Human Cell Cultures Research Articles

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Study of the metallome and transcriptome of a degenerating cellular model of the retinal pigment epithelium

Aims/Purpose: To study the changes on the metallome and transcriptome of a cellular model of the retinal pigment epithelium (RPE) during its in vitro degeneration after 4 months in culture.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts and cultured for 4 months. Cell pigmentation and barrier function was followed up using optical microscopy and measurement of the transepithelial electrical resistance (TEER). Samples were collected at 21, 91 and 133 days in culture and analysed for gene expression, metal homeostasis of Ca, Na, P, Mg and P, and extracellular deposit formation through RNA sequencing (RNA‐Seq), elemental mass spectrometry (ICP‐MS) and immunocytochemistry assays, respectively.Results: RPE cells formed a differentiated monolayer at 21 days in culture, expressing specific genes and proteins of RPE and giving increasing TEER values. However, after 133 days in culture, loss of tight junction proteins (i.e. CLDN19 and BEST1) and electrical resistance was seen, together with loss of pigmentation and increase of apolipoprotein E deposition in the extracellular space. Gene expression analysis showed an enrichment on apoptotic processes, biomolecule phosphorylation, and signs of epithelial‐to‐mesenchymal transition (EMT). Degeneration of RPE cells concurred with an increase of Na and P content in the cytosol and decrease of Mg, while a decrease of Mg, P and Cu was determined in the insoluble fraction of cells. Time‐dependent changes of metals were accompanied with the alteration of metal transporters and antioxidant enzymes using metals as cofactors.Conclusions: Degeneration of RPE cells in culture was characterized by depigmentation of cells, improper barrier function, downregulation of antioxidant protection, loss of epithelial phenotype, and changes on the levels of Na, P, Mg and Cu, as well as of the expression of their homeostatic proteins.

Adverse effects of BAK in glaucoma medications

Benzalkonium chloride (BAK) is a common preservative found in glaucoma medications used to increase shelf‐life, prevent bacterial contamination, and enhance ocular penetration. Its detergent properties kill microorganisms by disrupting cell membranes and causing cell lysis. Unfortunately BAK also has potentially harmful cytotoxic effects, particularly for the ocular surface, with evidence of BAK‐induced apoptosis of conjunctival and corneal epithelial cells, decreased density of conjunctival goblet cells, delayed corneal wound healing and reduced density of corneal nerved. It may also affect deeper ocular structures including the lens and trabecular meshwork, with BAK shown to cause apoptosis of human trabecular meshwork cells in culture. Clinical consequences may include increased risk of ocular surface disease, reduced adherence, and with long‐term use, reduced success of glaucoma surgery.

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Isolation of Membrane Bound Mucins from Human Bronchial Epithelial Cells.

Membrane-bound mucins constitute a large portion of the periciliary layer of lung epithelial surfaces, and thus play an important role in many aspects of innate defense. The biophysical and biochemical properties of the membrane-bound mucins have important implications for mucociliary clearance, viral penetration, and potential therapeutics delivered to the airway surface. Hence, isolating them and determining these properties is important in understanding airways disease and ultimately in developing treatments. Here, we describe a method using isopycnic centrifugation to enrich and isolate shed membrane-bound mucins from the washings of human bronchial epithelial cell cultures.

SARS-CoV-2-infected human airway epithelial cell cultures uniquely lack interferon and immediate early gene responses caused by other coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of a class of highly pathogenic coronaviruses. The large family of coronaviruses, however, also includes members that cause only mild symptoms, like human coronavirus-229E (HCoV-229E) or OC43 (HCoV-OC43). Unravelling how molecular (and cellular) pathophysiology differs between highly and low pathogenic coronaviruses is important for the development of therapeutic strategies. Here, we analysed the transcriptome of primary human bronchial epithelial cells (PBEC), differentiated at the air-liquid interface (ALI) after infection with SARS-CoV-2, SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV and HCoV-229E using bulk RNA sequencing. ALI-PBEC were efficiently infected by all viruses, and SARS-CoV, MERS-CoV and HCoV-229E infection resulted in a largely similar transcriptional response. The response to SARS-CoV-2 infection differed markedly as it uniquely lacked the increase in expression of immediate early genes, including FOS, FOSB and NR4A1 that was observed with all other coronaviruses. This finding was further confirmed in publicly available experimental and clinical datasets. Interfering with NR4A1 signalling in Calu-3 lung epithelial cells resulted in a 100-fold reduction in extracellular RNA copies of SARS-CoV-2 and MERS-CoV, suggesting an involvement in virus replication. Furthermore, a lack in induction of interferon-related gene expression characterised the main difference between the highly pathogenic coronaviruses and low pathogenic viruses HCoV-229E and HCoV-OC43. Our results demonstrate a previously unknown suppression of a host response gene set by SARS-CoV-2 and confirm a difference in interferon-related gene expression between highly pathogenic and low pathogenic coronaviruses.

Immunophenotyping, Part I: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures.

Nanoparticles are increasingly used in biomedical applications to influence the way the immune system reacts to tumors and infectious disease-causing agents. Nanoparticles not-intended for immunomodulation can also influence immune responses by affecting immune cell subsets' viability and/or activity. While immunophenotyping is commonly used to assess the effects of drugs and nanoparticles on immune cell subsets, no standardized approach exists due to the breadth of available cell models and instrumentation. In this chapter, we describe a protocol for flow cytometer calibration and reagent qualification prior to its use in the immunophenotyping experiment. The strategies described herein can be adapted to other instruments. The subsequent chapter-immunophenotyping part II (Chap. 25 )-provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood.

Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells.

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.

Rifampicin Inhibits TLR4 and IL1β Gene Expression and Enhances SH-SY5Y Cell Viability After Prolonged Ethanol Exposure in an In Vitro Experiment

Prolonged alcohol exposure activates TLR4-signaling pathways in the brain, responsible for the development of neuroinflammation. There is interest in pharmacologic correction of these mechanisms. The antibiotic rifampicin (Rif) is known as a potential neuroprotectant which can correct various pathologic conditions of the nervous system associated with the development of neuroinflammatory events. We performed a study on human neuroblastoma cell culture SH-SY5Y. Prolonged incubation of SH-SY5Y cells with ethanol (24 h, 100 mM) of induced activation the innate immune system genes Tlr4 and Il1β. Pre-treatment with Rif (25-100 mM) prior to incubation of cells with ethanol inhibited Tlr4 and Il1β gene expression, whereas addition of Rif after incubation of cells with ethanol dose-dependently reduced the increased expression of Tlr4 and Il1β genes, with the most significant effect observed at a concentration of 100 mM. In addition, the use of Rif increased cell survival in culture. Thus, the results of our experiment has shown that Rif is able to eliminate the increased expression of inflammation genes caused by prolonged alcohol exposure and to increase the survival rate of long-term incubated cells in ethanol solution.

Transforming Toxicity Assessment through Microphysiology, Bioprinting, and Computational Modeling

Background: Traditional toxicity testing emphasizes animal models with growing concerns regarding predictive capacity, throughput and ethics. Rapid innovation surrounding human cell platforms, bioengineered tissues, omics techniques and computational tools offers more modern alternatives aligned with expanding knowledge of chemical biological pathways. These disruptive approaches promise immense potential to transform next-generation chemical safety assessment and drug development pipelines. Purpose: This review provides clinical researchers an updated, comprehensive perspective across evolving areas of focus in new toxicity testing methods with analysis of latest advances and translational context. Main Body: We survey progress in two- and three-dimensional human cell cultures recapitulating tissue/organ complexity impossible in conventional assays. Complementing this, computational modeling integrates structure-activity relationships, physicochemical properties and physiological interactions to predict pharmacokinetics and toxicity in silico. Expanding model organisms add further dimensionality and demographic relevance. High-throughput omics and imaging technologies unravel mechanisms and illuminate biomarkers undetectable by standard measures. Specialized techniques show high promise addressing toxicodynamic intricacies within disease contexts like diabetes and NAFLD. Evaluating traditional medicines and expanding phytochemicals likewise represents an area of growth well-suited for contemporary platforms. Future outlook weighs remarkable potential advantages in reducing animal testing demands, enabling precision toxicology links to clinical medicine and overhauling core chemical risk assessment frameworks. Conclusion: This review intends to catalyze discourse on strategic optimization priorities and roadmaps towards fully unlocking the immense yet still emerging public health potential of these disruptive techniques poising transformation in toxicity sciences centered on human-focused models.

Drug Synergism of Anticancer Action in Combination with Favipiravir and Paclitaxel on Neuroblastoma Cells.

Background and Objectives: Favipiravir (FPV) is an antiviral medication and has an inhibitory effect on Cytochrome P450 (CYP2C8) protein, which is mainly involved in drug metabolism in the liver, and the expression of this gene is known to be enhanced in neuronal cells. The metabolization of Paclitaxel (PTX), a chemotherapeutic drug used in cancer patients, was analyzed for the first time in the human SH-SY5Y neuroblastoma cell line for monitoring possible synergistic effects when administered with FPV. Materials and Methods: Further, in vitro cytotoxic and genotoxic evaluations of FPV and PTX were also performed using wide concentration ranges in a human fibroblast cell culture (HDFa). Nuclear abnormalities were examined under a fluorescent microscope using the Hoechst 33258 fluorescent staining technique. In addition, the synergistic effects of these two drugs on cultured SH-SY5Y cells were determined by MTT cell viability assay. In addition, the death mechanisms that can occur in SHSY-5Y were revealed by using the flow cytometry technique. Results: Cell viability analyses on the HDFa healthy cell culture showed that both FPV and PTX have inhibitory effects at higher concentrations. On the other hand, there were no significant differences in nuclear abnormality numbers when both of the compounds were applied together. Cell viability analyses showed that FPV and PTX applications have higher cytotoxicity, which indicated synergistic toxicity against the SHSY-5Y cell line. Also, PTX exhibited higher anticancer properties against the neuroblastoma cell line when applied with FPV, as shown in both cytotoxicity and flow cytometry analyses. Conclusions: In light of our findings, the anticancer properties of PTX can be enhanced when the drug application is coupled with FPV exposure. Moreover, these results put forth that the anticancer drug dosage should be evaluated carefully in cancer patients who take COVID-19 treatment with FPV.

Changes in the mitochondrial function of fibroblast-like cells exposed to copper oxide nanoparticles

Introduction. Mitochondria are targets for almost all types of damaging agents, including toxins and oxidative stress. There is no doubt that possible effects on mitochondria should be taken into account in a comprehensive assessment of metal toxicity. Our objective was to establish changes in the mitochondrial function under the effect of copper oxide nanoparticles in vitro.
 Material and methods. A monolayer culture of human lung fibroblast-like cells of the FLECH-104 line were exposed to copper oxide nanoparticles (CuO NPs) 21±4 nm in size, final concentrations of which in the media were 25, 50, and 100 μg/mL. We measured the rate of oxygen consumption by the cells and its changes under the influence of modulators, such as oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and rotenone combined with antimycin A. We estimated parameters of mitochondrial function and the bioenergetics index.
 Results. At the concentration of 100 μg/mL in the incubation medium, CuO NPs induced changes in the culture of fibroblast-like cells that impeded further assessment of the mitochondrial function. At the lower concentrations of 25 and 50 μg/mL, we observed a dose-dependent trend toward a decrease in ATP-related respiration and bioenergetics index. It is noteworthy that the concentration of 25 μg/mL increased the maximum and reserve respiratory capacity of the cells, which was probably related to the dual effect of copper as a toxicant and an essential element.
 Limitations of the study. The study was conducted using only one cell line and three concentrations of CuO nanoparticles suspended in the culture medium.
 Conclusion. We established that copper oxide nanoparticles, when added to the incubation medium, have a multidirectional effect on the mitochondrial function of fibroblast-like cells potentially attributed to biotic properties of this metal.

Sandwich-type channeled chemical hydrogels manufactured by 3D ink-jet printing under freezing conditions using a photochemical process for human cell cultures

This work aims to present the technology of preparing chemical hydrogels for cell culture by printing. It consists in drop-on-drop 3D ink-jet printing under freezing conditions of a solution containing a diacrylic compound (poly(ethylene glycol) diacrylate (PEGDA)) with an initiator (hydrogen peroxide) of photochemically induced polymerization and cross-linking. 3D printing takes place at a low temperature, thanks to which droplets of the solution immediately freeze, allowing the desired structures of the frozen solution to be built. UVC radiation of frozen structures at room temperature and in the presence of oxygen induces simultaneous melting and radical polymerization and cross-linking of PEGDA, resulting in the formation of a firm chemical hydrogel with the same shape as the shape of the printed frozen solution. Discussed are: (i) technological parameters, (ii) reaction mechanism, (iii) sol–gel analysis, (iv) morphology of printed hydrogels and (v) biocompatibility studies with the applied human fibroblasts, human umbilical vein endothelial cells and human neuroblastoma cells. A method for printing sandwich-type channeled structures was developed and shown in application for cell growth. This work demonstrates the feasibility of 3D printing biocompatible chemical hydrogels for cell culture and opens new possibilities for printing hybrid structures for biomedical applications.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity.

Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.

A comparative survey of the influence of small self-cleaving ribozymes on gene expression in human cell culture

ABSTRACT Self-cleaving ribozymes are versatile tools for synthetic biologists when it comes to controlling gene expression. Up to date, 12 different classes are known, and over the past decades more and more details about their structure, cleavage mechanisms and natural environments have been uncovered. However, when these motifs are applied to mammalian gene expression constructs, the outcome can often be unexpected. A variety of factors, such as surrounding sequences and positioning of the ribozyme influences the activity and hence performance of catalytic RNAs. While some information about the efficiency of individual ribozymes (each tested in specific contexts) is known, general trends obtained from standardized, comparable experiments are lacking, complicating decisions such as which ribozyme to choose and where to insert it into the target mRNA. In many cases, application-specific optimization is required, which can be very laborious. Here, we systematically compared different classes of ribozymes within the 3’-UTR of a given reporter gene. We then examined position-dependent effects of the best-performing ribozymes. Moreover, we tested additional variants of already widely used hammerhead ribozymes originating from various organisms. We were able to identify functional structures suited for aptazyme design and generated highly efficient hammerhead ribozyme variants originating from the human genome. The present dataset will aide decisions about how to apply ribozymes for affecting gene expression as well as for developing ribozyme-based switches for controlling gene expression in human cells.

The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D3 (VD3) enhances legumain expression and function. In turn, legumain alters VD3 bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD3 (1,25(OH)2D3) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D3 (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn-/-), whereas VDBP was cleaved in wild-type control mice (Lgmn+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D3 and total VD3 and altered expression of key renal enzymes involved in VD3 metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD3 and legumain is suggested.

Isolation and Culture of Human Meibomian Gland Ductal Cells.

Hyperkeratinization of meibomian gland (MG) ducts is currently recognized as the primary pathologic mechanism of meibomian gland dysfunction (MGD). This research figured out a method to isolate the MG ducts and established a novel system to culture the human meibomian gland ductal cells (HMGDCs) for investigating the process of MGD. The MG ducts were obtained from the eyelids of recently deceased donors and subjected to enzymatic digestion. The acini were then removed to isolate independent ducts. These MG ducts were subsequently cultivated on Matrigel-coated wells and covered with a glass plate to obtain HMGDCs. The HMGDCs were further cultivated until passage 2, and when they reached 60% confluence, they were treated with IL-1β and rosiglitazone for a duration of 48 hours. Immunofluorescence staining and Western blot techniques were employed to identify ductal cells and analyze the effects of IL-1β on HMGDCs in an in vitro setting. Ophthalmic micro-forceps and insulin needles can be employed for the purpose of isolating ducts. Within this particular culture system, the rapid expansion of HMGDCs occurred in close proximity to the duct tissue. MG ducts specifically expressed keratin 6 (Krt6) and hardly synthesized lipids. Furthermore, the expression of Krt6 was significantly higher (P < 0.0001) in HMGDCs compared to human meibomian gland cells. Upon treatment with IL-1β, HMGDCs exhibited an overexpression of keratin 1, which was effectively blocked by the administration of rosiglitazone. The present study successfully isolated human MG ducts and cultured HMGDCs, providing a valuable in vitro model for investigating the mechanism of MGD. Additionally, the potential therapeutic efficacy of rosiglitazone in treating hyperkeratinization of ducts in patients with MGD was identified.

Synthesis of Cytotoxic Quino[4,3-b]carbazole Frameworks through an Intramolecular Diels-Alder Reaction.

An intramolecular Diels-Alder reaction of positionally isomeric indole-2/3-phenylvinyl-N-alkynylated (N-phenylsulfonyl)amines has been successfully exploited for the synthesis of quino[4,3-b]carbazole and its analogues. This reaction proceeds through a [4 + 2] cycloaddition followed by elimination and deprotection of phenylsulfonyl units to afford the quinocarbazoles in moderate to good yields. The reaction features a broad substrate scope and remarkable functional group forbearance. A preliminary in vitro cytotoxicity evaluation of representative quino[4,3-b]carbazoles was performed against NCI-H460 human cancer cell culture. Among the quino[4,3-b]carbazoles evaluated, five of the fluorine-containing quinocarbazoles displayed nano molar range (0.8-2.0 nm) GI50 values. The UV-vis and fluorescence spectral studies of representative quinocarbazoles were also performed. Like ellipticine, four of the quinocarbazoles displayed dual emissions confirming the existence of p-quinonoid like tautomeric forms in a polar protic solvent.

Assessing the antiviral potential of melatonin: A comprehensive systematic review.

This review assesses the antiviral potential of melatonin through comprehensive analysis of studies across human subjects, animal models, cell cultures, and in-silico simulations. The search strategy targeted relevant research until 22 June 2023, resulting in 20 primary studies after screening and deduplication. The findings highlight strong evidence supporting antiviral properties of melatonin. In silico studies identify melatonin as a candidate against SARS-CoV-2, reducing cytokine storm-related respiratory responses. Cell culture experiments reveal its multifaceted effects on different viruses including respiratory syncytial virus, anti-dengue virus, transmissible gastroenteritis virus, and encephalomyocarditis virus. Animal studies show melatonin reduces mortality and viral replication in various infections such as Venezuelan equine encephalomyelitis and COVID-19. Clinical trials show how it could be evaluated, but with no conclusive evidence of efficacy and safety so far from large, double-blind placebo-controlled trials. These insights showcase the potential of melatonin as a versatile antiviral agent with immunomodulatory, antioxidant, anti-inflammatory and antiviral properties. In summary, our review highlights melatonin's promising antiviral properties across diverse settings. Melatonin's immunomodulatory and antiviral potential makes it a compelling candidate for further investigation, emphasising the need for rigorous clinical trials to establish its safety and efficacy against viral infections.