Kidney Cell Line Research Articles

Cytotoxicity of vanadium dioxide nanoparticles to human embryonic kidney cell line: Compared with vanadium(IV/V) ions

Vanadium dioxide (VO2) is a class of thermochromic material with potential applications in various fields. Massive production and wide application of VO2 raise the concern of its potential toxicity to human, which has not been fully understood. Herein, a commercial VO2 nanomaterial (S-VO2) was studied for its potential toxicity to human embryonic kidney cell line HEK293, and two most common vanadium ions, V(IV) and V(V), were used for comparison to reveal the related mechanism. Our results indicate that S-VO2 induces dose-dependent cellular viability loss mainly through the dissolved V ions of S-VO2 outside the cell rather than S-VO2 particles inside the cell. The dissolved V ions of S-VO2 overproduce reactive oxygen species to trigger apoptosis and proliferation inhibition via several signaling pathways of cell physiology, such as MAPK and PI3K-Akt, among others. All bioassays indicate that the differences in toxicity between S-VO2, V(IV), and V(V) in HEK293 cells are very small, supporting that the toxicity is mainly due to the dissolved V ions, in the form of V(V) and/or V(IV), but the V(V)’s behavior is more similar to S-VO2 according to the gene expression analysis. This study reveals the toxicity mechanism of nanosized VO2 at the molecular level and the role of dissolution of VO2, providing valuable information for safe applications of vanadium oxides.

Development of Cholinium-Based API Ionic Liquids with Enhanced Drug Solubility: Biological Evaluation and Interfacial Properties.

We report an efficient sustainable two-step anion exchange synthetic procedure for the preparation of choline API ionic liquids (Cho-API-ILs) that contain active pharmaceutical ingredients (APIs) as anions combined with choline-based cations. We have evaluated the in vitro cytotoxicity for the synthesized compounds using three different cells lines, namely, HEK293 (normal kidney cell line), SW480, and HCT 116 (colon carcinoma cells). The solubility of APIs and Cho-API-ILs was evaluated in water/buffer solutions and was found higher for Cho-API-ILs. Further, we have investigated the antimicrobial potential of the pure APIs, ILs, and Cho-API-ILs against clinically relevant microorganisms, and the results demonstrated the promise of Cho-API-ILs as potent antimicrobial agents to treat bacterial infections. Moreover, the aggregation and adsorption properties of the Cho-API-ILs were observed by using a surface tension technique. The aggregation behavior of these Cho-API-ILs was further supported by conductivity and pyrene probe fluorescence. The thermodynamics of aggregation for Cho-API-ILs has been assessed from the temperature dependence of surface tension. The micellar size and their stability have been studied by dynamic light scattering, transmission electron microscopy, and zeta potential. Therefore, the duality in the nature of Cho-API-ILs has been explored with the upgradation of their physical, chemical, and biopharmaceutical properties, which enhance the opportunities for advances in pharmaceutical sciences.

Drug repurposing platform for deciphering the druggable SARS-CoV-2 interactome

The coronavirus disease 2019 (COVID-19) pandemic has heavily challenged the global healthcare system. Despite the vaccination programs, the new virus variants are circulating. Further research is required for understanding of the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and for discovery of therapeutic agents against the virus. Here, we took advantage of drug repurposing to identify if existing drugs could inhibit SARS-CoV-2 infection. We established an open high throughput platform for in vitro screening of drugs against SARS-CoV-2 infection. We screened ∼1000 drugs for their ability to inhibit SARS-CoV-2–induced cell death in the African green monkey kidney cell line (Vero-E6), analyzed how the hit compounds affect the viral N (nucleocapsid) protein expression in human cell lines using high-content microscopic imaging and analysis, determined the hit drug targets in silico, and assessed their ability to cause phospholipidosis, which can interfere with the viral replication. Duvelisib was found by in silico interaction assay as a potential drug targeting virus–host protein interactions. The predicted interaction between PARP1 and S protein, affected by Duvelisib, was further validated by immunoprecipitation. Our results represent a rapidly applicable platform for drug repurposing and evaluation of the new emerging viruses’ responses to the drugs. Further in silico studies help us to discover the druggable host pathways involved in the infectious cycle of SARS-CoV-2.

A Functional Assessment of Fetal Liver and Monocyte-Derived Macrophages in the Lung Alveolar Environment.

It is becoming clear that every organ is seeded by a population of fetal liver-derived macrophages that are replaced at different rates by monocyte-derived macrophages. Using the Ms4a3tdTomato reporter mouse that reports on monocyte-derived alveolar macrophages (Mo-AMs) and our ability to examine AM function using our multichannel intravital microscopy, we examined the fetal-liver derived alveolar macrophage (FL-AM) and Mo-AM populations within the same mouse under various environmental conditions. The experiments unveiled that AMs migrated from alveolus to alveolus and phagocytosed bacteria identically regardless of ontogenic origin. Using 50 PFU of influenza A virus (IAV) determined using the Madin-Darby canine kidney (MDCK) cell line, we noted that both populations were susceptible to IAV-induced immunoparalysis, which also led to impaired phagocytosis of secondary bacterial infections. Both FL-AMs and Mo-AMs were trained by β-glucan to resist IAV-induced paralysis. Over time (40 wk), Mo-AMs began to outperform FL-AMs, although both populations were still sensitive to IAV. Our data also show that clodronate depletion of AMs leads to replenishment, but by FL-AMs, and these macrophages do show some functional impairment for a limited time. Overall, the system is designed such that new macrophages rapidly assume the function of tissue-resident macrophages when both populations are examined in an identical environment. These data do differ from artificial depletion methods that compare Mo-AMs and FL-AMs.

Biopolymer hydrogel matrices for the stabilization and in-vitro delivery of anti-cancer polyoxometalate [CoW11O39(CpTi)]7−

In the present study, the polyoxometalate (POM), [CoW11O39(CpTi)]7−, known for its significant anti-cancer activity and relatively low toxicity compared to established organic drugs such as CP and 5FU, is investigated. However, it is acknowledged that POMs inherently exhibit toxicity to living cells due to the presence of heavy and toxic metal ions within their core structures and their limited selectivity toward biological targets. Consequently, a methodology to mitigate this toxicity is sought by encapsulating these POMs within hydrogel matrices, with the intention to facilitate slow, sustained release and enhanced target specificity. In this study, the hydrogel of the well-researched anti-cancer POM, K6H[CoW11O39(CpTi)] (hereafter abbreviated as CoW11CpTi), is synthesized through the electrostatic interaction of the positively charged carboxymethyl chitosan (CMC), carboxymethyl cellulose (CMCell), and gelatin, which serve as hydrogelators. Subsequent characterization of the resultant CMC-CoW11CpTi, CMCell-CoW11CpTi, and gelatin-CoW11CpTi gels is performed utilizing Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry/Thermogravimetric Analysis (DSC/TGA), Electron Spin Resonance (ESR) Spectroscopy, Scanning Electron Microscopy (SEM), and Tgel analysis. Among these, the gelatin-CoW11CpTi gel demonstrates the highest stability, retaining its structural integrity for in excess of 10 days. In addition, cytotoxicity assays are conducted on an assortment of cell lines including NRK52e (Rattus norvegicus kidney cell line), MDA-MB-231 (human breast adenocarcinoma), and MCF-7 (human breast epithelial adenocarcinoma). It is observed that the release of POM from the hydrogel matrices under physiological pH conditions exhibits a slow and sustained profile. This study therefore offers a promising approach for the effective delivery of POMs in cancer therapy.

Design, synthesis and biological evaluation of novel pyrido-[2,3-d]-pyrimidin-2-amine analogues as antimycobacterial agents

Thirty-eight novel Pyrido-[2,3-d]-pyrimidin-2-amine derivatives (P1-P38) were designed, synthesized, and their structures confirmed using analytical techniques viz.,1H NMR, 13C NMR, and mass spectrometry. Using the Microplate Alamar Blue Assay (MABA), the final derivatives, P1-P38, were tested for their anti-mycobacterial activity against the Mycobacterium tuberculosis (Mtb) H37Rv strain. Amongst all the assessed compounds, P35 displayed significant inhibition with MIC 7.18 μg/mL. The remaining compounds exhibited MIC from 18.11 to >100 μg/mL. The potent compounds with MIC 〈 40.96 μg/mL, were evaluated for toxicity on HEK293T a normal human embryonic kidney cell line. The most active compound P35 exhibited a selectivity index 〉 20, proving its selective behavior towards Mtb H37Rv strain than human cells. To further evaluate the binding pattern in the active site of Thymidylate Kinase (TMPK) from Mtb (PDB-1G3U), a molecular docking analysis of compound P35 was performed using the glide module of Schrodinger software. Finally, molecular dynamics simulations were performed for 100 ns in order to elucidate the stability, confirmation, and intermolecular interactions of the co-crystal ligand and significantly active compound P35 on the selected target protein.

Crocin-loaded Niosomal Nanoparticles Reversing Cytotoxicity and Oxidative Stress in HEK293 Cell Line Exposed to Paraquat: An In vitro Study.

Paraquat (PQ) is an effective herbicide which is widely used around the world to remove weeds in agriculture. As a water-soluble carotenoid, crocin is a pharmacologically active constituent of C. sativus L. (saffron). In the present study, we investigated the effects of crocin-loaded niosomes (Cro-NIO) compared to free crocin on PQ-induced toxicity in the eukaryotic human embryonic kidney (HEK293) cell line. The Cro-NIO was synthesized and characterized. Cell viability was determined using the MTT assay in PQ-exposed HEK293 cell lines. The activities of biochemical markers were quantitatively determined to reveal the potential mechanism of PQ-induced oxidative stress in HEK293 cell line. The particle size, zeta potential, polydispersity index (PDI), DL, and EE of Cro-NIO were 145.4 ± 19.5 nm, -22.3 ± 3.11 mV, 0.3 ± 0.03, 1.74 ± 0.01%, and 55.3 ± 7.1%, respectively. PQtreated HEK293 cell lines decreased cell viability. The results of oxidative status showed that PQ significantly could increase ROS accumulation, accompanied by a decreasing antioxidant defense system. However, treatment with Cro-NIO, compared to crocin, not only did dose-dependently improve the cell viability but also significantly attenuated the ROS accumulation and increased antioxidant markers. According to these results, Cro-NIO, compared to crocin, was superior to ameliorating PQ-induced cytotoxicity and oxidative damage in HEK293 cells.

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin-Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

7SK small nuclear RNA (Rn7SK) induces apoptosis through intrinsic and extrinsic pathways in human embryonic kidney cell line.

Rn7SK, a highly conserved small nuclear non-coding RNA, controls Polymerase II transcription machinery by activating of the Positive Transcriptional Elongation Factor b (P-TEFb). Apart from its role in transcriptional regulation, the potential functions of Rn7SK in cell apoptosis are poorly understood. In a previous study, we demonstrated that overexpression of 7SK induces apoptosis in HEK cells. However, it remains unclear whether 7SK-mediated apoptosis induction is exerted through the intrinsic or extrinsic pathways. Rn7SK was overexpressed in HEK 293T cell line using Lipofectamine 2000 reagent to investigate its potential apoptotic functions. The overexpression of Rn7SK resulted in reduced cell viability through the induction of apoptosis, as evidenced by MTT assay and Annexin V/PI staining. Concurrently, alterations in the expression levels of key apoptosis-related genes were observed, as determined by quantitative RT-PCR. Furthermore, Rn7SK overexpression led to a decrease in cell proliferation, as assessed by colony formation assay and growth curve analysis. This reduction was associated with downregulated expression of key proliferative-related genes. Additionally, the migration and invasion capabilities of cells were significantly inhibited upon upregulation of Rn7SK, as demonstrated by transwell assays. This study suggests the apoptotic role of 7SK through both intrinsic and extrinsic pathways, necessitating further investigation into its underlying mechanisms.

Acmella oleracea (L.) R.K. Jansen essential oils: Chemical composition, antioxidant, and cytotoxic activities

Acmella oleracea is an important food and medicinal plant in the Amazon; it is used to treat toothaches, hepatitis, malaria, fever is also described as an anticancer plant. This work reports for the first time the comparison of chemical profiles, antioxidant, and cytotoxic capacities of inflorescence (INF) and leaf (LEA) A. oleracea essential oils (EOs) obtained by hydrodistillation (HD) and steam distillation (SD). The essential oils were obtained by hydrodistillation and steam distillation and analyzed by GC and GC-MS. The antioxidant potential was evaluated by the co-oxidation method of the β-carotene/linoleic acid, and the cytotoxic activity against healthy human embryonic kidney (HEK-293), gastric ascites (AGP-01), melanoma (SK-MEL-19), lung carcinoma (A549) malignant cell lines by the MTT method. The essential oils obtained from INF by HD and SD showed the highest yields (HD = 0.68; SD = 0.5%). The monoterpene hydrocarbons (0–49.97%) and sesquiterpene hydrocarbons (25.79–50.63%) were predominant in the analyzed oils. The main constituents (>10%) identified in the EO samples were caryophyllene oxide (0.3–28.11%), E-caryophyllene (6.71–27.31%), myrcene (0–25.03%), germacrene D (0.06–19.56%), guaiol (0–14.23%), 1-pentadecene (0–13.88%), and β-pinene (0–10.04%). The chemometric analysis displayed the formation of three distinct groups of chemical compositions as follows: group I, LEA EOs (HD and SD); group II, INF EO (HD); and group III, INF EO (SD). The INF (14.3%) and LEA (9.0%) EOs showed antioxidant action with no significant difference (p < 0.05). The INF EO (HD) showed higher cytotoxicity against the malignant lineages of gastric ascites (IC50 5.31 μg/mL), melanoma (IC50 6.43 μg/mL), while the LEA EO (SD) was more cytotoxic against the lung carcinoma cell line (13.39 μg/mL), and higher toxicity was also observed against the healthy embryonic kidney cell line (IC50 5.14 μg/mL). Acmella oleracea is chemically influenced by the volatile extraction methods, and this influences on the antioxidant or cytotoxic capacity of the species.

Atorvastatin increases autophagic flux and p62/SQSTM1 of kidney cells in hyperglycemic conditions and treatment in combination with insulin improves renal function of streptozotocin (STZ)-induced diabetic rats

Background: Although atorvastatin is commonly used as a hypolipidemic agent, it confers many health benefits in which the underlying mechanisms are not fully understood. We have previously shown that combined treatment of atorvastatin and insulin effectively restored renal function of streptozotocin (STZ)-induced diabetic rats; nevertheless, the underlying mechanism was not known. Objective: To determine whether the reno-protective effect of atorvastatin and insulin is mediated through its impact on autophagy. Materials and methods: Markers of autophagy, LC3, and p62/SQSTM1, in rat kidney tissues and cell lines treated with atorvastatin and/or insulin were determined by Western blot analysis. Results: Levels of both LC3-I and LC3-II proteins in kidney tissues of STZ-diabetic rats treated with atorvastatin and insulin were significantly increased. The autophagic flux was examined in vitro and showed that high glucose culture conditions suppressed the autophagic flux in kidney cells. Treatment with insulin moderately increased the conversion of LC3-I to LC3-II. Interestingly, atorvastatin increased autophagic flux only in the hyperglycemic but not in the normoglycemic condition. p62/SQSTM1 protein level was decreased in response to high glucose treatment but increased with the addition of insulin and/or atorvastatin. Conclusion: This study has demonstrated that atorvastatin may represent a novel regimen in providing prevention and protection for diabetic nephropathy through the underlying mechanisms of inducing autophagy and p62/SQSTM1.

Anticancer Study on IrIII and RhIII Half-Sandwich Complexes with the Bipyridylsulfonamide Ligand.

Organometallic half-sandwich complexes [(η5-Cp)IrCl(L)]PF6 (1) and [(η5-Cp)RhCl(L)]PF6 (2) were prepared using pentamethylcyclopentadienyl chloride dimers of iridium(III) or rhodium(III) with the 4-amino-N-(2,2'-bipyridin-5-yl)benzenesulfonamide ligand (L) and ammonium hexafluorophosphate. The crystal structures of L, 1, and 2 were analyzed in detail. The coordination reactions of the ligand with the central ions were confirmed using various spectroscopic techniques. Additionally, the interactions between sulfaligand, Ir(III), and Rh(III) complexes with carbonic anhydrase (CA), human serum albumin (HSA), and CT-DNA were investigated. The iridium(III) complex (1) did not show any antiproliferative properties against four different cancer cell lines, i.e., nonsmall cell lung cancer A549, colon cancer HCT-116, breast cancer MCF7, lymphoblastic leukemia Nalm-6, and a nonmalignant human embryonic kidney cell line HEK293, due to high binding affinity to GSH. The sulfonamide ligand (L) and rhodium(III) complex (2) were further studied. L showed competitive inhibition toward CA, while complexes 1 and 2, uncompetitive. All compounds interacted with HSA, causing a conformational change in the protein's α-helical structure, suggesting the induction of a more open conformation in HSA, reducing its biological activity. Both L and 2 were found to induce cell death through a caspase-dependent pathway. These findings position L and 2 as potential starting compounds for pharmaceutical, therapeutic, or medicinal research.

Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor β/Smad3 Signaling Pathway.

Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-β-treated renal cells in vitro. Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-βstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-β-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-β secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. Alantolactone improves renal fibrosis by inhibiting the TGF-β/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

Box A of HMGB1 Maintains the DNA Gap and Prevents DDR-induced Kidney Injury in D-galactose Induction Rats.

Disability and mortality rates for renal failure are still increasing. DNA damage and oxidative stress intoxication from body metabolism, high blood glucose, or the environment cause significant kidney damage. Recently, we reported that Box A of HMGB1 (Box A) acts as molecular scissors, producing DNA gaps that prevent DNA damage in kidney cell lines and ultimately reverse aging phenotypes in aging rat models. The present study aimed to demonstrate the potency of Box A in preventing D-galactose (D-gal)-induced kidney injury. A Box A expression plasmid was constructed and administered to a rat model. D-gal was injected subcutaneously for eight weeks. Serum was collected to study renal function, and white blood cells were collected for DNA gap measurement. Kidney tissue was also collected for γ-H2AX and NF-κB immunostaining; Senescence-associated (SA)-beta-gal staining; and analysis of the mRNA expression of p16INK4A, TNF-α, and IL-6. Moreover, histopathology analysis was performed using hematoxylin & eosin and Masson trichome staining. Pretreatment with Box A administration prevented the reduction of DNA gaps and the consequences of the DNA damage response, which include elevated serum creatinine; high serum BUN; an increased positive SA-beta-gal staining area; overexpression of p16INK4A, NF-κB and senescence-associated secretory phenotype molecules, including IL-6, TNF-α; and histological alterations, including tubular dilation and collagen accumulation. Box A effectively prevents DNA gap reduction and all D-gal-induced kidney pathological changes at the molecular, histological, and physiological levels. Therefore, Box A administration is a promising novel therapeutic strategy to prevent DNA-damaging agent-induced kidney failure.

Molecular bowls for inclusion complexation of toxic anticancer drug methotrexate.

We describe the preparation and study of novel cavitands, molecular bowls 16+ and 26+, as good binders of the anticancer drug methotrexate (MTX). Molecular bowls are comprised of a curved tribenzotriquinacene (TBTQ) core conjugated to three macrocyclic pyridinium units at the top. The cavitands are easily accessible via two synthetic steps from hexabromo-tribenzotriquinacene in 25% yield. As amphiphilic molecules, bowls 16+ and 26+ self-associate in water by the nucleation-to-aggregation pathway (NMR). The bowls are preorganized, having a semi-rigid framework comprising a fixed bottom with a wobbling pyridinium rim (VT NMR and MD). Further studies, both experimental (NMR) and computational (DFT and MCMM), suggested that a folded MTX occupies the cavity of bowls wherein it forms π-π, C-H-π, and ion pairing intermolecular contacts but also undergoes desolvation to give stable binary complexes (μM) in water. Moreover, a computational protocol is introduced to identify docking pose(s) of MTX inside molecular bowls from NMR shielding data. Both molecular bowls have shown in vitro biocompatibility with liver and kidney cell lines (MTS assay). As bowl 26+ is the strongest binder of MTX reported to date, we envision it as an excellent candidate for further studies on the way toward developing an antidote capable of removing MTX from overdosed cancer patients.

Construction of an IFNAR1 knockout MDBK cell line using CRISPR/Cas9 and its effect on bovine virus replication.

The type I interferon (IFN) pathway is important for eukaryotic cells to resist viral infection, as well as an impediment to efficient virus replication. Therefore, this study aims to create an IFNAR1 knockout (KO) Madin-Darby bovine kidney (MDBK) cell line using CRISPR/Cas9 and investigate its application and potential mechanism in increasing viral replication of bovines. The IFNAR1 KO cells showed increased titers of bovine viral diarrhea virus (BVDV) (1.5 log10), with bovine enterovirus and bovine parainfluenza virus type 3 (0.5-0.8 log10). RNA-seq revealed reduced expression of the genes related IFN-I pathways including IFNAR1, STAT3, IRF9, and SOCS3 in IFNAR1 KO cells compared with WT cells. In WT cells, 306 differentially expressed genes (DEGs) were identified between BVDV-infected and -uninfected cells. Of these, 128 up- and 178 down-regulated genes were mainly associated with growth cycle and biosynthesis, respectively. In IFNAR1 KO cells, 286 DEGs were identified, with 82 up-regulated genes were associated with signaling pathways, and 204 down-regulated genes. Further, 92 DEGs were overlapped between WT and IFNAR1 KO cells including ESM1, IL13RA2, and SLC25A34. Unique DEGs in WT cells were related to inflammation and immune regulation, whereas those unique in IFNAR1 KO cells involved in cell cycle regulation through pathways such as MAPK. Knocking down SLC25A34 and IL13RA2 in IFNAR1 KO cells increased BVDV replication by 0.3 log10 and 0.4 log10, respectively. Additionally, we constructed an IFNAR1/IFNAR2 double-knockout MDBK cell line, which further increased BVDV viral titers compared with IFNAR1 KO cells (0.6 log10). Overall, the IFNAR1 KO MDBK cell line can support better replication of bovine viruses and therefore provides a valuable tool for bovine virus research on viral pathogenesis and host innate immune response.

Considerations for Glycoprotein Production.

This chapter is intended to provide insights for researchers aiming to choose an appropriate expression system for the production of recombinant glycoproteins. Producing glycoproteins is complex, as glycosylation patterns are determined by the availability and abundance of specific enzymes rather than a direct genetic blueprint. Furthermore, the cell systems often employed for protein production are evolutionarily distinct, leading to significantly different glycosylation when utilized for glycoprotein production. The selection of an appropriate production system depends on the intended applications and desired characteristics of the protein. Whether the goal is to produce glycoproteins mimicking native conditions or to intentionally alter glycan structures for specific purposes, such as enhancing immunogenicity in vaccines, understanding glycosylation present in the different systems and in different growth conditions is essential. This chapter will cover Escherichia coli, baculovirus/insect cell systems, Pichia pastoris, as well as different mammalian cell culture systems including Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) cell lines, and baby hamster kidney (BHK) cells.

Recent sequencing and phylogenetic analysis of equine herpesviruses 1 and 4 among different equine populations in Egypt.

Equine herpes viruses (EHVs) are considered one of the most important respiratory pathogens in equids, resulting in serious outcomes for equine health worldwide. The objectives of the current research were the detection, molecular characterization, and isolation of EHV-1 and EHV-4 circulating within different equine populations in Egypt, either clinically or in apparently healthy horses. A total of 120 field samples were collected, and DNA was extracted. Screening and typing of extracted DNA were done by consensus and conventional PCR assays for detection of EHV-1 and EHV-4, followed by sequencing and phylogenetic analysis to confirm the virus identity. Selected positive samples for both EHV-1 and EHV-4 were subjected to Madin-Darby bovine kidney (MDBK) cell lines for virus isolation. The obtained results revealed that 58/120 (48%) samples were positive for EHVs. Typing of positive samples showed that EHV-1 was detected in (48/120) 40% of samples and EHV-4 was detected in (15/120) 12% of samples, while dual infection by both EHV-1 and 4 was detected in five samples. The current study revealed new data on the continuous circulation of EHV-1 and EHV-4 within equine populations in Egypt, and individual horses could be infected by multiple EHVs. In addition, latently infected horses are acting as potential reservoirs for frequent virus reactivation.

Cortisol controls endoplasmic reticulum stress and hypoxia dependent regulation of insulin receptor and related genes expression in HEK293 cells.

Objective. Glucocorticoids are important stress-responsive regulators of insulin-dependent metabolic processes realized through specific changes in genome function. The aim of this study was to investigate the impact of cortisol on insulin receptor and related genes expression in HEK293 cells upon induction the endoplasmic reticulum (ER) stress by tunicamycin and hypoxia. Methods. The human embryonic kidney cell line HEK293 was used. Cells were exposed to cortisol (10 µM) as well as inducers of hypoxia (dimethyloxalylglycine, DMOG; 0.5 mM) and ER stress (tunicamycin; 0.2 µg/ml) for 4 h. The RNA from these cells was extracted and reverse transcribed. The expression level of INSR, IRS2, and INSIG2 and some ER stress responsive genes encoding XBP1n, non-spliced variant, XBP1s, alternatively spliced variant of XBP1, and DNAJB9 proteins, was measured by quantitative polymerase chain reaction and normalized to ACTB. Results. We showed that exposure of HEK293 cells to cortisol elicited up-regulation in the expression of INSR and DNAJB9 genes and down-regulation of XBP1s, XBP1n, IRS2, and INSIG2 mRNA levels. At the same time, induction of hypoxia by DMOG led to an up-regulation of the expression level of most studied mRNAs: XBP1s and XBP1n, IRS2 and INSIG2, but did not change significantly INSR and DNAJB9 gene expression. We also showed that combined impact of cortisol and hypoxia introduced the up-regulation of INSR and suppressed XBP1n mRNA expression levels. Furthermore, the exposure of HEK293 cells to tunicamycin affected the expression of IRS2 gene and increased the level of XBP1n mRNA. At the same time, the combined treatment of these cells with cortisol and inductor of ER stress had much stronger impact on the expression of all the tested genes: strongly increased the mRNA level of ER stress dependent factors XBP1s and DNAJB9 as well as INSR and INSIG2, but down-regulated IRS2 and XBP1n. Conclusion. Taken together, the present study indicates that cortisol may interact with ER stress and hypoxia in the regulation of ER stress dependent XBP1 and DNAJB9 mRNA expression as well as INSR and its signaling and that this corticosteroid hormone modified the impact of hypoxia and especially tunicamycin on the expression of most studied genes in HEK293 cells. These data demonstrate molecular mechanisms of glucocorticoids interaction with ER stress and insulin signaling at the cellular level.

Synthesis of biaryl-based carbazoles via C-H functionalization and exploration of their anticancer activities.

The synthesis of a library of new biaryl-based carbazoles via bidentate directing group-assisted C-H functionalization and preliminary screening of the anticancer properties of biaryl-based carbazoles is reported. While various classes of modified carbazoles are known for their applications in materials and medicinal chemistry, to our knowledge, the biological activities of designed biaryl-based carbazoles have been rarely known. Given the prominence of carbazoles in research in medicinal chemistry, we envisioned the scope for new scaffolds of carbazole-based biaryl structures. We screened the synthesized biaryl-based carbazoles for their anticancer properties against various cancer cell lines such as HeLa (cervical cancer), HCT116 (colon cancer), MDA-MB-231 and MDA-MB-468 (breast cancer). In addition, the hits were also tested in the human embryonic kidney cell line HEK293T to assess their impact on the viability of normal human cells in the presence of these compounds. In this preliminary study, we identified some of the biaryl-based carbazoles as lead compounds with anticancer activities.