fluorescent optical microscopy. In summary, no significant differences were observed in viability, cell morphology, or attachment capabilities between control cells and cells grown on the EPI or QUI surfaces or cells cultivated in medium harvested from EPI- or QUI-coated wells. Our results suggest that both pigments are highly biocompatible. The absence of adverse biological effects of EPI and QUI together with their low cost and availability indicate their high application potential in next-generation bioelectronic devices.

Amine functionalised graphene embedded polyvinyl alcohol (PVA) and PVA-chitosan hydrogel composites

A novel amine-functionalized graphene oxide (AFG) doped polyvinyl alcohol (PVA)/chitosan (PVA-Ch) composite film was developed using an eco-synthesis approach, eliminating the need for halogenated compounds. The resulting AFG-doped PVA/Chitosan (PVA-Ch/AFG) polymer film exhibited promising properties for controlled delivery and biosensing applications. The investigation included assessing the swelling behaviour, dissolution percent, gel fraction, and mechanical properties of the polymer film. The swelling characteristics of PVA-Ch and PVA-Ch/AFG were found to be pH and temperature-dependent across various pH ranges (3, 5, 7, and 9). Interestingly, PVA-Ch/AFG demonstrated a stable swelling pattern at pH 5 and 7, unaffected by changes in chitosan concentration, indicating enhanced stability compared to PVA-Ch. The study also explored the use of PVA-Ch/AFG in a drug delivery system, revealing controlled release of the model antibiotic amphicillin, emphasizing its potential in medical applications. Furthermore, the eco-friendly synthesis route underscored the safety of PVA-Ch/AFG for use in food and medical applications. Biocompatibility assessments, including biodegradability studies and cytotoxicity tests on fibroblasts (3T3 cells), confirmed the safety profile of PVA-Ch/AFG. In conclusion, the study suggests that PVA-Ch/AFG holds promise for bio-sensing applications, offering a flexible and colorimetric platform capable of encapsulating, adsorbing, and desorbing biomolecules such as drugs and sensing compounds.

Decreased intranuclear cardiac troponin I impairs cardiac autophagy through FOS/ATG5 in ageing hearts.

In our previous study, intranuclear cardiac troponin I (cTnI) may function as a co-factor of Yin Yang 1(YY1). Here, we aimed to explore the role of intranuclear cTnI in ageing hearts. Nuclear translocation of cTnI was demonstrated using Western blot and immunofluorescence. The potential nuclear localization sequences (NLSs) of cTnI were predicted by a web server and then verified in 293T cells by putative NLS-eGFP-GST and NLS-mutant transfection. The ratio of Nuclear cTnI/ Total cTnI (Nu/T) decreased significantly in ageing hearts, accompanied with ATG5-decline-related impaired cardiac autophagy. RNA sequencing was performed in cTnI knockout hearts. The differential expressed genes (DEGs) were analysed by overlapping with YY1 ChIP-sequencing data. cTnI gain and loss experiments invitro determined those filtered DEGs' expression levels. A strong correlation was found between expression patterns cTnI and FOS. Using ChIP-q-PCR, we demonstrated that specific binding DNA sequences of cTnI were enriched in the FOS promoter -299 to -157 region. It was further verified that pcDNA3.1 (-)-cTnI could increase the promoter activity of FOS by using luciferase report assay. At last, we found that FOS can regulate the ATG5 (autophagy-related gene 5) gene by using a luciferase report assay. Taken together, our results indicate that decreased intranuclear cTnI in ageing hearts may cause impaired cardiac autophagy through the FOS/ATG5 pathway.

Microwave-Assisted Atom Transfer Radical Cyclization in the Synthesis of 3,3-Dichloro-γ- and δ-Lactams from N-Alkenyl-Tethered Trichloroacetamides Catalyzed by RuCl2(PPh3)3 and Their Cytotoxic Evaluation.

An expeditious synthesis of γ- and δ-lactams from tethered alkenyl trichloroacetamides in the presence of 5% of RuCl2(PPh3)3 is reported. In this investigation we have demonstrated that microwave activation significantly enhances reaction rates, leading to the formation of the corresponding lactams in yields ranging from good to excellent. Thus, we have been able to prepare a wide range of lactams, including indole and morphan bicyclic scaffolds, where the corresponding reactions were completely diastereoselective. This process was successfully extended to α,α-dichloroamides without affecting either their yield or their diastereoselectivity. Some of the lactams prepared in this work were evaluated for their hemolytic and cytotoxic responses. All compounds were found to be non-hemolytic at the tested concentration, indicating their safety profile in terms of blood cell integrity. Meanwhile, they exhibited interesting cytotoxicity responses that depend on both their lactam structure and cell line. Among the molecules tested, γ-lactam 2a exhibited the lowest IC50 values (100-250 µg/mL) as a function of its cell line, with promising selectivity against squamous carcinoma cells (A431) in comparison with fibroblasts (3T3 cell line).

STAT3 Contributes a Favorable Response to Pembrolizumab Through IFN-γ-induced Apoptosis in Urothelial Cancer.

Pembrolizumab, a second-line therapy for platinum-refractory advanced urothelial carcinoma (UC), is needed to improve objective response rate. Hence, it is crucial to identify optimal predictive biomarkers of responses. This study aimed to clarify the predictive value and role of signal transducer and activator of transcription 3 (STAT3) in selecting patients with advanced UC who might benefit clinically from pembrolizumab therapy. We retrospectively analyzed 31 patients who received pembrolizumab therapy for UC. STAT3, phosphorylated STAT3 (p-STAT3), and PD-L1 expression were determined using tissue microarrays constructed from patient-derived specimens, and the association of these expression levels with overall survival was analyzed. We assessed the functional role of STAT3 in bladder cancer cell lines in response to interferon-gamma (IFN-γ). Patients with high STAT3 or p-STAT3 expression, and high platelet-to-lymphocyte ratio (PLR) (n=6) had a significantly shorter OS; in the other patients (n=25), high STAT3 or p-STAT3 expression was significantly associated with improved prognosis. IFN-γ-induced apoptosis was partially dependent on STAT3 in T24 cells but not in JMSU1 cells. In patients with advanced UC, STAT3 plays a key role in mediating the efficacy of pembrolizumab through apoptosis in response to IFN-γ.

Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.