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We have reported that D,L-thiol esters, including D-cysteine ethyl ester (D-CYSee), are effective at overcoming opioid-induced respiratory depression (OIRD) in rats. Our on-going studies reveal that co-injections of D-CYSee with multi-day morphine injections markedly diminish spontaneous withdrawal that usually occurs after cessation of multiple injections of morphine in rats. Chronically administered opioids are known (1) to alter cellular redox status, thus inducing an oxidative state, and (2) for an overall decrease in DNA methylation, therefore resulting in the transcriptional activation of previously silenced long interspersed elements (LINE-1) retrotransposon genes. The first objective of the present study was to determine whether D-CYSee and the one carbon metabolism with the methyl donor, betaine, would maintain redox control and normal DNA methylation levels in human neuroblastoma cell cultures (SHSY5Y) under overnight challenge with morphine (100 nM). The second objective was to determine whether D-CYSee and/or betaine could diminish the degree of physical dependence to morphine in male Sprague Dawley rats. Our data showed that overnight treatment with morphine reduced cellular GSH levels, induced mitochondrial damage, decreased global DNA methylation, and increased LINE-1 mRNA expression. These adverse effects by morphine, which diminished the reducing capacity and compromised the maintenance of the membrane potential of SH-SY5Y cells, was prevented by concurrent application of D-CYSee (100 μM) or betaine (300 μM). Furthermore, our data demonstrated that co-injections of D-CYSee (250 μmol/kg, IV) and to a lesser extent, betaine (250 μmol/kg, IV), markedly diminished the development of physical dependence induced by multi-day morphine injections (escalating daily doses of 10-30 mg/kg, IV), as assessed by the lesser number of withdrawal phenomena elicited by the injection of the opioid receptor antagonist, naloxone (1.5 mg/kg, IV). These findings provide evidence that D-CYSee and betaine prevent the appearance of redox alterations and epigenetic signatures commonly seen in neural cells involved in opioid physical dependence/addiction, and lessen development of physical dependence to morphine. [ABSTRACT FROM AUTHOR]

This paper describes the development, design and characterization of a resistive pulse sensing (RPS) system for the analysis of size distributions of extracellular vesicles (EVs). The system is based on microfluidic chips fabricated using soft-lithography and operated in pressure-driven mode. This fabrication approach provided reproducible pore dimensions and the best performing chip design enabled, without calibration, sizing of both 252 nm and 460 nm test particles within 8% of theoretically calculated values, based on the size specifications provided by suppliers. The number concentration measurement had higher variations and without calibration provided estimates within an order of magnitude, for sample concentrations across 4 orders of magnitude. The RPS chips could also measure successfully EVs and other biological nanoparticles in purified samples from cell culture media and human serum. A compact, fast and inexpensive RPS system based on this design could be an attractive alternative to current gold-standard techniques for routine characterization of EV samples. [ABSTRACT FROM AUTHOR]

Purpose: Glaucoma, one of the leading causes of irreversible blindness, is a common progressive optic neuropathy characterised by visual field defects and structural changes to the optic nerve head (ONH). There is extracellular matrix (ECM) accumulation and fibrosis of the lamina cribrosa (LC) in the ONH, and consequently increased tissue stiffness of the LC connective tissue. Integrins are cell surface proteins that provide the key molecular link connecting cells to the ECM and serve as bidirectional sensors transmitting signals between cells and their environment to promote cell adhesion, proliferation, and remodelling of the ECM. Here, we investigated the expression of αVβ3 integrin in glaucoma LC cell, and its effect on stiffness-induced ECM gene transcription and cellular proliferation rate in normal (NLC) and glaucoma (GLC) LC cells, by down-regulating αVβ3 integrin expression using cilengitide (a known potent αVβ3 and αVβ5 inhibitor) and β3 integrin siRNA knockdown. Methods: GLC cells were compared to age-matched controls NLC to determine differential expression levels of αVβ3 integrin, ECM genes (Col1A1, α-SMA, fibronectin, vitronectin), and proliferation rates. The effects of αVβ3 integrin blockade (with cilengitide) and silencing (with a pool of four predesigned αVβ3 integrin siRNAs) on ECM gene expression and proliferation rates were evaluated using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting in the human NLC cells cultured on soft (4 kPa) and stiff (100 kPa) substrate and in GLC cells grown on standard plastic plates. Results: αVβ3 integrin gene and protein expression were enhanced (p < 0.05) in GLC cells as compared to NLC. Both cilengitide and siRNA significantly reduced αVβ3 expression in GLC. When NLC were grown in the stiff substrate, cilengitide and siRNA also significantly reduced the increased expression in αVβ3, ECM components, and proliferation rate. Conclusions: Here, we provide evidence of cilengitide- and siRNA-mediated silencing of αVβ3 integrin expression, and inhibition of ECM synthesis in LC cells. Therefore, αVβ3 integrin may be a promising target for the development of novel anti-fibrotic therapies for treating the LC cupping of the ONH in glaucoma. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV‐2), responsible for the COVID-19, may lead to multiple organ dysfunctions and long-term complications. The induction of microvascular dysfunction is regarded as a main player in these pathological processes. To investigate the possible impact of SARS-CoV-2-induced endothelial-to-mesenchymal transition (EndMT) on fibrosis in "long-COVID" syndrome, we used primary cultures of human microvascular cells derived from the lungs, as the main infection target, compared to cells derived from different organs (dermis, heart, kidney, liver, brain) and to the HUVEC cell line. To mimic the virus action, we used mixed SARS-CoV-2 peptide fragments (PepTivator®) of spike (S), nucleocapsid (N), and membrane (M) proteins. TGFβ2 and cytokine mix (IL-1β, IL-6, TNFα) were used as positive controls. The percentage of cells positive to mesenchymal and endothelial markers was quantified by high content screening. We demonstrated that S+N+M mix induces irreversible EndMT in all analyzed endothelial cells via the TGFβ pathway, as demonstrated by ApoA1 treatment. We then tested the contribution of single peptides in lung and brain cells, demonstrating that EndMT is triggered by M peptide. This was confirmed by transfection experiment, inducing the endogenous expression of the glycoprotein M in lung-derived cells. In conclusion, we demonstrated that SARS-CoV-2 peptides induce EndMT in microvascular endothelial cells from multiple body districts. The different peptides play different roles in the induction and maintenance of the virus-mediated effects, which are organ-specific. These results corroborate the hypothesis of the SARS-CoV-2-mediated microvascular damage underlying the multiple organ dysfunctions and the long-COVID syndrome. [ABSTRACT FROM AUTHOR]

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT. [ABSTRACT FROM AUTHOR]

Introduction: Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation. Methods: Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts. Results: Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h. Conclusion: In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS. [ABSTRACT FROM AUTHOR]

Introduction: The COVID-19 pandemic caused by the SARS-CoV-2 virus has underscored the urgent necessity for the development of antiviral compounds that can effectively target coronaviruses. In this study, we present the first evidence of the antiviral efficacy of hyperforin, a major metabolite of St. John's wort, for which safety and bioavailability in humans have already been established. Methods: Antiviral assays were conducted in cell culture with four human coronaviruses: three of high virulence, SARS-CoV-2, SARS-CoV, and MERS-CoV, and one causing mild symptoms, HCoV-229E. The antiviral activity was also evaluated in human primary airway epithelial cells. To ascertain the viral step inhibited by hyperforin, time-of-addition assays were conducted. Subsequently, a combination assay of hyperforin with remdesivir was performed. Results: The results demonstrated that hyperforin exhibited notable antiviral activity against the four tested human coronaviruses, with IC50 values spanning from 0.24 to 2.55 µM. Kinetic studies indicated that the observed activity occur at a post-entry step, potentially during replication. The antiviral efficacy of hyperforin was additionally corroborated in human primary airway epithelial cells. The results demonstrated a reduction in both intracellular and extracellular SARS-CoV-2 viral RNA, confirming that hyperforin targeted the replication step. Finally, an additive antiviral effect on SARS-CoV-2 was observed when hyperforin was combined with remdesivir. Discussion: In conclusion, hyperforin has been identified as a novel pancoronavirus inhibitor with activity in human primary airway epithelial cells, a preclinical model for coronaviruses. These findings collectively suggest that hyperforin has potential as a candidate antiviral agent against current and future human coronaviruses. [ABSTRACT FROM AUTHOR]

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus, allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus, C. albicans, and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus, and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus, addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions. [ABSTRACT FROM AUTHOR]

Mesoporous bioactive glass nanoparticles (MBGNs) doped with therapeutical ions present multifunctional systems that enable a synergistic outcome through the dual delivery of drugs and ions. The aim of this study was to evaluate influence of co-doping with strontium and magnesium ions (SrMg-MBGNs) on the properties of MBGNs. A modified microemulsion-assisted sol–gel synthesis was used to obtain particles, and their physicochemical properties, bioactivity, and drug-loading/release ability were evaluated. Indirect biological assays using 2D and 3D cell culture models on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and endothelial EA.hy926 cells, respectively, were used to determine biocompatibility of MBGNs, their influence on alkaline phosphatase (ALP) production, calcium deposition, and cytoskeletal organization. Results showed that Sr,Mg-doping increased pore volume and solubility, and changed the mesoporous structure from worm-like to radial–dendritic, which led to a slightly accelerated drug release compared to pristine MBGNs. Biological assays confirmed that particles are biocompatible, and have ability to slightly induce ALP production and calcium deposition of hBM-MSCs, as well as to significantly improve the proliferation of EA.hy926 compared to biochemical stimulation via vascular endothelial growth factor (VEGF) administration or regular media. Fluorescence staining revealed that SrMg-MBGNs had a similar effect on EA.hy926 cytoskeletal organization to the VEGF group. In conclusion, Sr,Mg-MBGNs might be considered promising biomaterial for biomedical applications. [ABSTRACT FROM AUTHOR]

Mast cells are the major effector cells that mediate IgE-dependent allergic reactions. We sought to use integrated network analysis to identify genomic biomarkers associated with high response in IgE-mediated activation of primary human mast cells. Primary human mast cell cultures derived from 262 normal donors were categorized into High, Average and Low responder groups according to their activation response profiles. Transcriptome analysis was used to identify genes that were differentially expressed in different responder cultures in their baseline conditions, and the data were analyzed by constructing a personalized perturbed profile (PEEP). For upregulated genes, the construction of PEEP for each individual sample of all three responder groups revealed that High responders exhibited a higher percentage of "perturbed" samples whose PEEP values lay outside the normal range of expression. Moreover, the integration of PEEP of four selected upregulated genes into distinct sets of combinatorial profiles demonstrated that the specific pattern of upregulated expression of these four genes, in a tandem combination, was observed exclusively among the High responders. In conclusion, this combinatorial approach was useful in identifying a set of genomic biomarkers that are associated with high degranulation response in human mast cell cultures derived from the blood of a cohort of normal donors. [ABSTRACT FROM AUTHOR]

Regeneration of articular cartilage remains a challenge for patients who have undergone cartilage injury, osteochondritis dissecans and osteoarthritis. Here, we describe a new recombinant silk fibroin with basic fibroblast growth factor (bFGF) binding peptide, which has a genetically introduced sequence PLLQATLGGGS, named P7. In this study, we cultured a human mesenchymal cell line derived from bone marrow, UE6E7-16, in wild-type fibroin sponge (FS) and recombinant silk fibroin sponge with P7 peptide (P7 FS). We compared cell proliferation, chondrogenic differentiation and cartilaginous tissue formation between the two types of sponge. After stimulation with bFGF at 3 ng/mL, P7 FS showed significantly higher cell growth (1.2-fold) and higher cellular DNA content (5.6-fold) than did wild-type FS. To promote chondrogenic differentiation, cells were cultured in the presence of TGF-β at 10 ng/mL for 28 days. Immunostaining of P7 FS showed SOX9-positive cells comparable to wild-type FS. Alcian-Blue staining of P7 FS also showed cartilaginous tissue formation equivalent to wild-type FS. A significant increase in cell proliferation in P7 FS implies future clinical application of this transgenic fibroin for regeneration of articular cartilage. To produce cartilaginous tissue efficiently, transgenic fibroin sponges and culture conditions must be improved. Such changes should include the selection of growth factors involved in chondrogenic differentiation and cartilage formation. [ABSTRACT FROM AUTHOR]

Multifunctional materials that combine antimicrobial properties with the ability to stimulate bone formation are needed to overcome the problem of infected bone defects. As a novel approach, a new composite based on bioactive glass nanoparticles in a simple system of SiO2–CaO (BG) coated with NH4[Cu3(μ3-OH)(μ3-4-carboxypyrazolato)3] (Cu-MOF) with additionally anchored silver nanoparticles (AgNPs) was proposed. Ag@Cu-MOF@BG obtained by the spin coating approach in the form of a disc was characterized using PXRD, ATR-FTIR, XPS, ICP-OES, and TEM. Importantly, the material retained its bioactivity, although ion exchange in the bioactive glass administered as a disc is limited. Hydroxyapatite (HA) formation was identified in TEM images after 7 days of immersion of the composite in a physiological-like buffer (pH 7.4, 37 °C). The Cu and Ag contents of Ag@Cu-MOF@BG were as low as 0.013 and 0.018 wt% respectively, but the slow release of the AgNPs ensured its antibacterial nature. Ag@Cu-MOF@BG exhibited antibacterial activity against all tested bacteria (E. coli, S. aureus, P. aeruginosa, and K. pneumoniae) with the diameter of the inhibition zones of their growth between 8 and 10 mm and the reduction index determined to be ≥3. Moreover, the biocompatibility of the new composite has been demonstrated, as shown by cell culture assays with human dermal fibroblasts (HDFs). The results from the migration test also proved that the HDF cell's phenotypic properties were not changed, and the cell adhesion and migration ability were the same as in control indirect assays. [ABSTRACT FROM AUTHOR]

Key features of chronic kidney disease (CKD) include tubulointerstitial inflammation and fibrosis. Protease activated receptor-2 (PAR2), a G-protein coupled receptor (GPCR) expressed by the kidney proximal tubular cells, induces potent proinflammatory responses in these cells. The hypothesis tested here was that PAR2 signalling can contribute to both inflammation and fibrosis in the kidney by transactivating known disease associated pathways. Using a primary cell culture model of human kidney tubular epithelial cells (HTEC), PAR2 activation induced a concentration dependent, PAR2 antagonist sensitive, secretion of TNF, CSF2, MMP-9, PAI-1 and CTGF. Transcription factors activated by the PAR2 agonist 2F, including NFκB, AP1 and Smad2, were critical for production of these cytokines. A TGF-β receptor-1 (TGF-βRI) kinase inhibitor, SB431542, and an EGFR kinase inhibitor, AG1478, ameliorated 2F induced secretion of TNF, CSF2, MMP-9, and PAI-1. Whilst an EGFR blocking antibody, cetuximab, blocked PAR2 induced EGFR and ERK phosphorylation, a TGF-βRII blocking antibody failed to influence PAR2 induced secretion of PAI-1. Notably simultaneous activation of TGF-βRII (TGF-β1) and PAR2 (2F) synergistically enhanced secretion of TNF (2.2-fold), CSF2 (4.4-fold), MMP-9 (15-fold), and PAI-1 (2.5-fold). In summary PAR2 activates critical inflammatory and fibrotic signalling pathways in human kidney tubular epithelial cells. Biased antagonists of PAR2 should be explored as a potential therapy for CKD. [ABSTRACT FROM AUTHOR]

Introduction: Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS). High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19 patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. Methods: We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. Results: We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Discussion: Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19. [ABSTRACT FROM AUTHOR]

Injectable hydrogels for cancer immunotherapy are effective for both active and passive approaches. Tumor‐infiltrating lymphocyte (TIL) immunoshaping can change the tumor microenvironment to favor tumor cell elimination. The primary objective of immunoshaping is to reduce regulatory T‐cells (Tregs), which can enhance the effectiveness of ex vivo immune cell therapy in solid tumors. A shear‐thinning injectable hydrogel that consists of gelatin and Laponite (Gel‐Lap) is used in this study. By optimizing the formulation, their immunotherapeutic and anti‐tumor properties are examined. Gemcitabine (GEM), an anti‐metabolite cancer chemotherapy agent, is loaded into a Gel‐Lap hydrogel (immunogel). The study compares the effects of immunogel on 4T1 inoculated breast cancer animal models. Results show that immunogel increases survival rates and significantly inhibits metastasis. The Treg cell population reduction is observed up to 70% in TILs and splenocyte population in line with CD8+ T‐cells population increment in inguinal lymph nodes near the tumor region; the CD8+ T‐cells function may be mediated through overexpression of eomesodermin (EOMES) as cytotoxic T lymphocyte (CTL) activation transcription factor. The human 3D cell culture model confirmed results in animal data demonstrating T‐cell migration through the hydrogel and anticancer efficacy. Local delivery of GEM using our silicate‐based hydrogel holds promise for editing tumor microenvironment in favor of systemic immune responses. [ABSTRACT FROM AUTHOR]

As electronic cigarettes (e-cigarettes, vaping, etc.) are considered a safer alternative to traditional smoking, they are gaining popularity, especially among adolescents (10–14 and 14–18 years) and young adults (18–25 years). They are used by millions of users worldwide, and new generations of e-cigarette devices are being introduced. Some reports have suggested that e-cigarettes have harmful effects on human health, which is why it is important to introduce restrictions on the use of e-cigarettes by young people. The aerosols produced when the e-liquid is heated contain a complex of gases, toxic substances and various types of flavoring chemicals, which are then inhaled by users. In addition, when the products in the e-liquid are thermally degraded, more harmful reactive substances are produced. Exposure to e-cigarette aerosols appears to have harmful effects on human health, but it is critical that our understanding of these effects be expanded and that data be collected on the long-term effects of the substances in e-cigarette aerosols on the human body. Data on the various health risks associated with the use of e-cigarettes are mainly based on in vitro studies using established cell lines or cultured human cells, or on animal models. The substances contained in e-cigarette liquids and their aerosol – including the solvents propylene glycol and vegetable glycerin – are known to cause organ and tissue irritation in the upper respiratory tract when inhaled. The aim of this narrative review is to present our current knowledge on the effects of the chemical components of e-cigarette aerosols on the nasal cavity, trachea, bronchi, and organs of the conducting part of the respiratory system. [ABSTRACT FROM AUTHOR]

Background: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. Methods: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. Results: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. Conclusions: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors. [ABSTRACT FROM AUTHOR]

A genetically engineered analog of human kappa casein was developed and obtained. It has been found to have the ability to induce apoptosis of human cancer cells in culture and inhibit the growth and metastasis of animal and human tumors. Based on the recombinant protein obtained, a new antitumor drug RAHMP (recombinant analogs of human milk peptides) has been developed, which is a concentrate for the preparation of an infusion solution. The main function of this drug is prevention and treatment of human breast cancer. The purpose of this work was to develop and validate a technique for the quantitative determination of impurities in the RAHMP substance by reverse-phase high-performance liquid chromatography. Criteria for assessing the quality of the substance were the area of the main peak, which should be at least 95% of the total area of all detected peaks, and the sum of the areas of additional peaks should be no more than 5% of the total area of all peaks. The permissible content of foreign impurities in the substance should be no more than 5%. The relative standard deviation of the impurity concentration at all levels of the concentrations under consideration does not exceed 2.0%, which indicates satisfactory convergence of the results. [ABSTRACT FROM AUTHOR]

Channa striatus, also known as haruan, is traditionally used in Malaysia for its wound-healing properties. This study evaluated the wound healing potential of Channa striatus water extract (CSWE) and identified key compounds promoting fibroblast cell growth. Channa striatus were deboned to maximize fillet retention. CSWE was obtained through aqueous extraction, and its physicochemical properties were analyzed, including pH, rheological characteristics, moisture content, amino acid composition, and arachidonic acid presence. The effective concentration (EC50) of the sample was determined using a 2-D cell culture system with human fibroblast cells (IMR-90) over three days. Results showed CSWE had a near-neutral pH (6.34 ± 0.01) and high moisture content (97.3 ± 0.01%). The extract displayed Newtonian fluid behavior with a viscosity of 1.50 ± 0.31 mPa.s. CSWE contained essential amino acid glycine and arachidonic acid, important for wound healing, but in low concentrations. These low concentrations did not significantly promote IMR-90 cell growth (p>0.05) compared to the control. Consequently, EC50 values for CSWE were invalid due to over-dilution from a high fish weight-to-solvent ratio. Despite this, IMR-90 cell growth rates remained consistent across different CSWE concentrations, with no observed mortality during the three-day incubation. Overall, IMR-90 cells exhibited insignificant growth, even with arachidonic acid and glycine at low concentrations during treatment. [ABSTRACT FROM AUTHOR]

The transcriptional response to hypoxia is largely regulated by the hypoxia-inducible factors (HIFs), which induce the expression of genes involved in glycolysis, angiogenesis, proliferation, and migration. Virtually all cell culture-based hypoxia experiments have used near-atmospheric (18% O2) oxygen levels as the baseline for comparison with hypoxia. However, this is hyperoxic compared with mammalian tissue microenvironments, where oxygen levels range from 2% to 9% O2 (physioxia). Thus, these experiments actually compare hyperoxia to hypoxia. To determine how the baseline O2 level affects the subsequent response to hypoxia, we cultured PC-3 prostate cancer cells in either 18% or 5% O2 for 2 wk before exposing them to hypoxia (∼1.1% pericellular O2) for 12–48 h. RNA-seq revealed that the transcriptional response to hypoxia was dependent on the baseline O2 level. Cells grown in 18% O2 before hypoxia exposure showed an enhanced induction of HIF targets, particularly genes involved in glucose metabolism, compared with cells grown in physioxia before hypoxia. Consistent with this, hypoxia significantly increased glucose consumption and metabolic activity only in cells previously cultured in 18% O2, but not in cells preadapted to 5% O2. Transcriptomic analyses also indicated effects on cell proliferation and motility, which were followed up by functional assays. Although unaffected by hypoxia, both proliferation and migration rates were greater in cells cultured in 5% O2 versus 18% O2. We conclude that an inappropriately hyperoxic starting condition affects the transcriptional and metabolic responses of PC-3 cells to hypoxia, which may compromise experiments on cancer metabolism in vitro. NEW & NOTEWORTHY: Although human cell culture models have been instrumental to our understanding of the mechanisms involved in the cellular response to hypoxia, in virtually all experiments, cells are routinely cultured in near-atmospheric (∼18% O2) oxygen levels, which are hyperoxic relative to physiological conditions in vivo. Here, we show for the first time that cells cultured in physiological O2 levels (5% O2) respond differently to subsequent hypoxia than cells grown at 18%. [ABSTRACT FROM AUTHOR]

In recent years, significant efforts have been made to understand oral drug absorption mechanisms and to develop new research models. These studies have been conducted using both in vitro and in vivo models and play a crucial role in determining the rate and extent of drug absorption in the intestines. Specifically, the permeability value (Peff value) is a common measure used to assess drug intestinal passage. Today, there is an increasing need for reliable gastrointestinal absorption models that can be used in preclinical studies to develop new drugs and appropriate dosages. However, collaboration and integration across different disciplines are important for further advancements in this field. Strengthening connections between pharmaceutical, biopharmaceutical, biochemical, and physiological research areas can contribute to a better understanding of drug absorption mechanisms and biopharmaceutical progress. Particularly, the use of animal models that mimic human intestinal drug permeability and the role of human cell culture models in investigating drug absorption in the intestine are highlighted as significant steps in this regard. Such research enables a more detailed examination of intestinal drug diffusion processes, which are crucial for the biopharmaceutical advancement of pharmaceutical compounds. Understanding factors such as the interactions between drug molecules and membrane transport molecules that affect the intestinal Peff value can further advance the assessment and improvement of drug absorption processes. [ABSTRACT FROM AUTHOR]

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Glioblastoma remains an incurable form of brain tumor. Existing therapeutic methods can only produce slight prolongation of the lives of patients with this diagnosis. There is therefore a need to seek new approaches and develop new methods of treating glioblastoma. We describe here the principle of an approach to glioblastoma tumor cells which consists of targeted inhibition of the proliferation of L1CAM-positive cells using aptamers. L1CAM is regarded as a marker of gliomastem cells, whose presence in a tumor may be responsible for resistance to treatment. The studies reported here led to selection of the yly12 aptamer from a panel of aptamers for L1CAM; this aptamer was shown to have antiproliferative effects which were more powerful in the case of human glioblastoma cells with elevated L1CAM expression. Thus, this approach may solve the issue of glioblastoma cell resistance and prevent tumor recurrence by acting on glioma stem cells. [ABSTRACT FROM AUTHOR]

Abstract Epilepsy is the most common chronic neurological disease, affecting nearly 1%–2% of the world's population. Current pharmacological treatment and regimen adjustments are aimed at controlling seizures; however, they are ineffective in one‐third of the patients. Although neuronal hyperexcitability was previously thought to be mainly due to ion channel alterations, current research has revealed other contributing molecular pathways, including processes involved in cellular signaling, energy metabolism, protein synthesis, axon guidance, inflammation, and others. Some forms of drug‐resistant epilepsy are caused by genetic defects that constitute potential targets for precision therapy. Although such approaches are increasingly important, they are still in the early stages of development. This review aims to provide a summary of practical aspects of the employment of in vitro human cell culture models in epilepsy diagnosis, treatment, and research. First, we briefly summarize the genetic testing that may result in the detection of candidate pathogenic variants in genes involved in epilepsy pathogenesis. Consequently, we review existing in vitro cell models, including induced pluripotent stem cells and differentiated neuronal cells, providing their specific properties, validity, and employment in research pipelines. We cover two methodological approaches. The first approach involves the utilization of somatic cells directly obtained from individual patients, while the second approach entails the utilization of characterized cell lines. The models are evaluated in terms of their research and clinical benefits, relevance to the in vivo conditions, legal and ethical aspects, time and cost demands, and available published data. Despite the methodological, temporal, and financial demands of the reviewed models they possess high potential to be used as robust systems in routine testing of pathogenicity of detected variants in the near future and provide a solid experimental background for personalized therapy of genetic epilepsies. Plain Language Summary Epilepsy affects millions worldwide, but current treatments fail for many patients. Beyond traditional ion channel alterations, various genetic factors contribute to the disorder's complexity. This review explores how in vitro human cell models, either from patients or from cell lines, can aid in understanding epilepsy's genetic roots and developing personalized therapies. While these models require further investigation, they offer hope for improved diagnosis and treatment of genetic forms of epilepsy.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability. [ABSTRACT FROM AUTHOR]

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis produces five classes of serine/glycine lipids that are recovered in lipid extracts from periodontitis‐afflicted teeth and diseased gingival tissues, particularly at sites of periodontitis. Because these lipids are recovered in diseased gingival tissues, the purpose of the present study was to evaluate the capacity of cultured human gingival fibroblasts (HGF), keratinocytes, and macrophages to hydrolyze these lipids. We hypothesize that one or more of these cell types will hydrolyze the serine/glycine lipids. The primary aim was to treat these cell types for increasing time in culture with individual highly enriched serine/glycine lipid preparations. At specified times, cells and culture media samples were harvested and extracted for hydrolysis products. The serine/glycine lipids and hydrolysis products were quantified using liquid chromatography–mass spectrometry (LC–MS) and free fatty acids were quantified using gas chromatograph–mass spectrometer. LC–MS analysis used two different mass spectrometric methods. This study revealed that treatment of HGF or macrophage (THP1) cells with lipid (L) 654 resulted in breakdown to L342 and subsequent release into culture medium. However, L654 was converted only to L567 in gingival keratinocytes. By contrast, L1256 was converted to L654 by fibroblasts and macrophages but no further hydrolysis or release into medium was observed. Gingival keratinocytes showed no hydrolysis of L1256 to smaller lipid products but because L1256 was not recovered in these cells, it is not clear what hydrolysis products are produced from L1256. Although primary cultures of gingival fibroblasts and macrophages are capable of hydrolyzing specific serine/glycine lipids, prior analysis of lipid extracts from diseased gingival tissues revealed significantly elevated levels of L1256 in diseased tissues. These results suggest that the hydrolysis of bacterial lipids in gingival tissues may reduce the levels of specific lipids, but the hydrolysis of L1256 is not sufficiently rapid to prevent significant accumulation at periodontal disease sites. [ABSTRACT FROM AUTHOR]

Epilepsy is the most common chronic neurological disease, affecting nearly 1%–2% of the world's population. Current pharmacological treatment and regimen adjustments are aimed at controlling seizures; however, they are ineffective in one‐third of the patients. Although neuronal hyperexcitability was previously thought to be mainly due to ion channel alterations, current research has revealed other contributing molecular pathways, including processes involved in cellular signaling, energy metabolism, protein synthesis, axon guidance, inflammation, and others. Some forms of drug‐resistant epilepsy are caused by genetic defects that constitute potential targets for precision therapy. Although such approaches are increasingly important, they are still in the early stages of development. This review aims to provide a summary of practical aspects of the employment of in vitro human cell culture models in epilepsy diagnosis, treatment, and research. First, we briefly summarize the genetic testing that may result in the detection of candidate pathogenic variants in genes involved in epilepsy pathogenesis. Consequently, we review existing in vitro cell models, including induced pluripotent stem cells and differentiated neuronal cells, providing their specific properties, validity, and employment in research pipelines. We cover two methodological approaches. The first approach involves the utilization of somatic cells directly obtained from individual patients, while the second approach entails the utilization of characterized cell lines. The models are evaluated in terms of their research and clinical benefits, relevance to the in vivo conditions, legal and ethical aspects, time and cost demands, and available published data. Despite the methodological, temporal, and financial demands of the reviewed models they possess high potential to be used as robust systems in routine testing of pathogenicity of detected variants in the near future and provide a solid experimental background for personalized therapy of genetic epilepsies. Plain Language Summary: Epilepsy affects millions worldwide, but current treatments fail for many patients. Beyond traditional ion channel alterations, various genetic factors contribute to the disorder's complexity. This review explores how in vitro human cell models, either from patients or from cell lines, can aid in understanding epilepsy's genetic roots and developing personalized therapies. While these models require further investigation, they offer hope for improved diagnosis and treatment of genetic forms of epilepsy. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNAbinding protein 43 (TDP-43). We show here that coexpression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solventexposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43–induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease. [ABSTRACT FROM AUTHOR]

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. We identified plasma insulin-like growth factor 1 (IGF1) as an independent risk factor in patients with AAA by correlating plasma IGF1 with risk. Smooth muscle cell– or fibroblast-specific knockout of Igf1r, the gene encoding the IGF1 receptor (IGF1R), attenuated AAA formation in two mouse models of AAA induced by angiotensin II infusion or CaCl2 treatment. IGF1R was activated in aortic aneurysm samples from human patients and mice with AAA. Systemic administration of IGF1C, a peptide fragment of IGF1, 2 weeks after disease development inhibited AAA progression in mice. Decreased AAA formation was linked to competitive inhibition of IGF1 binding to its receptor by IGF1C and modulation of downstream alpha serine/threonine protein kinase (AKT)/mammalian target of rapamycin signaling. Localized application of an IGF1C-loaded hydrogel was developed to reduce the side effects observed after systemic administration of IGF1C or IGF1R antagonists in the CaCl2-induced AAA mouse model. The inhibitory effect of the IGF1C-loaded hydrogel administered at disease onset on AAA formation was further evaluated in a guinea pig-to-rat xenograft model and in a sheep-to-minipig xenograft model of AAA formation. The therapeutic efficacy of IGF1C for treating AAA was tested through extravascular delivery in the sheep-to-minipig model with AAA established for 2 weeks. Percutaneous injection of the IGF1C-loaded hydrogel around the AAA resulted in improved vessel flow dynamics in the minipig aorta. These findings suggest that extravascular administration of IGF1R antagonists may have translational potential for treating AAA. Editor's summary: An abdominal aortic aneurysm (AAA) is life threatening and has limited therapeutic options. Wei et al. showed that localized inhibition of insulin-like growth factor 1 receptor (IGF1R) protected against AAA. Humans with AAA had increased IGF1 and IGF1R in aortic tissue. In mice treated with angiotensin II infusion or calcium chloride to induce AAA, IGF1R was increased, and genetic deletion of Igf1r in smooth muscle cells or fibroblasts decreased AAA pathology. IGF1C, a peptide fragment of IGF1, inhibited IGF1R in human cells in culture and in the two mouse models, resulting in decreased AAA pathology. In both a rat and minipig model of aorta xenotransplantation to induce AAA, the application of a hydrogel containing IGF1C to the aorta decreased AAA pathology. Inhibiting IGF1R in AAA may be a treatment for slowing AAA. —Brandon Berry [ABSTRACT FROM AUTHOR]

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer (QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish (Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications. [ABSTRACT FROM AUTHOR]

Background: Dysregulation of the endo-lysosomal–autophagy pathway has been identified as a critical factor in the pathology of various demyelinating neurodegenerative diseases, including peripheral neuropathies. This pathway plays a crucial role in transporting newly synthesized myelin proteins to the plasma membrane in myelinating Schwann cells, making these cells susceptible to lysosome-related dysfunctions. Nevertheless, the specific impact of lysosomal dysfunction in Schwann cells and its contribution to neurodegeneration remain poorly understood. Methods: We aim to mimic lysosomal dysfunction in Schwann cells using chloroquine, a lysosomal dysfunction inducer, and to monitor lysosomal leakiness, Schwann cell viability, and apoptosis over time. Additionally, due to the ethical and experimental issues associated with cell isolation and the culturing of human Schwann cells, we use human dental pulp stem cell-derived Schwann cells (DPSC-SCs) as a model in our study. Results: Chloroquine incubation boosts lysosomal presence as demonstrated by an increased Lysotracker signal. Further in-depth lysosomal analysis demonstrated an increased lysosomal size and permeability as illustrated by a TEM analysis and GAL3-LAMP1 staining. Moreover, an Alamar blue assay and Caspase-3 staining demonstrates a reduced viability and increased apoptosis, respectively. Conclusions: Our data indicate that prolonged lysosomal dysfunction leads to lysosomal permeability, reduced viability, and eventually apoptosis in human DPSC-SCs. [ABSTRACT FROM AUTHOR]

Empagliflozin (EMPA) is a potent, competitive and selective sodium glucose cotransporter-2 (SGLT-2) inhibitor that ameliorates blood glucose with the insulin-independent manner. EMPA reduces weight and blood pressure of patients with type 2 diabetes mellitus (T2DM) without developing hypoglycemic risk. To the best of our knowledge, its safety profiling has not been evaluated on human blood cell cultures yet. Again, the embryotoxicity potential by EMPA is still unclear. Therefore, in this investigation we aimed to evaluate the in vitro cytotoxic, genotoxic and embryotoxic damage potential as well as antioxidative/oxidative effects by EMPA in cultured human blood and human pluripotent embryonal carcinoma NT2 cells for the first time. Cell cultures (n = 5) were exposed to different concentrations ranging from 3.25 to 100 mg/L of EMPA for 48 and 72 h. Cell viability was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays. The alterations in antioxidant/oxidant activity were monitored via measuring the total antioxidant capacity (TAC) and total oxidative stress (TOS) levels. For evaluating the genotoxicity of EMPA chromosomal aberration (CA) assay was performed. The present results revealed that EMPA did not induce cytotoxic or genotoxic damage on healthy human blood cells. Moreover, EMPA exerted non-embryotoxic property and supported antioxidative capacity and decreased the oxidative stress in cultured human blood cells. Our results supported the safe and advantageous use of EMPA for the treatment of T2DM. [ABSTRACT FROM AUTHOR]

In recent years, increasing interest has been accorded to polyester-based polymer microstructures, driven by their promising potential as advanced drug delivery systems. This study presents the preparation and characterization of new polymeric microparticles based on poly(ethylene brassylate-co-squaric acid) loaded with norfloxacin, a broad-spectrum antibiotic. Polymacrolactone was synthesised in mild conditions through the emulsion polymerization of bio-based and renewable monomers, ethylene brassylate, and squaric acid. The microparticles were obtained using the precipitation technique and subsequently subjected to comprehensive characterization. The impact of the copolymer/drug ratio on various properties of the new system was systematically evaluated, confirming the structure of the copolymer and the encapsulation of norfloxacin. The microspheres are approximately spherical and predominantly homogeneously distributed. The average hydrodynamic diameter of the microparticles falls between 400 and 2000 nm, a decrease that is observed with the increase in norfloxacin content. All samples showed good encapsulation efficiency and drug loading capacity, with the highest values obtained for microparticles synthesised using an equal ratio of copolymer and drug. In vitro drug release results disclose that norfloxacin molecules are released in a sustained biphasic manner for up to 24 h. Antimicrobial activity was also studied, with samples showing very good activity against E. coli and moderate activity against S. aureus and E. faecalis. In addition, HDFA human fibroblast cell cultures demonstrated the cytocompatibility of the microparticles. [ABSTRACT FROM AUTHOR]

The complement system is a family of proteins that facilitate immune resistance by attacking microbes to decrease pathogen burden. As a result, deficiencies of certain complement proteins result in recurrent bacterial infections, and can also result in acute lung injury (ALI). We and others have shown that C3 is present in both immune and nonimmune cells, and modulates cellular functions such as metabolism, differentiation, cytokine production, and survival. Although the emerging roles of the complement system have implications for host responses to ALI, key questions remain vis-a-vis the lung epithelium. In this review, we summarize our recent article in which we reported that during Pseudomonas aeruginosa-induced ALI, lung epithelial cell-derived C3 operates independent of liver-derived C3. Specifically, we report the use of a combination of human cell culture systems and global as well as conditional knockout mouse models to demonstrate the centrality of lung epithelial cell-derived C3. We also summarize recent articles that have interrogated the role of intracellular and/or locally derived C3 in host defense. We propose that C3 is a highly attractive candidate for enhancing tissue resilience in lung injury as it facilitates the survival and function of the lung epithelium, a key cell type that promotes barrier function. [ABSTRACT FROM AUTHOR]

Being the green gold of the future, cyanobacteria have recently attracted considerable interest worldwide. This study investigates the adaptability and biocompatibility of the cyanobacterial strain Synechococcus sp. PCC 7002 with human dermal cells, focusing on its potential application in biomedical contexts. First, we investigated the adaptability of Synechococcus PCC 7002 bacteria to human cell culture conditions. Next, we evaluated the biocompatibility of cyanobacteria with common dermal cells, like 3T3 fibroblasts and HaCaT keratinocytes. Therefore, cells were directly and indirectly cocultured with the corresponding cells, and we measured metabolic activity (AlamarBlue assay) and proliferation (cell count and PicoGreen assay). The lactate dehydrogenase (LDH) assay was performed to determine the cytotoxic effect of cyanobacteria and their nutrition medium on human dermal cells. The cyanobacteria exhibited exponential growth under conventional human cell culture conditions, with the temperature and medium composition not affecting their viability. In addition, the effect of illumination on the proliferation capacity was investigated, showing a significant impact of light exposure on bacterial growth. The measured oxygen production under hypoxic conditions demonstrated a sufficient oxygen supply for further tissue engineering approaches depending on the number of bacteria. There were no significant adverse effects on human cell viability and growth under coculture conditions, whereas the LDH assay assessed signs of cytotoxicity regarding 3T3 fibroblasts after 2 days of coculturing. These negative effects were dismissed after 4 days. The findings highlight the potential of Synechococcus sp. PCC 7002 for integration into biomedical approaches. We found no cytotoxicity of cyanobacteria on 3T3 fibroblasts and HaCaT keratinocytes, thus paving the way for further in vivo studies to assess long-term effects and systemic reactions. [ABSTRACT FROM AUTHOR]

Cardiac pacing with temporary epicardial pacing wires (TEPW) is used to treat rhythm disturbances after cardiac surgery. Occasionally, TEPW cannot be mechanically extracted and remain in the thorax, where they may rarely cause serious complications like migration and infection. We aim to develop bioresorbable TEPW that will dissolve over time even if postoperative removal is unsuccessful. In the present study, we demonstrate a completely bioresorbable design using molybdenum (Mo) as electric conductor and the resorbable polymers poly(D, L-lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) for electrically insulating double-coating. We compared the pacing properties of these Mo TEPW demonstrators to conventional steel TEPW in Langendorff-perfused rat hearts and observed similar functionality. In vitro , static immersion tests in simulated body fluid for up to 28 days elucidated the degradation behaviour of uncoated Mo strands and the influence of polymer coating thereon. Degradation was considerably reduced in double-coated Mo TEPW compared to the uncoated and the PLGA-coated condition. Furthermore, we confirmed good biocompatibility of Mo degradation products in the form of low cytotoxicity in cell cultures of human cardiomyocytes and cardiac fibroblasts. Temporary pacing wires are routinely implanted on the heart surface to treat rhythm disturbances in the days following cardiac surgery. Subsequently, these wires are to be removed. When removal attempts are unsuccessful, wires are cut at skin level and the remainders are left inside the chest. Retained fragments may migrate within the body or become a centre of infection. These complications may be prevented using resorbable pacing wires. We manufactured completely resorbable temporary pacing wires using molybdenum as electrical conductor and assessed their function, degradation and biological compatibility. Our study represents an important step in the development of a safer approach to the treatment of rhythm disturbances after cardiac surgery. [Display omitted] [ABSTRACT FROM AUTHOR]

Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with β-galactosidase (β-gal) ex vivo. Here, we describe a progressive accumulation of β-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of β-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating β-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated β-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes. [ABSTRACT FROM AUTHOR]

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology. [ABSTRACT FROM AUTHOR]

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-β estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy. [ABSTRACT FROM AUTHOR]

The Mpox (formerly named Monkeypox) virus is the etiological cause of a recent multi-country outbreak, with thousands of distinct cases detected outside the endemic areas of Africa as of December 2023. In this article, we analyze the sequences of full genomes of Mpox virus from Europe and compare them with all available Mpox sequences of historical relevance, annotated by year and geographic origin, as well as related Cowpox and Variola (smallpox) virus sequences. Our results show that the recent outbreak is most likely originating from the West African clade of Mpox, with >99 % sequence identity with sequences derived from historical and recent cases, dating from 1971 to 2017. We analyze specific mutations occurring in viral proteins between the current outbreak, previous Mpox and Cowpox sequences, and the historical Variola virus. Genome-wide sequence analysis of the recent outbreak and other Mpox/Cowpox/Variola viruses shows a very high conservation, with 97.9 % (protein-based) and 97.8 % (nucleotide-based) sequence identity. We identified significant correlation in human transcriptional responses as well, with a conserved immune pathway response induced in human cell cultures by the three families of Pox virus. The similarities identified between the major strains of Pox viruses, as well as within the Mpox clades, both at the genomic and transcriptomic levels, provide a molecular basis for the observed efficacy of Variola vaccines in other Poxviruses. [ABSTRACT FROM AUTHOR]

Cellular senescence and the senescence‐associated secretory phenotype (SASP) contribute to age‐related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP‐related inflammation were lower 1 week after the final dose of oral intermittent (1 week on—2 weeks off—1 weeks on dosing) fisetin supplementation. Old fisetin‐supplemented mice had higher endothelial function. Leveraging old p16‐3MR mice, a transgenic model allowing genetic clearance of p16INK4A‐positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle‐ but not fisetin‐treated mice increased endothelium‐dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular‐ and mitochondrial‐related oxidative stress. Arterial stiffness was lower in fisetin‐treated mice. Ex vivo genetic senolysis in aorta rings from p16‐3MR mice did not further reduce mechanical wall stiffness in fisetin‐treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age‐related arterial dysfunction. [ABSTRACT FROM AUTHOR]

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (K M and V max) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed. • Novel isoform-selective xylosyltransferase MS/MS-based activity assay. • Suitable for use with human serum and cell culture samples. • Broad spectrum of activity measurable. • Mathematical model allowing simultaneous determination of XT-I and XT-II. [ABSTRACT FROM AUTHOR]

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host–pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air–liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae , and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models. [ABSTRACT FROM AUTHOR]

In vitro cell culturing witnessed its applications in scientific research and industrial activities. Attempts to shorten the doubling time of cultured cells have never ceased. In plants, auxin is applied to promote plant growth, the synthetic derivative 1‐Naphthaleneacetic acid (NAA) is a good example. Despite the auxin's naturally occurring receptors are not present in mammalian cells, studies suggested they may affect cell culturing. Yet the effects and mechanisms are still unclear. Here, an up to 2‐fold increase in the yield of in vitro cultured human cells is observed. Different types of human cell lines and primary cells are tested and found that NAA is effective in all the cells tested. The PI staining followed by FACS suggested that NAA do not affect the cell cycling. Apoptosis‐specific dye staining analysis implicated that NAA rescued cell death. Further bulk RNA sequencing is done and it is identified that the lipid metabolism‐engaging and anti‐apoptosis gene, ANGPTL4, is enhanced in expression upon NAA treatment. Studies on ANGPTL4 knockout cells indicated that ANGPTL4 is required for NAA‐mediated response. Thus, the data identified a beneficial role of NAA in human cell culturing and highlighted its potency in in vitro cell culturing. [ABSTRACT FROM AUTHOR]

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm’s fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility. [ABSTRACT FROM AUTHOR]

Marine glycosaminoglycans (GAG) isolated from different invertebrates, such as molluscs, starfish or jellyfish, have been described as unique molecules with important pharmacological applications. Scarce information is available on GAG extract from Rapana venosa marine snail. The aim of this study was to isolate a GAG extract from R. venosa marine snail and to investigate its physicochemical, antioxidant and antiproliferative properties for further biomedical use. The morphology, chemical and elemental composition of the extract were established as well as the sulfate content and N- to O-sulfation ratio. Fourier transform infrared (FTIR) spectra indicated that GAG extract presented similar structural characteristics to bovine heparan sulfate and chondroitin sulfate. The pattern of extract migration in agarose gel electrophoresis and specific digestion with chondroitinase ABC and heparinase III indicated the presence of a mixture of chondroitin sulfate-type GAG, as main component, and heparan sulfate-type GAG. Free radical scavenging and ferric ion reducing assays showed that GAG extract had high antioxidant activity, which slightly decreased after enzymatic treatment. In vitro MTT and Live/Dead assays showed that GAG extract had the ability to inhibit cell proliferation in human Hep-2 cell cultures, at cytocompatible concentrations in normal NCTC clone L929 fibroblasts. This capacity decreased after enzymatic digestion, in accordance to the antioxidant activity of the products. Tumoral cell migration was also inhibited by GAG extract and its digestion products. Overall, GAG extract from R. venosa marine snail exhibited antioxidant and antiproliferative activities, suggesting its potential use as novel bioactive compound for biomedical applications. [ABSTRACT FROM AUTHOR]

Medicine today faces the combined challenge of an increasing number of untreatable diseases and fewer drugs reaching the clinic. While pharmaceutical companies have increased the number of drugs in early development and entering phase I of clinical trials, fewer actually successfully pass phase III and launch into the market. In fact, only 1 out of every 9 drugs entering phase I will launch. In vitro preclinical tests are used to predict earlier and better the potential of new drugs and thus avoid expensive clinical trial phases. The most recent developments favor 3D cell culture and human stem cell biology. These 3D humanized models known as organoids better mimic the 3D tissue architecture and physiological cell behavior of healthy and disease models, but face critical issues in production such as small-scale batches, greater costs (when compared to monolayer cultures) and reproducibility. To become the gold standard and most relevant biological model for drug discovery and development, organoid technology needs to integrate biological culture processes with advanced microtechnologies, such as microphysiological systems based on microfluidics technology. Microphysiological systems, known as organ-on-a-chip, mimic physiological conditions better than conventional cell culture models since they can emulate perfusion, mechanical and other parameters crucial for tissue and organ physiology. In addition, they reduce labor cost and human error by supporting automated operation and reduce reagent use inminiaturized culture systems. There is thus a clear advantage in combining organoid culture with microsystems for drug development. The main objective of this review is to address the recent advances in organoids and microphysiological systems highlighting crucial technologies for reaching a synergistic strategy, including bioprinting. [ABSTRACT FROM AUTHOR]

Lapatinib-(LPT) is a dual tyrosine kinase inhibitor that has important effects on the targeted therapy of various tumours. The current study is aimed to use a novel tracer strategy including Technetium-99 m-(99mTc) radiolabeled LPT and its poly(lactic-co-glycolic acid)-(PLGA) encapsulated formulation-(LPT-PLGA). In vitro bioaffinity of the [99mTc]Tc-LPT and [99mTc]Tc-LPT-PLGA was evaluated by using cell culture methods on human breast adenocarcinoma (MDA-MB-231), cervix adenocarcinoma and human ovarian (MDAH-2774) cancer cell lines. Radiochemical yield were over 95% and both compounds were examined high incorporation values on HeLa and MDAH cell lines. Current results may contribute to develope as the encapculated agents including radiotracer. [ABSTRACT FROM AUTHOR]