Mouse Embryonic Fibroblast Cells Research Articles

Nuclear mTORC1 Live-Cell Sensor nTORSEL Reports Differential Nuclear mTORC1 Activity in Cell Lines

The mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is activated on the surface of lysosomes and phosphorylates substrates at various subcellular locations, including the lysosome, cytosol, and nucleus. However, the signaling and biological functions of nuclear mTORC1 (nmTORC1) are not well understood, primarily due to limited tools for monitoring mTORC1 activity in the nucleus. In this study, we developed a genetically encoded nmTORC1 sensor, termed nTORSEL, based on the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) by mTORC1 within the nucleus. nTORSEL, like its predecessor TORSEL, exhibits a fluorescent punctate pattern in the nucleus through multivalent protein–protein interactions between oligomerized 4EBP1 and eIF4E when nmTORC1 activity is low. We validated nTORSEL using biochemical analyses and imaging techniques across representative cell lines with varying levels of nmTORC1 activity. Notably, nTORSEL specifically detects physiological, pharmacological, and genetic inhibition of nmTORC1 in mouse embryonic fibroblast (MEF) cells but not in HEK293T cells. Therefore, nTORSEL is an effective tool for investigating nuclear mTORC1 signaling in cell lines.

Cytotoxicity of norstictic acid derivatives, a depsidone from Ramalina anceps Nyl.

Structural modifications in lichen phenolic compounds have been one of the tools to potentiate their biological activity. In the present work, seven alkyl derivatives of norstictic acid were prepared and evaluated against eight cell lines. Norstictic acid was isolated from the lichen Ramalina anceps and the alkyl derivatives were obtained through reactions with alcohols. Cytotoxicity was evaluated against the 786-0 (kidney carcinoma), MCF7 (breast carcinoma), HT-29 (colon carcinoma), PC-03 (prostate carcinoma), HEP2 (laryngeal carcinoma), B16-F10 (murine melanoma), UACC-62 (human melanoma), and NIH/3T3 (mouse embryonic fibroblast) cell lines using the sulforhodamine B assay. Norstictic acid exhibited poor activity, while the 8'-O-n-butyl-norstictic acid and 8'-O-sec-butyl-norstictic acid derivatives showed potential activity (GI50 values of 6.37-45.0 μM and 6.8-52.40 μM, respectively) and high selectivity (selectivity index (SI) values of 13.88-98.11 and SI 11.30-87.40, respectively) against all tumor cells. The 8'-O-n-hexyl-norstictic acid showed good activity (5.96-9.53 μM) and moderate selectivity (SI 9.2-5.76) against MCF7, HT-29, PC-03, and HEP2 cells, while 8'-O-isopropyl-norstictic acid demonstrated high activity and selectivity against PC-03 cells (GI50 1.28 μM and SI 33.8), and was highly active but moderately selective against UACC, HEP2, and B16-F10 cells (GI50 6.2, 7.78, and 9.65 μM; SI 7.0, 5.5, and 4.5, respectively). Additionally, 8'-O-n-pentyl- and 8'-O-tert-butyl-norstictic acids were active and selective against PC-03 cells (GI50 8.77 and 7.60 μM; SI 6.53 and 5.0, respectively). Chemometric analysis revealed a clear relationship between all compounds and their biological activities. The insertion of a four-carbon alkyl chain (n-butyl and sec-butyl) produced potentially active compounds on all tested tumor cells.

Sorting differentiated mammalian cells using deterministic lateral displacement microfluidic devices.

Separation of differentiated and undifferentiated cells without labeling is required for cell analyses and clinical application of cultured differentiated cells in vitro. To proceed with the passive separation of differentiated cells inside a clean bench, we developed a system of deterministic lateral displacement (DLD) microfluidic devices and applied this system to sort differentiated cells in vitro. The fluid flow is driven by compressed air to the buffer. Priming and sorting can be completed by air pressure control. We use this system to separate C2C12 mononuclear myocytes from multinuclear myotubes. Additionally, using a DLD microfluidic channel of Dc = 20μm, multinuclear myotubes can be effectively sorted as larger particles. We prepared differentiated adipocytes from mouse embryonic fibroblast (MEF) cells and sorted those containing lipid droplets. The diameters of these sorted adipocytes considered larger particles, exceeded 20μm, similar to the Dc of the DLD microfluidic channel. Differentiated cell sorting by cell size will contribute to single-cell analyses and in vitro tissue model preparation for drug discovery.

Investigation of the anticancer effect of newly synthesized palladium conjugate Schiff base metal complexes on non-small cell lung cancer cell line and mouse embryonic fibroblast cell line

Lung cancer remains one of the leading causes of death worldwide. Due to the side effects of chemotherapeutic agents on normal cells and the development of resistance by cancer cells, there is an urgent need for alternative new pharmacological agents. Palladium (Pd)-conjugated Schiff base (SB) compounds represent an alternative approach with promising potential applications in cancer treatment. This study aims to identify novel therapeutic agents on A549 cells through the synthesis and characterization of Schiff base conjugated-Palladium complexes (Pd-L1 and Pd-L2). Additionally, it seeks to elucidate the mechanism of action of these compounds on both the A549 and NIH/3T3 cell lines. In the present study, two new Pd-L1 and Pd-L2 were synthesized for the first time and characterized mainly by single crystal X-ray diffraction and 1H, 13C, 31P NMR techniques. The cytotoxic effect of the compounds was evaluated by MTT assay on A549 and NIH/3T3 cell lines for 24 and 48 h. Cisplatin was used as a positive control group. Based on the cytotoxicity results, the complexes were evaluated for their anticancer activities against A549 cell lines for 48 h through reactive oxygen species (ROS), cell cycle, apoptotic, and necrotic cell analyses. The most potent cytotoxic effects were determined for Pd-L1 (IC50: 23.33 μM), Pd-L2 (IC50: 3.19 μM), and cisplatin (IC50: 33.27 μM) on A549 cells (p < 0.05). The compounds exhibited a significant cytotoxic effect at lower concentrations on A549 cells compared to NIH/3T3 cells (p < 0.05). All compounds showed a significant increase in ROS levels in A549 cells compared to the control group (p < 0.05). While necrosis and apoptosis was observed in A549 cells treated with cisplatin, induction of apoptosis was effective in cell death for A549 cells treated with Pd-L1 and Pd-L2 (p < 0.05). Additionally, it was observed that the compounds inhibited cell proliferation in the G0/G1 and G2/M cell cycle phases (p < 0.05). All compounds induced cell cycle arrest and cell death in A549 cells by increasing ROS levels. The results obtained in the present study could advance the utilization of the compounds as anticancer agents.

In vitro cytotoxic study of synthesized Ag and Ce dual-doped α-Fe2O3 nanoparticles on NIH/3T3 and U87 cell lines

The significant potential of iron oxide nanoparticles in biomedical fields, particularly in the delivery of anticancer drugs and in sono-chemodynamic therapy, can be attributed to their remarkable physicochemical properties. Consequently, this study aimed to organize artificially produced hematite nanospheres (α-Fe2O3 NS) that were doped with silver (Ag) and cerium (Ce) using an environmentally sustainable method utilizing taranjabin. Following this, an assessment of their cytotoxicity was conducted. The structure and morphology characteristics of nanoparticles were analyzed using powder X-ray diffraction (PXRD), Field Emission Scanning Electron Microscope (FESEM), energy dispersive X-ray(EDX), and Raman techniques. The FESEM images revealed that the α-Fe2O3 NS exhibited a spherical shape; however, the morphology of the α-Fe2O3 NS doped with Ag and Ce transitioned to a conical form. The doping of Ag and Ce within the α-Fe2O3 structure was thoroughly validated through EDX and Raman analysis. Based on the cytotoxic outcomes, the impacts of Ag and Ce-doped α-Fe2O3 against human glioblastoma(U87) and mouse embryonic fibroblast (NIH/3T3) cells were studied using MTT assay. The results indicated that doped nanoparticles exhibited greater cytotoxic than α-Fe2O3 in both NIH/3T3 and U87 cells. The IC50 values were determined to be 188.33 μg ml−1 for Ag-doped α-Fe2O3 and 308.58 μg ml−1 for Ce-doped α-Fe2O3 against U87 and NIH/3T3 cells, respectively, both of which were lower than the IC50 of α-Fe2O3. Consequently, the doping process significantly enhanced the cytotoxic effect of α-Fe2O3 NS. This study represents the first documentation of the synthesis and anticancer activity of Ag and Ce-doped α-Fe2O3 on NIH/3T3 and U87 cells. However, further investigation into the antitumor efficacy of Ag and Ce-doped α-Fe2O3 in appropriate in vivo models is recommended.

Expression of Concern: Naringin prevents Ultraviolet-B radiation-induced oxidative damage and inflammation through activation of peroxisome proliferator-activated receptor γ in mouse embryonic fibroblast (NIH-3T3) cells.

R. NilamberLal Das, S. Muruhan, R. P. Nagarajan and A. Balupillai, "Naringin Prevents Ultraviolet-B Radiation-induced Oxidative Damage and Inflammation Through Activation of Peroxisome Proliferator-activated Receptor γ in Mouse Embryonic Fibroblast (NIH-3T3) Cells," Journal of Biochemical and Molecular Toxicology 33, no. 3 (2019): e22263, https://doi.org/10.1002/jbt.22263. This Expression of Concern for the above article published online on 4 December 2018 in Wiley Online Library (wileyonlinelibrary.com), has been published by agreement between the journal Editor-in-Chief, Hari K. Bhat; and Wiley Periodicals, Inc. The Expression of Concern has been agreed following concerns raised by a third party regarding the standard deviations presented in Table 2, which are unusually low and show an unexpected even distribution. The authors admitted they made a mistake and wished to correct Table 2. However, since the new data provided could not be substantiated, the editors did not consider the data appropriate to correct the article. The editors would like to issue an Expression of Concern to alert the readers.

Fabrication of TiO2 Nanoparticle Coating on Stainless Steel 316L and Its Assessment for Orthopaedic Applications

The study aims to investigate the efficacy of titanium dioxide (TiO2) nanoparticle coating on stainless steel 316L (SS 316L) orthopaedic implants to enhance their biocompatibility, osseointegration, and durability. The TiO2 nanoparticles were synthesized via the hydrothermal method and extensively characterized for composition, crystallinity, and morphology using techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), corroborated by elemental mapping. SEM and XRD analyses revealed the synthesized nanoparticles have a spherical shape and an average size of approximately 23 nanometres. The synthesized TiO2 nanoparticles were uniformly coated on SS 316L substrates using the spin coating technique, as confirmed by SEM images. Cell viability of the synthesized TiO2 nanoparticles, as well as uncoated and TiO2 nanoparticle-coated SS 316L substrates, was evaluated using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay against the NIH-3T3 mouse embryonic fibroblast cell line. The results demonstrated that the TiO2 nanoparticle-coated SS 316L substrate showed a significant increase of 22.87% in cell viability as compared to the uncoated SS 316L substrate. A ball-on-disc tribometer was employed to assess wear and friction resistance at various speeds, viz., 150 rpm, 300 rpm, and 450 rpm, under 30N load conditions for five minutes. The results collectively indicate a substantial improvement in the performance of TiO2 nanoparticle-coated SS 316L substrates for orthopaedic applications.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress.

The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small non-catalytic subunit of the calpain 1 and 2 proteases, regulates the production of TF independent of the proteolytic activity of the larger subunits. Capn4 was later found to facilitate tyrosine phosphorylation and secretion of the lectin-binding protein galectin-3 (Gal3). In this study, recombinant Gal3 (rGal3) was added to the media-enhanced TF generated by capn4-/- mouse embryonic fibroblasts (MEFs). Extracellular Gal3 also rescued defects in the distribution, morphology, and adhesive strength of focal adhesions present in capn4-/- MEF cells. Surprisingly, extracellular Gal3 does not influence mechanosensing. c-Abl kinase was found to affect Gal3 secretion and the production of TF through phosphorylation of Y107 on Gal3. Our study also suggests that Gal3-mediated regulation of TF occurs through signaling pathways triggered by β1 integrin but not by focal adhesion kinase (FAK) Y397 autophosphorylation. Our findings provide insights into the signaling mechanism by which Capn4 and secreted Gal3 regulate cell migration through the modulation of TF distinctly independent from a mechanosensing mechanism.

Enhancement in Anticancer Activity of Chitosan Tailored Imidazole-2-Thiosemicarbazones against MCF-7 Cancer Cell Line by Coordination with Copper(II) Ions

The coordination behavior of new batch synthesized chitosan imidazole-2-thiosemicarbazones tailored from both the low molecular weight commercial chitosan and high molecular weight crab shell chitosan was confirmed by Fourier Transform Infrared (FT-IR) and magnetic susceptibility measurements. Chlorine content in the complexes was estimated by potentiometric titration technique. The study showed the tridentate NNS tridentate coordination with copper (II) ion in a square planar orientation with the remaining valence satisfied by coordination with chloride ion. The MTT assay studies showed an enhancement in the anticancer activity of these chitosan thiosemicarbazone ligands upon their coordination with copper (II) ions against the human breast cancer (MCF-7) cell line in vitro. The minimal cytotoxicity of both the ligands and complexes against the normal mouse embryonic fibroblast (NIH3T3) cells revealed the selective attraction of these biomaterial chitosan derivatives towards the cancer cells with non-toxicity to healthy cells. There was only a marginal effect of molecular weight (Mw) and degree of deacetylation (DDA) of chitosan upon the anticancer activity of these chitosan derivatives.

The role of Atg5 gene in tumorigenesis under autophagy deficiency conditions.

Autophagy is a self-recycling machinery to maintain cellular homeostasis by degrading harmful materials in the cell. Autophagy-related gene 5 (Atg5) is required for autophagosome maturation. However, the role of Atg5 in tumorigenesis under autophagy deficient conditions remains unclear. This study focused on the autophagy-independent role of Atg5 and the underlying mechanism in tumorigenesis. We demonstrated that knockout of autophagy-related genes including Atg5, Atg7, Atg9, and p62 in mouse embryonic fibroblast (MEF) cells consistently decreased cell proliferation and motility, implying that autophagy is required to maintain diverse cellular functions. An Atg7 knockout MEF (Atg7-/- MEF) cell line representing deprivation of autophagy function was used to clarify the role of Atg5 transgene in tumorigenesis. We found that Atg5-overexpressed Atg7-/-MEF (clone A) showed increased cell proliferation, colony formation, and migration under autophagy deficient conditions. Accordingly, rescuing the autophagy deficiency of clone A by overexpression of Atg7 gene shifts the role of Atg5 from pro-tumor to anti-tumor status, indicating the dual role of Atg5 in tumorigenesis. Notably, the xenograft mouse model showed that clone A of Atg5-overexpressed Atg7-/- MEF cells induced temporal tumor formation, but could not prolong further tumor growth. Finally, biomechanical analysis disclosed increased Wnt5a secretion and p-JNK expression along with decreased β-catenin expression. In summary, Atg5 functions as a tumor suppressor to protect the cell under normal conditions. In contrast, Atg5 shifts to a pro-tumor status under autophagy deprivation conditions.

Epoxyeicosatrienoic Acids Attenuate LPS-Induced NIH/3T3 Cell Fibrosis through the A2AR and PI3K/Akt Signaling Pathways.

Inflammation plays a crucial role in progression of fibrosis. Epoxyeicosatrienoic acids (EET) have multiple protective effects in different diseases, but their ability to inhibit the development of LPS-induced fibrosis remains unknown. The potential therapeutic effects of 11,12-EET were studied in in vitro model of LPS-induced fibrosis. Mouse embryonic fibroblast cells NIH/3T3 were pre-incubated with 1 μM 11,12-EET and/or a structural analogue and selective EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid before exposing to LPS. The effect of EET was evaluated by the protein and mRNA expression of NF-κB, collagens I and III, and α-smooth muscle actin by Western blotting and quantitative reverse transcription PCR, respectively. LPS provoked inflammation and fibrosis-like changes accompanied by elevated expression of NF-κB and collagens in NIH/3T3 cells. We also studied the effects of 11,12-EET on the A2AR and PI3K/Akt signaling pathways in intact and LPS-treated NIH/3T3 cells. 11,12-EET prevented inflammation and fibrosis-like changes through up-regulation of A2AR and PI3K/Akt signaling pathways. Our findings demonstrate the potential antifibrotic effects of 11,12-EET, which can be natural antagonists of tissue fibrosis.

DNMT1 driven by mouse amniotic fluid mesenchymal stem cell exosomes improved corneal cryoinjury via inducing microRNA-33 promoter DNA hypermethylation modification in corneal epithelium cells.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice.

Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress.Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-β-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.

Physicochemical and biological characterization of propolis-loaded composite polyamide-6/soybean protein nanofibers for wound healing applications

Dressings are pivotal in aiding wound recovery, yet they are burdened by significant constraints. In addition to possessing antibacterial properties, the effectiveness of a wound dressing must be rigorously assessed for its potential utility across diverse medical applications. Propolis (Pro) is widely acknowledged for its antioxidant, antibacterial, and anti-inflammatory properties, positioning it as a promising candidate for wound healing and tissue engineering applications. This study presents a novel composite nanofiber (NF) material comprising polyamide-6 (PA6) and soybean protein as matrix components, incorporating Pro as a payload. We aimed to mimic the extracellular matrix, thus promotion the proliferation and adhesion of mouse embryonic fibroblast cells (NIH3T3) on the scaffold's surface. The morphology and composition of the NFs, examined through scanning electron microscopy (SEM) imaging and Fourier-transform infrared (FTIR) spectroscopy. The Pro-loaded fibers exhibited diameters ranging from 102 to 270 nm. Notably, the NFs demonstrated a dose-dependent increase in porosity, water vapor transmission rate, and fluid uptake capacity. However, the hydrophilic categorized nanofibers (NFs) did not exhibit a concentration-dependent effect on contact angle measurements. After a 15-day period, the Pro-loaded fibers experienced degradation within the range of 39–53 %. Using UV–visible spectrophotometry, the amount of pro released was measured. The rates of release were found to be 29.93 %, 41.02 %, and 60.06 % within 24 h for PA6/Soy/Pro5 %, PA6/Soy/Pro10 %, and PA6/Soy/Pro20 % NFs, respectively. Importantly, our study showcased the significant inhibitory effects of Pro-loaded fibers on the growth of E. coli and S. aureus, with inhibition increasing proportionally with higher Pro concentrations. This observation underscores the potential of Pro-based composite NFs as effective antimicrobial agents for wound dressings and tissue scaffolds. Furthermore, the biocompatibility of the fabricated nanofibrous mats was confirmed through the MTT test, affirming the non-toxic nature of all formulations. SEM imaging further demonstrated the optimal adhesion of cells on the NFs, confirming the findings of the MTT test and highlighting the suitability of Pro-loaded NFs for cell attachment and proliferation.

Synthesis and anticancer activity evaluation of some novel imidazo[1,2-a]pyridine based heterocycles containing S-alkyl/aryl moiety

Here we report the synthesis and anticancer activities of fourteen novel imidazo[1,2-a]pyridine derivatives (6a-6n) containing S-alkyl/aryl moiety. The target compounds were obtained via a six-step synthetic route. 1-(4-Aminophenyl)ethan-1-one was the starting material. In the last step, 2-chloro-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]acetamide (5) was allowed to react with various aliphatic/aromatic thiol derivatives, affording the final compounds 6a-6n. The cytotoxicity of the compounds was evaluated against A549 (human non-small cell lung cancer), C6 (rat glioma), MCF-7 (human breast carcinoma) and HepG2 (human liver carcinoma) tumor cells and NIH/3T3 (mouse embryonic fibroblast cells) healthy cells. First step MTT assay reported that compounds 6a, 6d, 6e and 6i exhibited antiproliferative activity against all tested tumor lines. Second step BrdU cell proliferation assay and flow cytometric analysis revealed that compounds 6d and 6i inhibited DNA synthesis on HepG2 cell line time-dependently by apoptotic pathway. Molecular docking study of compounds 6d and 6i with caspase-3 and caspase-9 revealed their binding interactions with the enzyme’s active site, confirming the experimental findings.

Methylene blue treatment with zero-valent iron/pyrite/H2O2 system under static and continuous flow conditions: Reaction mechanism and toxicity evaluation

Laboratory batch and continuous flow studies, coupled with surface and toxicity analysis, were performed to evaluate methylene blue treatment by heterogeneous Fenton process using zero-valent iron (ZVI) and pyrite as co-catalyst. The use of pyrite in batch reactors significantly enhanced methylene blue treatment by the ZVI/H2O2 system because of improved iron redox cycling. The continuous-flow experiments revealed that the reactor performance increased in the order of: ZVI/H2O2 < pyrite/H2O2 < ZVI/pyrite/H2O2 under dynamic flow conditions. The methylene blue treatment by the ternary ZVI/pyrite/H2O2 system was described by an initial degradation of methylene blue with *OH radicals, followed by the adsorption and/or co-precipitation of degradation intermediates with some spherical particles. The surface analysis showed that these spherical particles formed, even at pH less than 4. The genotoxicity and cytotoxicity tests performed on mouse embryonic fibroblast (3 T3-L1) and human embryonic kidney (HEK293) cell lines showed that the Fenton treatment of methylene blue using the ZVI/pyrite/H2O2 system resulted in the formation of degradation species with much lower toxicity levels relative to methylene blue. Moreover, the Fenton degradation species of methylene blue significantly enhanced the metabolic activity of several bacterial strains, including E. coli ATCC 8739 and P. aeruginosa PAO1.

The Metabolic and Lipidomic Fingerprint of Torin1 Exposure in Mouse Embryonic Fibroblasts Using Untargeted Metabolomics.

Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5'-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators.

Synthesis and evaluation of tryptanthrin derivatives as promising anticancer agents: In vitro, in silico, and SAR insights

In this study, with the aim of identifying new anticancer agents, we synthesized a series of tryptanthrin derivatives. All the synthesized compounds were investigated for their anticancer potential in-vitro, using an MTT-based assay against a panel of three different cancer cell lines (A-549, MCF-7, and MDA-MB-231). Amongst the synthesized derivatives, compound 3 showed excellent anticancer activity, with GI50 values of 5.03 ± 0.77 µM, 3.76 ± 0.55 µM, and 4.20 ± 0.50 µM against A-549, MDA-MB-231 and MCF-7 cells, respectively. Furthermore, these molecules did not show any cytotoxic effect against normal mouse embryonic fibroblast (NIH/3T3) cell lines suggesting no intrinsic cytotoxicity. In this study, ADME and toxicity profiles of the synthesized compounds were also predicted using in silico techniques. According to the findings, all of the synthesized derivatives are nontoxic and non-carcinogenic in the biological system and showed good ADME properties. Finally, potent compounds were subjected to in silico molecular docking and simulation studies were carried out against maternal embryonic leucine zipper kinase (MELK) to uncover potential protein-ligand interactions and affinity at the binding site. This study will help in the future development of novel anticancer agents.