Deregulated JNK signaling enhances apoptosis during hyperthermia.

PURPOSE: c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. METHODS AND MATERIALS: The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. RESULTS: Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1-SEK1-JNK pathway and apoptosis. CONCLUSION: JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1-SEK1-JNK pathway and subsequent apoptosis.

Cytotoxic, acute oral toxicity, genotoxic and mutagenic assessment of the essential oil from fresh leaves of Croton argyrophyllus (Kunth.).

ETHNOPHARMACOLOGICAL RELEVANCE: Croton argyrophyllus Kunth., commonly known as "marmeleiro" or "cassetinga," is widely distributed in the Brazilian Northeast region. Its leaves and flowers are used in traditional medicine as tranquilizers to treat flu and headaches. AIM OF THE STUDY: This study was conducted to determine the chemical composition and toxicological safety of essential oil from C. argyrophyllus leaves using in vitro and in vivo models. MATERIALS AND METHODS: The chemical composition of the essential oil was determined using a gas chromatograph coupled to a mass spectrometer. Cytotoxicity was tested in the HeLa, HT-29, and MCF-7 cell lines derived from human cells (Homo sapiens) and Vero cell lines derived from monkeys (Cercopithecus aethiops) using the MTT method. Acute toxicity, genotoxicity. Mutagenicity tests were performed in Swiss mice (Mus musculus), which were administered essential oil orally in a single dose of 2000 mg/kg by gavage. RESULTS: The main components of the essential oil were p-mentha-2-en-1-ol, α-terpineol, ß-caryophyllene, and ß-elemene. The essential oil exhibited more than 90% cytotoxicity in all cell lines tested. No deaths or behavioral, hematological, or biochemical changes were observed in mice, revealing no acute toxicity. In genotoxic and mutagenic analyses, there was no increase in micronuclei in polychromatic erythrocytes or in the damage and index in the comet assay. CONCLUSIONS: The essential oil was cytotoxic towards the tested cell lines but did not exert toxic effects or promote DNA damage when administered orally at a single dose of 2000 mg/kg in mice.

Comparative analysis of chemical profiles, antioxidant, antibacterial, and anticancer effects of essential oils of two Thymus species from Montenegro.

The essential oils of Thymus vulgaris (TVEO) and Thymus serpyllum (TSEO) show different biological activities. The aim of the study was to evaluate the biological activities of TVEO and TSEO from Montenegro. The main components of TVEO were p-cymene (29.52%), thymol (22.8%) and linalool (4.73%) while the main components of TSEO were p-cymene (19.04%), geraniol (11,09%), linalool (9.16%), geranyl acetate (6.49%) and borneol (5.24%). Antioxidant activity determined via DPPH for TVEO was 4.49 and FRAP 1130.27, while for TSEO it was estimated that DPPH was 4.88 µL/mL and FRAP was 701.25 µmol FRAP/L. Both essential oils were active against all tested bacteria, with the highest level of sensitivity of E. coli with MIC of 1.5625 µL/mL. Essential oils showed strong cytotoxic effects on human cancer cell lines, with IC50 values ranging from 0.20 to 0.24 µL/mL for TVEO and from 0.32 to 0.49 µL/mL for TSEO. TVEO caused apoptosis in cervical adenocarcinoma HeLa cells through activation of caspase-3 and caspase-8, while TSEO caused apoptosis through caspase-3. EOs decreased levels of oxidative stress in normal MRC-5 cells. HeLa cells treated with TVEO had reduced MMP2 expression levels, while cells treated with TSEO had lowered MMP2 and MMP9 levels. The treatment of HeLa cells with TVEO increased the levels of miR-16 and miR-34a, indicating potential tumor-suppressive properties. Our findings suggest that Thymus essential oils may be considered as good candidates for further investigation as cancer-chemopreventive and cancer-therapeutic agents.

In vitro anticancer activity of Hirudinaria manillensis methanolic extract and its validation using in silico molecular docking approach.

Cancer has emerged as a potentially lethal illness, which recently upsurged in the mortality rate. Animal-derived compounds could be promising targets with higher efficacy and low toxicity in anticancer therapy. The present study aimed to explore the presence of anticancer potential compounds in Hirudinaria manillensis methanolic extract and their anticancer potential against various cancer cell types and target identification by Auto dock method. Initially, the identification of bioactive compounds was achieved by GC-MS analysis followed by the anticancer activity by MTT assay against A549, HeLa, MDA-MB-231, MG-63, and MOLT-4. Further, the effect of a lead compound on the cancer cell target was analyzed by the Auto dock method. GC-MS analysis results revealed the presence of 25 different bioactive compounds including anticancer potential compounds, such as Lupeol, Carvacrol, and Demecolcine. Interestingly, MTT assay results demonstrated the anticancer potential of Hirudinaria manillensis extract (LE) against various cancer cell lines, such as A549 (54.60 µg/ml), HeLa (19.93 µg/ml), MDA-MB-231 (20.23 µg/ml), MG-63 (20.04 µg/ml), and MOLT-4 (171.8 µg/ml), respectively. Among these cell types, the maximum inhibition was observed against HeLa with the IC50 concentration of 19.93 µg/ml. Furthermore, Demecolcine compound was docked with the EGFR tyrosine kinase showed the binding affinity of the docked complex was predicted to be - 6.2 kcal/mol. Thus, we conclude that H. manillensis has a significant anticancer effect on human cancer cell lines and could be used as a natural target which paves the way for further studies on biomedical applications in cancer therapeutics.

Preparation, characterization and cytotoxicity assessment of a novel selenized polysaccharide from Morchella sextelata.

Selenylation modification has been widely developed to improve the biological effects of natural polysaccharides. In this study, a purified new polysaccharide (MSP-4) was isolated from Morchella Sextelata, and selenized into SeMSP-4 using the HNO3-Na2SeO3 method. The selenium (Se) content of SeMSP-4 was 101.81 ± 9.90 mg/kg, and the molecular weight of SeMSP-4 was 1.23 × 105 Da. The FT-IR, XRD and AFM results showed that MSP-4 was successfully combined with the Se element. The structure characters of SeMSP-4 were analyzed by methylation analysis combined with 1D and 2D NMR spectroscopy. And, the radical scavenging test revealed that SeMSP-4 exhibited higher antioxidant capacities in vitro than MSP-4. The cytotoxicity analysis indicated that SeMSP-4 could dose-dependently inhibit the proliferation of HepG2 and HeLa cells, but did not show a cytotoxic effect on normal cells (HEK293). Furthermore, SeMSP-4 stimulation significantly increased the macrophage viability and enhanced NO production in macrophage cells. This study suggested that SeMSP-4 could be utilized as a potential selenium source with antioxidant, antitumor, and immunostimulatory activities.

Red blood cell membrane-camouflaged gold-core silica shell nanorods for cancer drug delivery and photothermal therapy.

Gold core mesoporous silica shell (AuMSS) nanorods are multifunctional nanomedicines that can act simultaneously as photothermal, drug delivery, and bioimaging agents. Nevertheless, it is reported that once administrated, nanoparticles can be coated with blood proteins, forming a protein corona, that directly impacts on nanomedicines' circulation time, biodistribution, and therapeutic performance. Therefore, it become crucial to develop novel alternatives to improve nanoparticles' half-life in the bloodstream. In this work, Polyethylenimine (PEI) and Red blood cells (RBC)-derived membranes were combined for the first time to functionalize AuMSS nanorods and simultaneously load acridine orange (AO). The obtained results revealed that the RBC-derived membranes promoted the neutralization of the AuMSS' surface charge and consequently improved the colloidal stability and biocompatibility of the nanocarriers. Indeed, the in vitro data revealed that PEI/RBC-derived membranes' functionalization also improved the nanoparticles' cellular internalization and was capable of mitigating the hemolytic effects of AuMSS and AuMSS/PEI nanorods. In turn, the combinatorial chemo-photothermal therapy mediated by AuMSS/PEI/RBC_AO nanorods was able to completely eliminate HeLa cells, contrasting with the less efficient standalone therapies. Such data reinforce the potential of AuMSS nanomaterials to act simultaneously as photothermal and chemotherapeutic agents.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Combining cisplatin with Pinellia pedatisecta Schott lipid-soluble extract induces tumor immunogenic cell death in cervical cancer.

BACKGROUND: Pinellia pedatisecta Schott extract (PE) is extracted from Pinellia pedatisecta Schott (PPS), a traditional Chinese medicinal plant with the potential for direct anticancer effects or eliciting an anti-tumor response by activating the immune system. PURPOSE: To explore PE's ability and mechanism to reconstruct cisplatin's immunogenicity. METHODS: Cervical cancer cells were treated with cisplatin (CDDP) and/or PE. The exposure of calreticulin (CRT) on cell membrane was investigated by flow cytometry. The extracellular of ATP and HMGB1 was investigated by Western blot analysis, immunofluorescence and ELISA assay. Changes in immune profiles were using flow cytometry in vaccination and anti-tumor assays in vivo. Lastly, the mechanism of PE influenced the ROS/ERS pathway was examined by ROS assay kit, flow cytometry and Western blotting. RESULTS: PE treatment induced translocation of CRT from the endoplasmic reticulum to the cell membrane of tumor cells, concomitantly triggering immunogenic cell death (ICD). In terms of mechanisms, endoplasmic reticulum (ER) stress relievers could impede the ability of PE to induce immunogenicity. This indicates that PE is activated by ER stress, leading to subsequent induction of ICD. Upon analyzing RNA-seq data, it was observed that PE primarily induces programmed cell death in tumors by impeding upstream antioxidant mechanisms. Additionally, it transforms dying tumor cells into vaccines, activating a series of immune responses. CONCLUSIONS: This study observed for the first time that PE-induced CRT exposure on the membrane of cervical cancer cells compensates for the defect of nonimmunogenic cell death inducer CDDP thereby stimulating potent ICD. This ability restores the immunogenicity of CDDP through ER stress induced by the ROS signal. ROS played a role in PE's ability to induce ICD, leading to increased expression of ER stress-related proteins, including ATF3 and IRE-1α. PE exerted anti-cancer effects by increasing the ROS levels, and ROS/ERS signaling may be a potential avenue for cervical cancer treatment. Hence, the synergistic use of PE and CDDP holds potential for enhancing immunochemotherapy in cancer treatment.

Comprehensive Analysis of 11 Species of Euodia (Rutaceae) by Untargeted LC-IT-TOF/MS Metabolomics and In Vitro Functional Methods.

The Euodia genus comprises numerous untapped medicinal plants that warrant thorough evaluation for their potential as valuable natural sources of herbal medicine or food flavorings. In this study, untargeted metabolomics and in vitro functional methods were employed to analyze fruit extracts from 11 significant species of the Euodia genus. An investigation of the distribution of metabolites (quinolone and indole quinazoline alkaloids) in these species indicated that E. rutaecarpa (Euodia rutaecarpa) was the most widely distributed species, followed by E. compacta (Euodia compacta), E. glabrifolia (Euodia glabrifolia), E. austrosinensis (Euodia austrosinensis), and E. fargesii (Euodia fargesii). There have been reports on the close correlation between indole quinazoline alkaloids and their anti-tumor activity, especially in E. rutaecarpa fruits which exhibit effectiveness against various types of cancer, such as SGC-7901, Hela, A549, and other cancer cell lines. Additionally, the E. rutaecarpa plant contains indole quinazoline alkaloids, which possess remarkable antibacterial properties. Our results offer novel insights into the utilization of Euodia resources in the pharmaceutical industry.

Theoretical evaluation of the impact of diverse treatment conditions by calculation of the tumor control probability (TCP) of simulated cervical cancer Hyperthermia-Radiotherapy (HT-RT) treatments in-silico.

INTRODUCTION: Hyperthermia (HT) induces various cellular biological processes, such as repair impairment and direct HT cell killing. In this context, in-silico biophysical models that translate deviations in the treatment conditions into clinical outcome variations may be used to study the extent of such processes and their influence on combined hyperthermia plus radiotherapy (HT + RT) treatments under varying conditions. METHODS: An extended linear-quadratic model calibrated for SiHa and HeLa cell lines (cervical cancer) was used to theoretically study the impact of varying HT treatment conditions on radiosensitization and direct HT cell killing effect. Simulated patients were generated to compute the Tumor Control Probability (TCP) under different HT conditions (number of HT sessions, temperature and time interval), which were randomly selected within margins based on reported patient data. RESULTS: Under the studied conditions, model-based simulations suggested a treatment improvement with a total CEM43 thermal dose of approximately 10 min. Additionally, for a given thermal dose, TCP increased with the number of HT sessions. Furthermore, in the simulations, we showed that the TCP dependence on the temperature/time interval is more correlated with the mean value than with the minimum/maximum value and that comparing the treatment outcome with the mean temperature can be an excellent strategy for studying the time interval effect. CONCLUSION: The use of thermoradiobiological models allows us to theoretically study the impact of varying thermal conditions on HT + RT treatment outcomes. This approach can be used to optimize HT treatments, design clinical trials, and interpret patient data.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Rutin ameliorated lipid metabolism dysfunction of diabetic NAFLD via AMPK/SREBP1 pathway.

BACKGROUND: In diabetic liver injury, nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease. Rutin is a bioflavonoid produced by the hydrolysis of glucosidases to quercetin. Its biological activities include lowering blood glucose, regulating insulin secretion, regulating dyslipidemia, and exerting anti-inflammatory effects have been demonstrated. However, its effect on diabetic NAFLD is rarely reported. PURPOSE: Our study aimed to investigate the protective effects of Rutin on diabetic NAFLD and potential pharmacological mechanism. METHODS: We used db/db mice as the animal model to investigate diabetic NAFLD. Oleic acid-treated (OA) HeLa cells were examined whether Rutin had the ability to ameliorate lipid accumulation. HepG2 cells treated with 30 mM/l d-glucose and palmitic acid (PA) were used as diabetic NAFLD in vitro models. Total cholesterol (TC) and Triglycerides (TG) levels were determined. Oil red O staining and BODIPY 493/503 were used to detect lipid deposition within cells. The indicators of inflammation and oxidative stress were detected. The mechanism of Rutin in diabetic liver injury with NAFLD was analyzed using RNA-sequence and 16S rRNA, and the expression of fat-synthesizing proteins in the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway was investigated. Compound C inhibitors were used to further verify the relationship between AMPK and Rutin in diabetic NAFLD. RESULTS: Rutin ameliorated lipid accumulation in OA-treated HeLa. In in vitro and in vivo models of diabetic NAFLD, Rutin alleviated lipid accumulation, inflammation, and oxidative stress. 16S analysis showed that Rutin could reduce gut microbiota dysregulation, such as the ratio of Firmicutes to Bacteroidetes. RNA-seq showed that the significantly differentially genes were mainly related to liver lipid metabolism. And the ameliorating effect of Rutin on diabetic NAFLD was through AMPK/SREBP1 pathway and the related lipid synthesis proteins was involved in this process. CONCLUSION: Rutin ameliorated diabetic NAFLD by activating the AMPK pathway and Rutin might be a potential new drug ingredient for diabetic NAFLD.

Liquid chromatography-tandem mass spectrometry profile and antioxidant, antimicrobial, antiproliferative, and enzyme activities of Thymus pectinatus and Thymus convolutus: in vitro and in silico approach.

BACKGROUND: The objective of this study is to investigate the antiproliferative, antioxidant, antimicrobial, and enzyme activity capacities and phytochemical compositions of Thymus pectinatus (TP), Thymus convolutus (TC), which are endemic to Türkiye. Quantitative analysis of phenolic compounds in the extracts was conducted using liquid chromatography-tandem mass spectrometry, targeting 53 phenolic compounds. RESULTS: Rosmarinic acid, quinic acid, and cynaroside were identified as the major compounds, exhibiting quantitative variation in both extracts. The extracts had a high total phenolic content, with 113.57 ± 0.58 mg gallic acid equivalents (GAE)/g extract for TP and 130.52 ± 1.05 mg GAE/g extract for TC. Furthermore, although both extracts exhibited high total flavonoid content; the TP extract (75.12 ± 1.65 mg quercitin equivalents (QE)/g extract) displayed a higher flavonoid content than the TC extract (30.24 ± 0.74 mg QE/g extract) did. The extracts had a promising antiproliferative effect on C6, HeLa, and HT29 cancer cell lines with a less cytotoxic effect (10.5-14.2%) against normal cells. Both extracts exhibited very potent inhibitory activity against the xanthine oxidase enzyme, with half-maximal inhibitory concentration values of respectively 2.07 ± 0.03 µg mL-1 and 2.76 ± 0.06 µg mL-1 and moderate activity against tyrosinase and α-glucosidase. Docking simulations proved that rosmarinic acid and cynaroside, the major components of the extracts, were the most potent inhibitors of xanthine oxidase. According to antimicrobial activity results, the TC extract exhibited moderate activity against Staphylococcus aureus, and the TP extract had strong activity against both Enterococcus faecium and S. aureus. CONCLUSION: These findings emphasize the beneficial effects of the two endemic Thymus species on human health and suggest their potential use as plant-derived bioactive agents. © 2024 Society of Chemical Industry.

Investigation of in-vitro Anti-Cancer and Apoptotic Potential of Garlic-Derived Nanovesicles against Prostate and Cervical Cancer Cell Lines.

OBJECTIVE: Investigate the anti-cancerous potential of garlic-derived nanovesicles (GDNVs), exploring their cytotoxic effects on HeLa and PC-3 cell lines, and elucidate the underlying mechanisms, including apoptosis induction and inhibition of epithelial-mesenchymal transition (EMT). METHODS: GDNVs were isolated using differential centrifugation and ultracentrifugation. Characterization was performed through dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM), and Fourier-transform infrared spectroscopy (FTIR). Cytotoxicity assessments on HeLa and PC-3 cell lines using MTT assay. Apoptosis induction was evaluated through nuclear morphology changes and quantification of apoptotic cells using DAPI and PI/annexin V analysis. Western blot of apoptosis-related proteins (bcl-2, bax, caspase-3) was analysed. Anti-metastatic potential was assessed using wound healing assay and EMT transition inhibition. RESULTS: Garlic-derived nanovesicles (GDNVs), characterized by a size of 134.2 nm, demonstrated a substantial and dose- as well as time-dependent anti-proliferative impact on HeLa and PC-3 cell lines. The induction of apoptosis was unequivocally established through discernible modifications in nuclear morphology. The apoptotic cell count in HeLa and PC-3 cells increased by 42.4 ± 4.2% and 38.2 ± 3.2%, respectively. Comprehensive Western blot demonstrated alterations in the expression of key apoptotic regulators, namely bcl-2, bax, and caspase-3, providing robust evidence for the initiation of apoptosis. Furthermore, GDNVs exerted a significant inhibitory effect (p < 0.001) on the migratory potential of both HeLa and PC-3 cells. Moreover, there was a discernible association between GDNVs and the suppression of Epithelial-Mesenchymal Transition (EMT), emphasizing their role in impeding the metastatic potential of these cancer cell lines. CONCLUSION: This study establishes, for the first time, the anti-cancerous potential of GDNVs. The observed dose- and time-dependent anti-proliferative effects, selective cytotoxicity, apoptosis induction, and anti-migratory potential highlight GDNVs as a promising candidate for cancer treatment.

Kinetics of Cellular Cobalamin Uptake and Conversion: Comparison of Aquo/Hydroxocobalamin to Cyanocobalamin.

Cyanocobalamin (CNCbl) and aquo/hydroxocobalamin (HOCbl) are the forms of vitamin B12 that are most commonly used for supplementation. They are both converted to methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), which metabolize homocysteine and methylmalonic acid, respectively. Here, we compare the kinetics of uptake and the intracellular transformations of radiolabeled CNCbl vs. HOCbl in HeLa cells. More HOCbl was accumulated over 4-48 h, but further extrapolation indicated similar uptake (>90%) for both vitamin forms. The initially synthesized coenzyme was MeCbl, which noticeably exceeded AdoCbl during 48 h. Yet, the synthesis of AdoCbl accelerated, and the predicted final levels of Cbls were MeCbl ≈ AdoCbl ≈ 40% and HOCbl ≈ 20%. The designed kinetic model revealed the same patterns of the uptake and turnover for CNCbl and HOCbl, apart from two steps. First, the "activating" intracellular processing of the internalized HOCbl was six-fold faster. Second, the detachment rates from the cell surface (when the "excessive" Cbl-molecules were refluxed into the external medium) related as 4:1 for CNCbl vs. HOCbl. This gave a two-fold faster cellular accumulation and processing of HOCbl vs. CNCbl. In medical terms, our data suggest (i) an earlier response to the treatment of Cbl-deficiency with HOCbl, and (ii) the manifestation of a successful treatment initially as a decrease in homocysteine.

Nitrate reductase involves in selenite reduction in Rahnella aquatilis HX2 and the characterization and anticancer activity of the biogenic selenium nanoparticles.

BACKGROUND: Biogenic selenium nanoparticles (SeNPs) show numerous advantages including their high stability, low toxicity, and high bioactivity. While metabolism of SeNPs remains not well studied and need more investigation to reveal the process. PURPOSE: The objective of the study was to investigate the relationship between nitrate reductase and selenite reduction in Rahnella aquatilis HX2, characterize the properties of HX2 produced SeNPs, and explore their potential applications, particularly their anticancer activity. PROCEDURES: Selenium species were measured by high-performance liquid chromatography coupled to inductively coupled plasma - Mass spectrometry (HPLC-ICP-MS). Transcription level of nitrate reductase was determined by Real-time quantitative PCR. Morphology, particle size, crystal structure and surface chemistry of SeNPs were determined by electron microscopy, dynamic light scattering method, Raman scattering, X-ray photoelectron spectroscopy, respectively. Anti cancer cell activity was measured by CCK-8 assay. MAIN FINDINGS: SeNP production in R. aquatilis HX2 was correlated with the cell growth. The products of selenite reduction in HX2 detected by HPLC-ICP-MS included SeNPs, selenocysteine (SeCys), Se-Methylselenocysteine (MeSeCys), and 7 unknown compounds. Nitrate addition experiments suggested the involvement of nitrate reductase in selenite reduction in HX2. Both the cellular membrane and cytoplasm of HX2 exhibited selenite-reducing ability, indicating that membrane-associated nitrate reductase was not the sole selenite reductase in HX2. Characterization of the biogenic SeNPs revealed a spherical morphology and amorphous structure of them. Surface chemistry analysis implicated the binding of extracellular polymeric substances to the biogenic SeNPs, and the presence of Se0, Se2-, and electron-rich Se atoms on the surface of SeNPs. Finally, the IC50 values of the biogenic SeNPs were 36.49 µM for HepG2 and 3.70 µM for HeLa cells. CONCLUSIONS: The study first revealed that the nitrate reductase is involving in selenite reduction in R. aquatilis HX2. The biogenic SeNPs coordinated with organic substances in the surface. And SeNPs produced by R. aquatilis HX2 showed excellent anticancer activities on HepG2 and HeLa cells.

Exploring In-Silico, In-Vitro Antioxidant, and Cytotoxic Potential of Valerian wallichii by On Cervical Epithelial Carcinoma (HeLa) Cell Lines.

Traditional medicinal practices often utilize herbal remedies for treating various diseases. This study focuses on exploring the phytochemical constituents, in-silico, in-vitro antioxidant, and anticancer activities of Valerian wallichii root extracts on human cervical epithelial carcinoma (HeLa) cell lines. The molecular docking approach was employed to predict the ligand molecule's orientation within the receptor like Epidermal growth factor receptor tyrosine kinase domain (PDB ID: 1M17) using Schrodinger's GLIDE model. Among the selected phytocompounds, hesperidin exhibited promising inhibitory activity against EGFR (Epidermal Growth Factor Receptor) domain with -8.701 kcal/mol docking score and interactions with MET 769, ASP 831, ASP776, LEU694 and ASN818 residues as compared to standard doxorubicin with -7.6 kcal/mol docking score and interactions with ASP 831, ASN818 and ASP776 residues and further, various antioxidant activity was assessed and In-vitro anticancer activity against HeLa cell lines was evaluated by hydroalcoholic root extracts demonstrated antioxidant capacities, and selective cytotoxicity was observed, with IC50 : 45.759±0.42 µg/mL for the overall extract and IC50 : 30.245±0.58 µg/mL for the EAF fraction as compared to standard doxorubicin with IC50 : 25.9891±0.25 µg/mL, respectively. The present study concluded that Valerian wallichii L possesses potential human cervical epithelial carcinoma cell line inhibition properties based on the computer aided drug design models and in-vitro activity.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

Phytofabricated bimetallic synthesis of silver-copper nanoparticles using Aerva lanata extract to evaluate their potential cytotoxic and antimicrobial activities.

In this study, we demonstrate the green synthesis of bimetallic silver-copper nanoparticles (Ag-Cu NPs) using Aerva lanata plant extract. These NPs possess diverse biological properties, including in vitro antioxidant, antibiofilm, and cytotoxic activities. The synthesis involves the reduction of silver nitrate and copper oxide salts mediated by the plant extract, resulting in the formation of crystalline Ag-Cu NPs with a face-centered cubic structure. Characterization techniques confirm the presence of functional groups from the plant extract, acting as stabilizing and reducing agents. The synthesized NPs exhibit uniform-sized spherical morphology ranging from 7 to 12 nm. They demonstrate significant antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, inhibiting extracellular polysaccharide secretion in a dose-dependent manner. The Ag-Cu NPs also exhibit potent cytotoxic activity against cancerous HeLa cell lines, with an inhibitory concentration (IC50) of 17.63 µg mL-1. Additionally, they demonstrate strong antioxidant potential, including reducing capability and H2O2 radical scavenging activity, particularly at high concentrations (240 µg mL-1). Overall, these results emphasize the potential of A. lanata plant metabolite-driven NPs as effective agents against infectious diseases and cancer.

Clerodendrum chinense Stem Extract and Nanoparticles: Effects on Proliferation, Colony Formation, Apoptosis Induction, Cell Cycle Arrest, and Mitochondrial Membrane Potential in Human Breast Adenocarcinoma Breast Cancer Cells.

Breast cancer stands out as the most widespread form of cancer globally. In this study, the anticancer activities of Clerodendrum chinense (C. chinense) stem ethanolic extract were investigated. High-performance liquid chromatography (HPLC) analysis identified verbascoside and isoverbascoside as the major bioactive compounds in the C. chinense stem extract. Successfully developed nanoparticles exhibited favorable hydrodynamic diameter, polydispersity index, and surface charge, thus ensuring stability after four months of storage. The total phenolic content and total flavonoid contents in the nanoparticles were reported as 88.62% and 95.26%, respectively. The C. chinense stem extract demonstrated a dose-dependent inhibitory effect on MCF-7, HeLa, A549, and SKOV-3 cancer cell lines, with IC50 values of 109.2, 155.6, 206.9, and 423 µg/mL, respectively. C. chinense extract and NPs exhibited dose-dependent cytotoxicity and the highest selectivity index values against MCF-7 cells. A dose-dependent reduction in the colony formation of MCF-7 cells was observed following treatment with the extract and nanoparticles. The extract induced cytotoxicity in MCF-7 cells through apoptosis and necrosis. C. chinense stem extract and nanoparticles decreased mitochondrial membrane potential (MMP) and induced G0/G1 phase arrest in MCF-7 cells. In conclusion, use of C. chinense stem extract and nanoparticles may serve as a potential therapeutic approach for breast cancer, thus warranting further exploration.