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Background: Apoptotic agents from natural products like phenolic compounds can be used effectively in the treatment of cancer. Chlorogenic acid (CGA) is one of the phenolic compounds in medicinal plants with anti-cancer properties. In this research, we aimed to explore the anti-cancer mode of action of CGA on colorectal cancer (CRC) cells in vitro conditions. Methods: HT-29 and HEK-293 cells were cultured after MTT assay for 24 h with CGA 100 µM, and without CGA. Then, flow cytometry assays and the expression of apoptosis-related genes including caspase 3 and 9, Bcl-2 and Bax, and cell cycle-related genes including P21, P53 and NF-κB at mRNA and protein levels were examined. Finally, we measured the amount of intracellular reactive oxygen species (ROS). Results: The cell viability of all two-cell lines decreased in a dose-dependent manner. Moreover, CGA induces cell cycle arrest in HT-29 cells by increasing the expression of P21 and P53. It also induces apoptosis in HT-29 cells by mitigating Bcl-2 and NF-κB expression and elevating caspase 3 and 9 expression and ROS levels. Conclusions: Considering the cytotoxicity and cell cycle arrest and induction of apoptosis in the colon cancer cell line by CGA, it can be concluded that CGA is a suitable option for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-β-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD. [ABSTRACT FROM AUTHOR]

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2 NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2 NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2 NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2 NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2 NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2 NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2, indicating that toxicity is partially driven via non-oxidative pathways. TiO2 NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions. [ABSTRACT FROM AUTHOR]

Despite the significant advancements in complex anticancer therapy, the search for new and more efficient specific anticancer agents remains a top priority in the field of drug discovery and development. Here, based on the structure-activity relationships (SARs) of eleven salicylaldehyde hydrazones with anticancer activities, we designed three novel derivatives. The compounds were tested in silico for drug-likeness, synthesized, and evaluated in vitro for anticancer activity and selectivity on four leukemic cell lines (HL-60, KE-37, K-562, and BV-173), one osteosarcomic cell line (SaOS-2), two breast adenocarcinomic cell lines (MCF-7 and MDA-MB-231), and one healthy cell line (HEK-293). The designed compounds were found to have appropriate drug likeness and showed anticancer activities in all cell lines tested; particularly, two of them exhibited remarkable anticancer activity in nanomolar concentrations on the leukemic cell lines HL-60 and K-562 and the breast cancer MCF-7 cells and extraordinary selectivity for the same cancer lines ranging between 164- and 1254-fold. The study also examined the effects of different substituents on the hydrazone scaffold and found that the 4-methoxy salicylic moiety, phenyl, and pyridinyl rings are the most appropriate for anticancer activity and selectivity of this chemical class. [ABSTRACT FROM AUTHOR]

Background: Exacum lawii C.B. Clarke (Gentianaceae) bitter medicinal herb, has been extensively applied for the treatment of kidney diseases and ailments by Indigenous medical practitioners and local communities in Maharashtra and Karnataka states of India. The pharmacognostical and phytochemical standardization has been performed, but its therapeutic mechanism is still unknown. Swertiamerin, a secoiridoid glycoside established as marker component. The aim of the current research is to investigate the cytoprotective ability and examine targets of action of Exacum lawii standardised extract against cisplatin exposed Human embryonic kidney cell line (HEK-293). Methods: Swertiamerin was isolated from standardised extract using column chromatography and characterized by various analytical techniques. In-vitro cellular assay (MTT cytotoxicity assay, iNOS expression, proinflammatory cytokine level, ROS estimation and cell cycle progression, DNA fragmentation assay and morphology of cells) were employed to understand the efficacy and mechanism of action of standardised extract and swertiamerin against cisplatin induced toxicity in HEK-293 cells. Results: Extract and swertiamerin summed up to be nontoxic to HEK-293 cells and the IC50 for cisplatin was 2.261 µg/ml. Molecular docking study showed the binding capacity of swertiamerin for iNOS enzyme (responsible for the nitrosative stress in cells). Various Scientific techniques validates the potential of extract and swertiamerin to reduce the nitrosative stress, oxidative stress, and inflammation in HEK-293 cells significantly (P

In this study, firstly, novel vitamin K3 analogs were synthesized by the reactions of vitamin K3 (2-methyl-1,4-naphthoquinone, also known as menadione) with some aromatic and heterocyclic ring substituted nucleophiles such as 2,4-dimethoxyaniline, 4-methoxyaniline, 4-benzylpiperidine and 1-(2-aminoethyl)piperazine in ethanol/Na2CO3, and 1-(diphenylmethyl)piperazine in chloroform/triethylamine (TEA) at room temperature. Their structures were elucidated by Fourier transform infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H NMR), attached proton test nuclear magnetic resonance (APT-NMR) and mass spectrometry (MS). Secondly, in vitro cytotoxic effects of vitamin K3 analogs were investigated by MTT assay against three cancer cell lines (HeLa, U87 MG, and MCF-7) to evaluate their anticancer activity and a human embryonic kidney cell line (HEK-293) to check their cancer cell selectivity. One of the compounds, namely 2-((2,4-dimethoxyphenyl)amino)-3-methylnaphthalene-1,4-dione(5), was found to inhibit the growth of HeLa cervical cancer cells selectively, even better than vitamin K3, at a non-toxic concentration for healthy cells. The selectivity index of this compound for HeLa cells was calculated approximately as "3". Vitamin K3 was more effective against U87 MG and MCF-7 cells than its derivatives, moreover it was the only compound, which was significantly toxic to breast cancer cells, but its selectivity was poor. Furthermore, anticancer properties of piperazine derivatives of vitamin K3 were investigated by us for the first time in this study. [ABSTRACT FROM AUTHOR]

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[Display omitted] The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders. [ABSTRACT FROM AUTHOR]

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

Ribwort plantain (Plantago lanceolata L.), which belongs to the Plantaginaceae family, has been widely used as a herbal plant in traditional medicine across the globe. The present study aimed to investigate the biologically active substances of P. lanceolata root fractions, as well as the cytotoxic and antibacterial activities of extracts. The cytotoxic activity of ethyl acetate, dichloromethane, and n-butanol extracts of P. lanceolata root was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The P.lanceolata root extracts were also evaluated on gram-positive and negative bacteria by disc diffusion and microtiter broth dilution methods. The phytochemical content was also examined by gas chromatography-mass spectrometry. The P.lanceolata root extracts were cytotoxic; IC50 values against HCT-116 at 72 h were 168.553 μg/mL, 167.458 μg/mL, and 205.004 μg/mL for ethyl acetate, dichloromethane, and n-butanol root extracts, respectively. The dichloromethane extract of P. lanceolata root had the highest inhibitory effect against S. paratyphi (14.00±1.0 mm) at the concentration of 100 mg/mL. The minimum MIC and MBC (5 and 15 mg/mL) were observed for dichloromethane extract of P. lanceolata root against S. paratyphi. The main composition of ethyl acetate extract was 1,2-Benzenedicarboxylic acid and mono(2-ethylhexyl) ester (60.93%). The major compositions in dichloromethane and n-butanol extracts were 1,2-Benzenedicarboxylic acid, mono(2- ethylhexyl) ester (60.64%) and 2-Methyl-1-butanol (.+/-.)- (17.85%). As evidenced by the results of the present research, P. lanceolata extracts are a significant source of bioactive metabolites. Therefore, they can play a prominent role in the production of pharmaceutical materials. [ABSTRACT FROM AUTHOR]

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose. [ABSTRACT FROM AUTHOR]

Xiongwei Liu,1,* Kuizhong Shan,2,* Xiaxia Shao,1 Xianqing Shi,3 Yun He,4 Zhen Liu,1 Joe Antony Jacob,5 Lichun Deng1 1Department of Oncology, Affiliated Jiangyin Hospital of Medical College, Southeast University, Jiangyin, People’s Republic of China; 2Department of Oncology, The Second People’s Hospital of Kunshan, Kunshan, People’s Republic of China; 3Department of Oncology, Liyang People’s Hospital, Liyang, People’s Republic of China; 4Department of Oncology, Affiliated Hospital of Chinese Medicine of Changshu City, Nanjing University of Chinese Medicine, Changshu, People’s Republic of China; 5Nanosynthesis Unit, Nanome Consulting, Salem, Tamil Nadu, India*These authors contributed equally to this workCorrespondence: Lichun DengDepartment of Oncology, Affiliated Jiangyin Hospital of Medical College, Southeast University, No. 163 Shoushan Road, Jiangyin, 214400, Jiangsu Province, People’s Republic of ChinaTel +86 510 8061 5131Email 18921233265@163.comJoe Antony JacobNanosynthesis Unit, Nanome Consulting, No. 11, Kannankurichi Road, Salem, 636008, Tamil Nadu, IndiaTel +91-9443861940Email joeantonyjacob@gmail.comPurpose: Biomimetic approaches for the synthesis of silver nanoparticles (AgNPs) had created a substantial impression among the research community that focuses on nano-bio interactions. In this study, an eco-friendly method using Rhizophora apiculata aqueous leaf extract as a reductant-rich hydrosol was followed to synthesize AgNPs and test its cytotoxicity.Methods: To optimise the parameters for the synthesis of AgNPs, central composite design based on response surface methodology was used. The particles synthesized at a nano-scale were characterized in our previously published report. The present report further characterizes the nanoparticles by X-ray diffraction, SEM and TEM at varying sites and magnifications. The characterized AgNPs were tested for their cytotoxic effects on HEK-293 and HeLa cells.Results: The cytotoxicity on the cell lines was dose-dependent. At a concentration of 2.5 μL/mL of the AgNPs-containing hydrosol, 100% inhibition of HEK-293 cells and 75% inhibition of the HeLa cells were observed. The IC50 value for AgNPs on HEK-293 was 0.622 μL/mL (12.135 ng), whereas, for HeLa cells, it was 1.98 μL/mL (38.629 ng).Conclusion: The nanoparticles were three-fold toxic towards the HEK-293 cells in comparison to the HeLa cells. Therefore, the therapeutic index is low for R. apiculata derived AgNPs on HeLa cells when tested in comparison with the HEK-293 cells. The nanotoxicity profile of the synthesized AgNPs seems more prominent than the nanotherapeutic index. According to our knowledge, this is the first-ever report on the optimization of synthesis of AgNPs using response surface methodology and identifying the therapeutic index of mangrove leaf-derived AgNPs.Keywords: AgNPs, XRD, HEK-293, HeLa, nanotoxicity

Sharbat-e-Bazoori Motadil (SBM) is a traditional Unani syrupy formulation, widely used for the management of kidney diseases. Due to lack of scientific evidence, the present work prospective is aimed to evaluate the protective effect of sugar-free SBM against cisplatin (CP)-induced nephrotoxicity using in vitro and in vivo followed by phytochemical studies. The sugar-free SBM formulation was chromatographically characterized for chemical analysis through HPTLC and evaluated for its metabolic contents. Thereafter, in vitro phytochemicals and free radical scavenging assays were performed to evaluate the total phenols and flavonoid content and antioxidant potential of sugar-free SBM. Human embryonic kidney-293 (HEK-293) cell was used to assess nephroprotective and antioxidant studies of sugar-free SBM. Further, in vivo nephroprotective studies were performed in female Wistar albino rats at different dose (32.1, 64.2, 128.4 mg/kg/day, p.o.) of SBM by assessment of biochemical markers, antioxidants status, inflammatory cytokines, and histopathological analysis. Qualitative and quantitative HPTLC analysis of SBM revealed eleven and eight metabolites at 254 and 366 nm, respectively, while the content of caffeic acid and trans ferulic acid was found as 5.63 ± 0.29 and 12.64 ± 0.71 µg/mg, respectively. In vitro free radical scavenging assays showed the significant antioxidant potential of sugar-free SBM. The in vitro assay for nephroprotective and cellular antioxidant analysis of SBM showed significant (p

Although lithium has long been one of the most widely used pharmacological agents in psychiatry, its mechanisms of action at the cellular and molecular levels remain poorly understood. One of the targets of Li+ is the phosphoinositide pathway, but whereas the impact of Li+ on inositol lipid metabolism is well documented, information on physiological effects at the cellular level is lacking. We examined in two mammalian cell lines the effect of acute Li+ exposure on the mobilization of internal Ca2+ and phospholipase C (PLC)-dependent membrane conductances. We first corroborated by Western blots and immunofluorescence in HEK293 cells the presence of key signaling elements of a muscarinic PLC pathway (M1AchR, Gq, PLC-β1, and IP3Rs). Stimulation with carbachol evoked a dose-dependent mobilization of Ca, as determined with fluorescent indicators. This was due to release from internal stores and proved susceptible to the PLC antagonist U73122. Li+ exposure reproducibly potentiated the Ca response in a concentration-dependent manner extending to the low millimolar range. To broaden those observations to a neuronal context and probe potential Li modulation of electrical signaling, we next examined the cell line SHsy5y. We replicated the potentiating effects of Li on the mobilization of internal Ca, and, after characterizing the basic properties of the electrical response to cholinergic stimulation, we also demonstrated an equally robust upregulation of muscarinic membrane currents. Finally, by directly stimulating the signaling pathway at different links downstream of the receptor, the site of action of the observed Li effects could be narrowed down to the G protein and its interaction with PLC-β. These observations document a modulation of Gq/PLC/IP3-mediated signaling by acute exposure to lithium, reflected in distinct physiological changes in cellular responses. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia and insulin resistance. 4-hydroxyisoleucine (4-HIL) is a non-proteinogenic amino acid isolated from the fenugreek seeds and has enormous pharmacological activities. The present study was undertaken to investigate the antihyperglycemic effect of 4-HIL in streptozotocin (STZ)-induced diabetic rats. Moreover, its toxicity was evaluated in vitro and in vivo employing human embryonic kidney cells (HEK-293) and healthy rats, respectively. In experiment 1, STZ-induced diabetic male rats were subjected to an oral treatment of 4-HIL (100 mg/kg), while experiment 2 deals with the effects of 4-HIL on healthy male and female rats following oral administration. The treatment (experiment 1) declined the elevated blood glucose level, feed intake, and increased body weight(s). Additionally, blood glucose impairment was improved as observed by OGTT and IPGT tests. Pancreatic histopathology revealed mild changes in the 4-HIL group. Moreover, experiment 2 showed increased body weight, normal blood glucose levels (male—106.06 ± 7.49 mg/dl and female—100.06 ± 14.69 mg/dL), hematological parameters, and histopathological profiles in the treatment group. 4-HIL did not affect the viability of HEK-293 cells, and no signs of toxicity were observed in healthy rats. Therefore, the study concludes that 4-HIL has potential antihyperglycemic activity without any toxic effects. [ABSTRACT FROM AUTHOR]

BACKGROUND: Butyrylcholineesterase (BChE) is a therapeutic drug and its producing as a recombinant protein is an essential issue in biotechnology. One of the highlights in this regard is choosing the best host cells and plasmids. OBJECTIVES: The aim of this study is to evaluate the production of butyrylcholinesterase in Vero, HEK-293, and CHO cell lines using a dual promoter vector. MATERIAL AND METHODS: The dual-promoter construction (pBudCE dual BChE) was transfected into cell lines categorized in three experimental groups (pBudCE dual BChE, pCMV and negative control). BChE gene expression and enzyme activity was evaluated at different times. RESULTS: All three cell lines showed higher gene expression level in pBudCE dual BChE group. BChE enzyme activity level of this group in CHO cells decreased in sixth day and increased in ninth day. In HEK-293 cells it has a downward trend from sixth to ninth day and in Vero cells its level in the ninth day was the highest. CONCLUSION: The difference of pBudCE dual BChE and pCMV groups was more pronounced in the HEK-293 cell and the BChE gene expression level of this cells was higher than the others while, CHO cells showed higher level of BChE enzyme activity. [ABSTRACT FROM AUTHOR]

Abstract Background Paracetamol (PCM), being extensively adapted analgesic and anti-inflammatory drug all over the world, beyond therapeutic dosages, the oxidative stress-involved nephrotoxicity has been evidenced. However, herbal plants are the windfall for the humankind providing solution for most of the wellness breakdowns. Annona squamosa (AS) is one of such plants with enormous therapeutic and nutraceutical potencies. The main aspiration of the current investigation is to evaluate the nephroprotective ability of ethanolic extract of Annona squamosa (EEAS) leaves against paracetamol-induced nephrotoxicity using in vitro human embryonic kidney (HEK)-293 cells and in vivo experiments in Wistar rats through biochemical parameters, oxidative parameters, and histopathological findings. Results When HEK-293 cells were incubated with PCM, an increased cell death associated with alterations in the morphology of normal cells was observed. At variable concentrations, HEK-293 cells co-treated with PCM and EEAS extracts gave a significant improvement in cell growth on comparing with PCM treatment showing cytoprotective feature of EEAS with an IC50 28.75 μg/mL. In vivo nephroprotective property was assessed from the amount of blood urea nitrogen (BUN) along with creatinine and uric acid which were reduced (P < 0.001) within serum and compact levels of glutathione, catalase, and superoxide dismutase which were termed as GSH, CAT, and SOD, respectively, were increased (P < 0.001) in kidney tissue homogenate in the treated groups than the PCM alone group. Results were additionally supported by histopathological observations. Conclusion The results exhibited that EEAS has impending benefits against PCM-induced nephrotoxicity through in vitro and in vivo experiments.

Abstract Cisplatin, as one of the most effective chemotherapeutic agents, its clinical use is limited by serious side effect of nephrotoxicity. Cisplatin‐induced nephrotoxicity is closely related to apoptosis induction and activation of caspase. The present study aimed to explore the potential protective effect of ginsenoside Rk1 (Rk1), a rare ginsenoside generated during steaming ginseng, on cisplatin‐induced nephrotoxicity and the underlying mechanisms in human embryonic kidney 293 (HEK‐293) cells. Our results showed that the reduced cell viability induced by cisplatin could significantly recover by Rk1. Furthermore, glutathione (GSH) as an oxidative index, was elevated and the lipid peroxidation product malondialdehyde (MDA) was significantly decreased after Rk1 treatment compared to the cisplatin group. Additionally, Rk1 can also decrease the ROS fluorescence expression and increase the protein levels of nuclear factor erythroid 2‐related factor 2 (Nrf2) and heme oxygenase‐1 (HO‐1) compared to the cisplatin group, which suggested a suppression of oxidative response. More importantly, the cisplatin‐induced elevated protein levels of Bax, cleaved caspase‐3, cleaved caspase‐9, and decreased protein level of Bcl‐2 were reversed after treatment with Rk1. Our results elucidated the possible protective mechanism of Rk1 for the first time, which may involve in its anti‐oxidation and anti‐apoptosis effects.

Vida Mirzaie,1 Touba Eslaminejad,2 Homayoon Babaei,3 Seyed Noureddin Nematollahi-Mahani4,5 1Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 2Pharmaceutics Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran; 4Neuroscience Research Centre, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran; 5Afzal Research Institute (NGO), Kerman University of Medical Sciences, Kerman, IranCorrespondence: Seyed Noureddin Nematollahi-Mahani Tel +983 433257343Email nnematollahi@kmu.ac.irPurpose: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion.Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman’s method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector.Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman’s method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity.Conclusion: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Large-scale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.Keywords: dual-promoter, vector, recombinant protein, drug delivery, Ellman’s method, HEK-293

Although lithium has long been one of the most widely used pharmacological agents in psychiatry, its mechanisms of action at the cellular and molecular levels remain poorly understood. One of the targets of Li+ is the phosphoinositide pathway, but whereas the impact of Li+ on inositol lipid metabolism is well documented, information on physiological effects at the cellular level is lacking. We examined in two mammalian cell lines the effect of acute Li+ exposure on the mobilization of internal Ca2+ and phospholipase C (PLC)-dependent membrane conductances. We first corroborated by Western blots and immunofluorescence in HEK293 cells the presence of key signaling elements of a muscarinic PLC pathway (M1AchR, Gq, PLC-β1, and IP3Rs). Stimulation with carbachol evoked a dose-dependent mobilization of Ca, as determined with fluorescent indicators. This was due to release from internal stores and proved susceptible to the PLC antagonist U73122. Li+ exposure reproducibly potentiated the Ca response in a concentration-dependent manner extending to the low millimolar range. To broaden those observations to a neuronal context and probe potential Li modulation of electrical signaling, we next examined the cell line SHsy5y. We replicated the potentiating effects of Li on the mobilization of internal Ca, and, after characterizing the basic properties of the electrical response to cholinergic stimulation, we also demonstrated an equally robust upregulation of muscarinic membrane currents. Finally, by directly stimulating the signaling pathway at different links downstream of the receptor, the site of action of the observed Li effects could be narrowed down to the G protein and its interaction with PLC-β. These observations document a modulation of Gq/PLC/IP3-mediated signaling by acute exposure to lithium, reflected in distinct physiological changes in cellular responses.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

Citronellal, an acyclic monoterpene with an aldehyde group, is present in many plants, including species of Eucalyptus, Corymbia, Cymbopogon, Coriandrum. It is also known as rhodinol and possesses strong insect repellant and antifungal properties. In this study, we investigated the antiproliferative action of citronellal on hypopharyngeal carcinoma (FaDu) and normal (HEK-293) cell lines. Citronellal suppresses the activity of ornithine decarboxylase (ODC: 77.12±10.73 µM) and inhibits the proliferation of FaDu cells with an IC 50 of 71.81±3.37 µM. The qRT-expression analysis depicts citronellal down-regulate the expression of ODC, and a robust binding interaction of citronellal with ODC was observed in molecular docking studies. Further, it also arrests the G2/M phase of FaDu cells, followed by an increase in the number of sub-diploid cells but was non-toxic to normal cell line. The toxicity prediction studies revealed citronellal follows the Lipinski rule of 5 and the Ghose rule. These observations provide experimental evidence for citronellal to be used as a new prototype for designing ODC inhibitors. [Display omitted] • Citronellal inhibits ornithine decarboxylase activity in cell-free and cell-based test systems. • It possesses antiproliferative activity against FaDu cell line. • Citronellal arrest G2/M phase and increases the sub-diploid cells of FaDu cells. • It follows Lipinski and Ghose rule. [ABSTRACT FROM AUTHOR]

[Display omitted] Most of the chemical compound collection (currently about 200 000), assembled at the N. D. Zelinsky Institute of Organic Chemistry over the past 30 years, has been screened for antimicrobial activity against five ESKAPE pathogens and two fungi at the University of Queensland on a charitable basis. A total of 2517 active molecules (MIC ≤ 32 μg ml–1) were found, of which about 10% are active at very low concentrations (MIC ≤ 1 μg ml–1). Structures of 142 012 compounds and experimental data on their antimicrobial activity are publicly available through the demo version of the CheD software and the public database of the Community for Open Antimicrobial Drug Discovery. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (GNps) were successfully synthesized via 17 different l -Amino acids (AA-GNps) as reducing agents, in a one-pot reaction. Comparative characterization of AA-GNps revealed variations in their size, morphology, and structure, highly dependent on the nature of the employed amino acid. Aromatic amino acids proved to be fast-reducing agents directing the formation of GNps. Hydroxylic and basic amino acids were the slowest reducing agents for the synthesis of GNps. Aliphatic amino acids produced GNps of similar behavior, except Isoleucine which led to the formation of hollow nanospheres. GNps formulated by aspartic acid and asparagine have comparable properties to citrate-synthesized GNps, which is the golden standard in plasmonic nanoparticles. A new protocol was established for the determination of gold content in GNps' colloidal solutions via a simple and cost-effective standard curve method employing UV–Vis spectroscopy. Most AA-GNps are colloidally stable, in both aqueous dispersion and simulated body fluid, posing as excellent candidates for in vitro experimentation. Comparative screening for their biocompatibility demonstrated the non-hemolytic and cytocompatible profile of most AA-GNps, via different in vitro assay protocols, performed on blood samples and the healthy cell line of Human Embryonic Kidney cells (HEK-293). [Display omitted] • Evaluation of 17 different amino acids as reducing agents for one-pot synthesis of gold nanoparticles. • Morphology and colloidal stability are affected by the type of amino acid. • Most amino-acid generated gold nanoparticles were proven hemocompatible and the morphology of RBCs remained unaffected. • In vitro evaluation on Human Embryonic Kidney Cells (HEK-293) demonstrated overall biocompatibility with different profiles depending on the amino acid functionalization. [ABSTRACT FROM AUTHOR]

Abstract The increased multidrug resistance among antimalarial drugs produces the urgency of potent anti malarial to combat resistant malaria and the malaria burden worldwide. The protein which may prevent the growth or transmission of malaria parasite may be the great target for rational drug designing. Plasmodium falciparum phosphoethanolamine methyltransferase (Pfpmt) absent in human catalyzes triple methylation of ethanolamine into phosphocholine for phosphatidylcholine biosynthesis from serine decarboxylation phosphoethanolamine methyltransferase pathway for the membrane development at asexual as well as sexual stages of parasite. The Plasmodium requires production of membrane rapidly for growth and multiplication. Hence, the phosphoethanolamine methyltransferase of Plasmodium falciparum was selected as drug target for rational drug designing. Using Discovery studio 3.5 software the library of zinc compounds was screened against target and analyzed. The compounds with better druglike properties and docking affinity and binding interaction for target protein were procured for in vitro analysis against Plasmodium falciparum culture (IC50). Compounds ZINC02103914 and ZINC12882412 were found to have good druglike properties and affinity for Pfpmt also inhibited P. falciparum growth at very low µM IC50 concentration 3.0 µM and 2.1 µM respectively also found nontoxic in vitro against HEK-293 cells. Simulation study of best inhibitor revealed the specificity for the target protein. Hence, the compounds possessed the immense probability of being inhibitors of Pfpmt and may be optimized as antimalarial agent for combinational therapy to overcome the multidrug resistance and may also be used as template for optimization and rational drug designing.

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host’s cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

Docetaxel (DOC) is a chemotherapeutic that induces microtubule stabilization. It is often used in breast, prostate, lung and gastric cancers but severe side effects such as cardiotoxicity, neurotoxicity, hepatotoxicity, and nephrotoxicity limit its usage. Curcumin (CUR), a natural bioactive compound derived from turmeric. Here, the possible preventive effect of CUR against DOC-induced oxidative stress and apoptosis on HEK-293 immortalized human embryonic kidney cells. Viability was assessed via MTT assay. The generation of ROS was measured by CM-H2DCFDA dye. Phosphatidylserine externalization and caspase 3/7 activity were used to measure apoptosis. CUR pretreatment remarkably inhibited DOCinduced cell viability reduction, ROS generation, and cell apoptosis in HEK-293 cells. Moreover, this study revealed that CUR pretreatment decreased caspase-3 activity. Thus, this study highlights the novel pharmacological mechanisms of CUR and understanding the detailed mechanisms of CUR action. [ABSTRACT FROM AUTHOR]

Safe, efficient and cancer cell targeted delivery of CRISPR/Cas9 is important to increase the effectiveness of available cancer treatments. Although cancer derived exosomes offer significant advantages, the fact that it carries cancer related/inducing signaling molecules impedes them from being used as a reliable drug delivery vehicle. In this study, we report that normal epithelial cell-derived exosomes engineered to have HN3 (HN3LC9-293exo), target tumor cells as efficiently as that of the cancer cell-derived exosomes (C9HuH-7exo). HN3LC9-293exo were quickly absorbed by the recipient cancer cell in vitro. Anchoring HN3 to the membrane of the exosomes using LAMP2, made HN3LC9-293exo to specifically enter the GPC3+ HuH-7 cancer cells than the GPC3− LO2 cells in a co-culture model. Further, sgIQ 1.1 plasmids were loaded to exosomes and surprisingly, in combination with sorafenib, synergistic anti-proliferative and apoptotic effect of loaded HN3LC9-293exo was more than the loaded C9HuH-7exo. While cancer-derived exosomes might induce the drug resistance and tumor progression, normal HEK-293 cells-derived exosomes with modifications for precise cancer cell targeting like HN3LC9-293exo can act as better, safe and natural delivery systems to improve the efficacy of the cancer treatments.

Abstract Background Pyrimidine is an important pharmacophore in the field of medicinal chemistry and exhibit a broad spectrum of biological potentials. A study was carried out to identify the target protein of potent bis-pyrimidine derivatives using reverse docking program. PharmMapper, a robust online tool was used for identifying the target proteins based on reverse pharmacophore mapping. The murine macrophage (RAW 264.7) and human embryonic kidney (HEK-293) cancer cell line used for selectivity and safety study. Methods An open web server PharmMapper was used to identify the possible target of the developed compounds through reverse pharmacophore mapping. The results were analyzed and validated through docking with Schrodinger v9.6 using 10 protein GTPase HRas selected as possible target. The docking studies with Schrödinger validated the binding behavior of bis-pyrimidine compounds within GTP binding pocket. MTT and sulforhodamine assay were used as antiproliferative activity. Results and discussion The protein was found one of the top scored targets of the compound 18, hence, the GTPase HRas protein was found crucial to be targeted for competing cancer. Toxicity study demonstrated the significant selectivity of most active compounds, 12, 16 and 18 showed negligible cell toxicity at their IC50 concentration. Conclusion From the results, we may conclude that GTPase HRas as a possible target of studied bis-pyrimidine derivatives where the retrieved information may be quite useful for rational drug designing.

3‐Monochloropropane‐1,2‐diol (3‐MCPD) as a main source of food contamination has always been known as a carcinogenic agent. Kidney, liver, testis, and heart seem to be the main target organs for 3‐MCPD. Because oxidative stress and mitochondrial dysfunction have been realized to be involved in 3‐MCPD‐induced cytotoxicity, the present study aimed to investigate the probable toxicity mechanisms of 3‐MCPD in isolated mitochondria, HEK‐293 cell line, and cell isolated from the rats' liver and kidney through measuring multiparametric oxidative stress assay. Based on the data indicating no significant difference between 3‐MCPD‐treated groups and control group, metabolites of 3‐MCPD have a key role in organ toxicity caused by them. To further investigating the suggested hypothesis, the effect of 3‐MCPD toxicity on HEK‐293 cell line was examined. Although the proliferation declined after exposure to a low dose of 3‐MCPD (10 to 200 µM), controversial responses in higher concentration (2 to 10 mM) have led to studies on the effect of oxidative stress and cell death signaling on isolated kidney and liver cells. Treatment of the isolated kidney and liver cells with 3‐MCPD resulted in an increase in the level of reactive oxygen species (ROS), the collapse of mitochondrial membrane potential (MMP), and activation of cell death signaling without creating any significant difference in the amount of reduced glutathione. In fact, 3‐MCPD can disrupt the mitochondrial electron transfer in isolated cells, which is correlated with the impairment of mitochondrial oxidative phosphorylation system, the rise of ROS level, and the failure of MMP, leading to the release of cytochrome c from mitochondria to cytosol and finally the activation of cell death signaling. [ABSTRACT FROM AUTHOR]

Cisplatin, as one of the most effective chemotherapeutic agents, its clinical use is limited by serious side effect of nephrotoxicity. Cisplatin‐induced nephrotoxicity is closely related to apoptosis induction and activation of caspase. The present study aimed to explore the potential protective effect of ginsenoside Rk1 (Rk1), a rare ginsenoside generated during steaming ginseng, on cisplatin‐induced nephrotoxicity and the underlying mechanisms in human embryonic kidney 293 (HEK‐293) cells. Our results showed that the reduced cell viability induced by cisplatin could significantly recover by Rk1. Furthermore, glutathione (GSH) as an oxidative index, was elevated and the lipid peroxidation product malondialdehyde (MDA) was significantly decreased after Rk1 treatment compared to the cisplatin group. Additionally, Rk1 can also decrease the ROS fluorescence expression and increase the protein levels of nuclear factor erythroid 2‐related factor 2 (Nrf2) and heme oxygenase‐1 (HO‐1) compared to the cisplatin group, which suggested a suppression of oxidative response. More importantly, the cisplatin‐induced elevated protein levels of Bax, cleaved caspase‐3, cleaved caspase‐9, and decreased protein level of Bcl‐2 were reversed after treatment with Rk1. Our results elucidated the possible protective mechanism of Rk1 for the first time, which may involve in its anti‐oxidation and anti‐apoptosis effects. [ABSTRACT FROM AUTHOR]

Background: Pathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics.Methods: We conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O'Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019.Results: Our search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n = 39), followed by Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported.Conclusions: Over the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM. [ABSTRACT FROM AUTHOR]

Sharbat-e-bazoori Motadil (SBM) is a polyherbal formulation that have been used for centuries as a part of the Unani system of medicine for renal disease. The objective of this study was to explore and validate the nephroprotective potential of sugar-free SBM (SF-SBM) and its mechanisms of action against sodium fluoride (NaF)-induced nephrotoxicity in HEK-293 cells. Additionally, the study aimed to assess the quality control of SF-SBM and investigate its effects using an in vivo rat model with pattern recognition following oral administration of SF-SBM. The nephroprotective effect of SF-SBM was investigated using both an HEK-293 cell line and Wistar rats. Nephrotoxicity was induced in these models by administering NaF at a concentration of 600 ppm (parts per million) for a duration of seven days. The SF-SBM formulation was standardized using high-performance thin-layer chromatography (HPTLC) to assess the presence of marker compounds, namely gallic acid, quercetin, and ferulic acid. Metabolite characterization of SF-SBM was carried out using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS) with a monolithic capillary silica-based C18 column. This analytical technique allowed for the identification of bioactive substances and verification of the identified markers. Acute toxicity of SF-SBM was evaluated in Wistar rats by administering a single oral dose of 2000 mg/kg of SF-SBM. The nephroprotective efficacy of SF-SBM was further assessed at low (LD), medium (MD) and high (HD) doses of 32.1, 64.2, and 128.4 mg/kg, respectively, administered orally. Nephrotoxicity was induced in Wistar rats by adding NaF to their drinking water for seven days. Biochemical and urine markers were analyzed to evaluate the antioxidant, inflammatory, and apoptotic potential of SF-SBM. Additionally, histopathological analysis and immunohistochemical alterations in the expression of caspase-3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (NOX-4) in kidney tissue were performed to confirm the findings of the in vivo experiments. Furthermore, in vivo pattern recognition of SF-SBM metabolites, identified through GC-MS metabolomics, and in-silico docking analysis of major metabolites in plasma were conducted to gain further insights. Phytochemical analysis using HPTLC, TLC-bioautography, and UPLC-MS revealed the presence of several bioactive constituents in SF-SBM, including ferulic acid, gallic acid (GA), ellagic acid, quercetin, and apigenin. These compounds exhibit diverse pharmacological properties. In vitro studies demonstrated the protective effect of SF-SBM on HEK-293 cell line against nephrotoxicity. The acute toxicity study of SF-SBM at a dose of 2000 mg/kg showed no mortality or signs of toxicity throughout the 14-day observation period. In the in vivo studies, administration of NaF resulted in significant elevation (P < 0.001) of biochemical and urine parameters, indicating oxidative, inflammatory, and apoptotic stress. Histopathological examination revealed severe depletion of Bowman's capsule, and immunohistochemistry demonstrated negative immunostaining for caspase-3 and reduced NOX-4 reactions. Pre-treatment with SF-SBM significantly attenuated the elevated biochemical and urine markers, restored the antioxidant enzyme levels (such as SOD, CAT, GSH, GPx and NO), and regulated the expression of inflammatory cytokines (TNF-α, IL-1β, CASP-3) in kidney tissue at doses of SF-SBM-MD (64.2 mg/kg) and SF-SBM-HD (128.4 mg/kg), showing comparable results to those of α-Ketoanalogue. Histopathological assessment demonstrated improvements in tissue damage. Pattern recognition analysis of SF-SBM identified the presence of 56 metabolites at different time intervals. Additionally, in-silico studies revealed strong interactions of SF-SBM with a binding energy of −6.5 and −5.6 kcal for 4C2N. The phytoconstituents present in SF-SBM play a crucial role in its nephroprotective action by acting as potent antioxidants and reducing proinflammatory and apoptotic damage in rat cells. This indicates that SF-SBM has promising potential for the treatment of nephrotoxicity. [Display omitted] • Nephroprotective potential of sugar-free SBM is enormous. • Major bioactive compounds in sugar-free SBM include ferulic acid, quercetin, etc. • Sugar-free SBM ameliorates sodium fluoride-induced nephrotoxicity in Wistar rats. [ABSTRACT FROM AUTHOR]

Biogenic silver nanoparticles (Ag NPs) have supple platforms designed for biomedical and therapeutic intervention. Utilization of Ag NPs are preferred in the field of biomedicines and material science research because of their antioxidant, antimicrobial, and anticancerous activity along with their eco-friendly, biocompatible, and cost-effective nature. Here we present a novel fungus Piriformospora indica as an excellent source for obtaining facile and reliable Ag NPs with a high degree of consistent morphology. We demonstrated their cytotoxic property, coupled with their intrinsic characteristic that make these biogenic nanoparticles suitable for the anticancerous activity. In vitro cytotoxicity of biologically synthesized Ag NPs (BSNPs) and chemically synthesized Ag NPs (SNPs) was screened on various cancer cell lines, such as Human breast adenocarcinoma (MCF-7), Human cervical carcinoma (HeLa), Human liver hepatocellular carcinoma (HepG2) cell lines and embryonic kidney cell line (HEK-293) as normal cell lines. The antiproliferative outcome revealed that the BSNPs exhibited significant cytotoxic activity against MCF-7 followed by HeLa and HepG2 cell lines as compared to SNPs. The blend of cytotoxic properties, together with green and cost-effective characteristics make up these biogenic nanoparticles for their potential applications in cancer nanomedicine and fabrication coating of ambulatory and non-ambulatory medical devices.

HEK293 cells are gaining in interest for the production of recombinant proteins. However, further understanding and engineering of cell metabolism are still needed to improve protein titers. The importance of G6PDH has already been studied regarding redox balance, and a correlation has recently been established between the oxidative pentose phosphate pathway (PPP) and antibody peak production. In this work, HEK293 cells stably expressing interferon-α2b, a parental clone, and a further engineered clone expressing the cytosolic yeast pyruvate-carboxylase (PYC) were transiently transfected to overexpress glucose-6-phosphate dehydrogenase (G6PDH) and increase fluxes through the PPP. The aim of the study was thus to evaluate the effect of overexpressing G6PDH on the “pull” effect brought by the PYC phenotype. Results indicate that the cell metabolism is, however, highly robust, showing a highly regulated PPP damping the potential effects of overexpressing G6PDH. A metabolomic study also clearly demonstrates, by metabolites profiling, that the PYC clone has a highly robust and more efficient metabolic efficiency, compared to its parental clone.

Background:Envenomation by Macrovipera lebetina (M. lebetina) is characterized by prominent local tissue damage, hemorrhage, abnormalities in the blood coagulation system, necrosis, and edema. However, the main cause of death after a bite by M. lebetina has been attributed to acute renal failure (ARF). It is unclear whether the venom components have a direct or indirect action in causing ARF. To investigate this point, we looked at the in vitro effect of M. lebetina crude venom, using cultured human embryonic kidney (HEK-293) mono layers as a model. Methods: The effect of M. lebetina snake venom on HEK-293 growth inhibition was determined by the MTT assay and the neutral red uptake assay. The integrity of the cell membrane through LDH release was measured with the Cytotoxicity Detection Kit. Morphological changes in HEK-293 cells were also evaluated using an inverted microscope. Results: In the MTT assay, crude venom showed a significant cytotoxic effect on HEK-293 cells at 24 hours of exposure and was confirmed by the neutral red assay. Also, at 24 hours exposure, crude venom caused a non-significant increase in LDH activity of the culture medium at concentrations above 20 μg/ml. Various morphological abnormalities were observed in cells exposed to the venom and showed loss of their common polygonal shape, appearing as several roughly rounded cells of variable size. The M. lebetina crude venom induced detachment of cells from the plate. Conclusion: Based on the results obtained in this study, it can be concluded that the Iranian snake M. lebetina venom causes a cytotoxic effect on kidney tissue not by necrotic mechanism but rather by secondary effects, including hypotension, hemolysis, hemoglobinuria, rhabdomyolysis, myoglobinuria and disseminated intravascular coagulation (DIC), which may lead to ARF.

Purpose: This study focuses on the development of the method of introduction of magnetic microparticles in the cytoplasm of HEK-293 cell line with their subsequent fixation under the retina of the eye.Materials and Methods. Magnetic particles (d = 2,8 mm) were treated with pluronic and injected into the cytoplasm of HEK-293 cell line, expressing GFP. The surgery was made under general anesthesia. HEK-293 containing magnetic particles were injected into the subretinal space of rabbit eyes (eyes 96, 48 rabbits) using original dosing device. In the experimental group (48 eyes, 24 rabbits) we fixed episcleral magnetic implant to hold cells in local place. In the control group (48 eyes, 24 rabbit) magnetic implant was not fixed. After the surgery all animals were examined using biomicroscopy, ophthalmoscopy with photographic recording, ultrasound, computed tomography and morphological study in certain terms (1, 3, 5, 7, 14, 21 day and 1 month).Results: The introduction of the magnetic particles into the cytoplasm of HEK 293 cell line has no effect on cell viability. HEK-293 containing magnetic particles remains in the place of injection during 21 days in rabbit eyes, where the magnetic implants were fixed (in control group during 3 days). Conclusions: Using of cells containing magnetic particles with fixation of the magnetic implant can be a promising method for cell therapy for the treatment of retinal diseases.

Candida albicans-caused local and systemic diseases are a serious health issue worldwide, leading to high mycosis-associated morbidity and mortality. Efficient combinations of novel compounds with commonly used antifungals could be an important tool for fighting infections. The aim of this study was to evaluate the interaction of synthesized 4-(4-cyanostyryl)-1-dodecylpyridin-1-ium (CSDP+) bromide alone or in combination with fluconazole with yeast and mammalian cells. We investigated cytotoxicity of the tested agents to mammalian HEK-293 cells and the influence of CSDP+ on the ability of C. albicans wt and a clinical isolate to adhere to HEK-293. Accumulation of lipophilic cation ethidium (Et+) was used to monitor the activity of efflux pumps in HEK-293 cells. The effect of CSDP+ on the expression of the main efflux transporter genes and transcription factors in C.albicans cells as well as HEK-293 efflux pump gene ABCB1 was determined. The study showed that CSDP+ alone and in combination with fluconazole was nontoxic to HEK-293 cells and was able to reduce C.albicans adhesion. The treatment of C.albicans cells with CSDP+ in combination with fluconazole resulted in a considerable overexpression of the MDR1 and MRR1 genes. The findings suggest that these genes could be associated with efflux-related resistance to fluconazole. Measurements of Et+ fluorescence and analysis of ABCB1 gene expression demonstrated that mammalian cells were not sensitive to concentrations of CSDP+ affecting C. albicans.

With the pandemic emergence of SARS-CoV-2, the exposure of cell substrates used for manufacturing of medicines has become a possibility. Cell lines used in biomanufacturing were thus evaluated for their SARS-CoV-2 susceptibility, and the detection of SARS-CoV-2 in culture supernatants by routine adventitious virus testing of fermenter harvest tested. [ABSTRACT FROM AUTHOR]

Objective(s): Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and Material and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence. Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.

In this study, the cytotoxic effects of the extracts of methanol: water (80:20) prepared from the above-ground parts of the varieties of Onobrychis albiflora Hub.-Mor., Onobrychis argyrea Boiss. subsp. argyrea, Onobrychis galegifolia Boiss. and Onobrychis tournefortii (Willd.) Desv. species to HCT-116 cells were investigated. With cytotoxicity analysis that with these species inhibitor concentrations (IC50) which resulted in a 50% reduction in the proliferation of HCT-116 cells were identified. In continuation of the study; the antiproliferative and apoptotic effects of Onobrychis albiflora extract on HCT-116 cells were evaluated by Caspase 3, Annexin V / PI Apoptosis / Necrosis analysis, Apopxin Green and 7-AAD Apoptosis / Necrosis analysis. The antiproliferative, apoptotic and necrotic effects of Onobrychis albiflora extract on HCT-116 cells also were determined. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A series of N -hydroxycinnamamide derivatives were designed and synthesized. • The synthesized compounds were screened against various breast cancer cell lines using MTT assay. • The compound numbers 3b and 3h exhibit most potent activity with IC50 13μM and 5μM respectively. • Compound 3h act via ROS production and promotes DNA fragmentation induction of apoptosis. Abstract The present study demonstrates the identification of N -hydroxycinnamamide derivatives and their anticancer potential against human triple-negative breast cancer cell line MDA-MB‑231, MCF-7 and non-malignant origin cell line, HEK-293 (human embryonic kidney). MTT assay was studied with HEK-293 cell line. Anticancer potential of the N -hydroxycinnamamide derivatives were compared with marked drug Tamoxifen through in vitro study. The compound numbers 3b and 3h exhibit most potent activity against antagonistic breast cancer cells (MDA-MB-231) with IC 50 13μM and 5μM respectively. Compound 3h promotes DNA fragmentation and induction of apoptosis. Furthermore, loss of mitochondrial membrane potential induced by compound 3h. The major mechanism of compound 3h for anti-breast cancer activity was probably initiation of reactive oxygen species (ROS) in cancer cells thereby persuading apoptotic cell deaths in cancer cells. [ABSTRACT FROM AUTHOR]

Background: Pyrimidine is an important pharmacophore in the field of medicinal chemistry and exhibit a broad spectrum of biological potentials. A study was carried out to identify the target protein of potent bis-pyrimidine derivatives using reverse docking program. PharmMapper, a robust online tool was used for identifying the target proteins based on reverse pharmacophore mapping. The murine macrophage (RAW 264.7) and human embryonic kidney (HEK-293) cancer cell line used for selectivity and safety study. Methods: An open web server PharmMapper was used to identify the possible target of the developed compounds through reverse pharmacophore mapping. The results were analyzed and validated through docking with Schrodinger v9.6 using 10 protein GTPase HRas selected as possible target. The docking studies with Schrödinger validated the binding behavior of bis-pyrimidine compounds within GTP binding pocket. MTT and sulforhodamine assay were used as antiproliferative activity. Results and discussion: The protein was found one of the top scored targets of the compound 18, hence, the GTPase HRas protein was found crucial to be targeted for competing cancer. Toxicity study demonstrated the significant selectivity of most active compounds, 12, 16 and 18 showed negligible cell toxicity at their IC50 concentration. Conclusion: From the results, we may conclude that GTPase HRas as a possible target of studied bis-pyrimidine derivatives where the retrieved information may be quite useful for rational drug designing. [ABSTRACT FROM AUTHOR]

Papain is a protease enzyme with therapeutic properties that are very valuable for medical applications. Poly(ε-caprolactone) (PCL) is an ideal polymeric carrier for controlled drug delivery systems due to its low biodegradability and its high biocompatibility. In this study, the three-dimensional structure and action mechanism of papain were investigated by in vitro and in silico experiments using molecular dynamics (MD) and molecular docking methods to elucidate biological functions. The results showed that the size of papain-loaded PCL nanoparticles (NPs) and the polydispersity index (PDI) of the NPs were 242.9 nm and 0.074, respectively. The encapsulation efficiency and loading efficiency were 80.4 and 27.2%, respectively. Human embryonic kidney cells (HEK-293) were used for determining the cytotoxicity of papain-loaded PCL and PCL nanoparticles. The in vitro cell culture showed that nanoparticles are not toxic at low concentrations, while toxicity slightly increases at high concentrations. In silico studies, which were carried out with MD simulations and ADME analysis showed that the strong hydrogen bonds between the ligand and the papain provide stability and indicate the regions in which the interactions occur. [ABSTRACT FROM AUTHOR]