Search Results (342)

Bioactive glass materials have been used for decades in orthopedic surgery, traumatology, and oral and maxillofacial surgery to repair bone defects. This study aimed to evaluate in vitro the survival and proliferation of MG63 human osteosarcoma-derived cells on S53P4 bioactive glass (BonAlive^® granules). Microscopic visualization was performed to directly observe the interactions between the cells and the material. Osteoblast-like cells were examined on non-adherent test plates, on tissue culture (TC)-treated plates and on the surface of the bioglass to assess the differences. Cell survival and proliferation were monitored using a CCK-8 optical density assay. Comparing the mean OD of MG63 cells in MEM on TC-treated plates with cells on BG, we detected a significant difference (p < 0.05), over each time of observation. The sustained cell proliferation confirmed the non-cytotoxic property of the bioglass, as the cell number increased continuously at 48, 72, 96, and 168 h and even did not plateau after 168 h. Since the properties of bioglasses can vary significantly depending on their composition and environment, a thorough characterization of their biocompatibility is crucial to ensure their effective and appropriate application—for example, during hip and knee prosthesis insertion. Full article

Background and Objectives: Gender disparities in pain management persist, with women frequently receiving inadequate analgesia despite reporting similar or higher pain levels compared with men. This issue is particularly evident across various medical and gynecological procedures. Materials and Methods: This integrative literature review synthesizes recent empirical studies examining gender biases in pain perception and management, focusing specifically on procedural pain in women. It includes an analysis of clinical research, patient-reported outcomes, and healthcare provider behaviors. Results: The findings indicate that unconscious biases, a lack of gender-specific clinical protocols, and prevailing cultural stereotypes contribute to the undertreatment of pain in women during procedures such as intrauterine device insertion and diagnostic hysteroscopy. Additionally, communication gaps between patients and healthcare providers exacerbate these disparities. Conclusions: Addressing gender disparities in pain management necessitates systemic reforms, including the implementation of gender-sensitive clinical guidelines, enhanced provider education, and targeted policy changes. Personalized, gender-informed approaches are essential to improving equity and quality of care in pain treatment. Full article

With increasing popularity of food delivery services, the microbial safety of transported meals should be ensured. An effect of the type of a meal (cooked rice; mashed potatoes; mushroom sauce), inner primary packaging (sugarcane bagasse [SB] tray; polypropylene [PP] tray), secondary container (polyester/polyethylene foam/aluminum foil [PPA] bag; PP box) on the time interval of the internal hot ready-to-eat (RTE) meal temperature decrease to the value critical for Bacillus cereus growth (40 °C) was tested during a simulated delivery; in aliquot samples of the same meals, B. cereus growth was quantified presuming a natural contamination of the meals. Type of a meal had no effect on the tested time interval (p > 0.05). Packaging a meal in the PP tray as compared to the SB tray and inserting primary trays into the PP box instead of PPA bag delayed (p < 0.05) the internal meal temperature decrease by 50 and 15 min, respectively. Average B. cereus counts in the naturally contaminated meals after the four-hour culturing at 40 °C was 2.99 log CFU·g⁻¹. It was concluded that a hot RTE meal delivered up to four hours under the tested conditions is not likely to facilitate B. cereus growth above unacceptable levels. Full article

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein. Full article

Poloxamer (P) 188 attenuates myocardial ischemia/reperfusion injury through cell membrane stabilization. Cell–cell interactions between endothelial cells (ECs) and cardiomyocytes (CMs) further protect CMs: co-cultures showed that, at an optimal density, ECs protected CMs against hypoxia/reoxygenation (HR) injury. The mechanism of interaction with P188 still requires exploration. We examined if N(ω)-nitro-L-arginine methyl ester (LNAME), a non-specific nitric oxide (NO) synthase inhibitor, abolishes protection in the presence or absence of P188 and/or ECs. We co-cultured mouse coronary artery ECs in an insert atop mouse CMs plated at confluency on the bottom of a well. Normoxic controls remained in complete media while HR groups were exposed to 24 h hypoxia at 0.01% O₂ in serum- and glucose-free media, followed by 2 h reoxygenation in complete media. P188 (300 μM), LNAME (40 mM), or vehicle were administered upon reoxygenation. ECs at the used lower density did not decrease HR-triggered lactate dehydrogenase release or calcium overload in CMs by themselves. P188 reduced both indicators after HR by 16/18% without and by 22/25% with ECs, respectively. LNAME abrogated CM protection by P188. Neither intervention had an effect under normoxia. Our co-culture data indicates that P188 requires NO, not necessarily of endothelial origin, to elicit CM protection. Full article

Anti-infectives include molecules that target microbes in the context of infection but lack antimicrobial activity under conventional growth conditions. We previously described D66, a small molecule that kills the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) within cultured macrophages and murine tissues, with low host toxicity. While D66 fails to inhibit bacterial growth in standard media, the compound is bacteriostatic and disrupts the cell membrane voltage gradient without lysis under growth conditions that permeabilize the outer membrane or reduce efflux pump activity. To gain insights into specific bacterial targets of D66, we pursued two genetic approaches. Selection for resistance to D66 revealed spontaneous point mutations that mapped within the gmhB gene, which encodes a protein involved in the biosynthesis of the lipopolysaccharide core molecule. E. coli and S. Typhimurium gmhB mutants exhibited increased resistance to antibiotics, indicating a more robust barrier to entry. Conversely, S. Typhimurium transposon insertions in genes involved in outer membrane permeability or efflux pump activity reduced fitness in the presence of D66. Together, these observations underscore the significance of the bacterial cell envelope in safeguarding Gram-negative bacteria from small molecules. Full article

Cultures of primary human nasal epithelial cells (hNECs) differentiated at the air–liquid interface (ALI) represent a sophisticated and widely used model of the human upper respiratory epithelium. Despite the availability of various cell culture insert types and the well-established understanding that different culture media influence the cell culture characteristics, the possible impact of the insert brand remains rather underexplored. We cultured hNECs from nineteen healthy adult donors on three distinct brands of commercially available inserts—Corning^® Transwell^®, CELLTREAT^®, and ThinCert^®—and compared the ciliary activity and cellular composition of the cultures using high-speed video microscopy and flow cytometry, respectively. Additionally, we employed an alternative method of hNEC culture setup—the inverted condition—wherein the hNECs were seeded on the basal side of the insert with the idea to avoid mucus accumulation. Our results show that ciliary activity and cell type composition did not differ between insert types for both culture conditions. However, we found a higher ciliary beat frequency and a lower active (ciliated) area in the inverted setup compared to the conventional setup across all three insert brands. These findings indicate that all three mentioned insert types yield comparable cell cultures. Full article

Therapeutic gene editing strategies utilize endogenous DNA repair pathways—nonhomologous end joining (NHEJ) or homology-directed repair (HDR)—to introduce targeted genomic modifications. Because HDR is restricted to dividing cells, whereas NHEJ functions in both dividing and non-dividing cells, NHEJ-based approaches are better suited for in vivo gene editing in the largely post-mitotic airway epithelium. Homology-independent targeted insertion (HITI), an NHEJ-based method, offers a promising strategy for cystic fibrosis (CF) gene therapy. Here, we applied HITI to drive the expression of a promoterless reporter through an exon trap strategy in both proliferating airway basal cells and well-differentiated primary airway epithelial cultures derived from transgenic ROSA^mTmG ferrets. We also established a versatile human gene editing reporter (GER) airway basal cell line capable of multipotent differentiation, enabling real-time visualization of editing outcomes and the quantitative assessment of HDR- and NHEJ-based editing efficiencies. Together, these platforms provide easily accessible tools for optimizing genome editing strategies in the respiratory epithelium and advancing clinically relevant delivery strategies for CF gene therapy. Full article

Microalgae interact with mineral particles in an aqueous environment, yet how clay minerals affect physiological processes in algal cells remains unexplored. In this study, we compared the effects of palygorskite (Pal) and montmorillonite (Mt), which respectively represent fibrous and layered clay minerals, on the physiological processes of Chlamydomonas reinhardtii. It was observed that C. reinhardtii responded differently to the treatments of Pal and Mt. The Pal particles bound tightly to and even inserted themselves into cells, resulting in a significant decrease in cell numbers from 27.35 to 21.02 × 10⁷ mL⁻¹. However, Mt was only loosely attached to the cell surface. The photosynthesis in the algal cells was greatly inhibited by Pal, with the rETR_max significantly reduced from 103.80 to 56.67 μmol electrons m⁻²s⁻¹ and the downregulation of IF2CP, psbH and OHP1, which are key genes involved in photosynthesis. In addition, Pal reduced the quantities of proteins and polysaccharides in extracellular polymeric substances (EPSs) and the P uptake by C. reinhardtii when the P level in the culture was 3.15 mg/L. However, no significant changes were found regarding the above EPS components or the amount of P in algal cells upon the addition of Mt. Together, the impacts of fibrous Pal on C. reinhardtii was more profound than those of layered Mt. Full article

The interaction between immune regulatory cells, such as regulatory B cells (Breg) and pro-resolving macrophages (M2 macrophages), plays an important role in the restoration of immune homeostasis during inflammation. PD-L1 is one of the effector molecules that mediates the immune regulation function of M2 macrophages. The activation of PD-L1/PD-1 signaling promotes the differentiation of Breg. Previous studies have shown that Breg promoted M2 macrophage polarization and enhanced their function, but little is known about the regulatory function of M2 macrophages on Breg differentiation. This study aims to determine the effect of M2 macrophages on Breg induction and the potential mechanism in vitro. Bone-marrow-derived macrophages were isolated from wild-type (WT) mice and polarized into M2 using IL-4/IL-13. To investigate the role of PD-L1/PD-1 in M2 macrophage-induced Breg differentiation, spleen B cells were isolated from WT or PD-1 knockout (KO) mice and co-cultured with either naïve (M0) or M2 macrophages for 48 h with or without trans-well inserts. The expression of IL-10, IL-35, and TGF-β1 in B cells was evaluated by flow cytometry and immunofluorescence staining. Recombinant PD-L1 was used to stimulate activated B cells, followed by the detection of IL-35 and TGF-β1. The results show that there was no significant difference in IL-10 expression among all groups. However, IL-35 and TGF-β1 expression in B cells was significantly increased in the M2+B, but not in M0+B, compared to B cells alone. Notably, such increases were diminished when M2 and B cells were separated by trans-well inserts. IL-35 expression was not significantly changed when B cells from PD-1 KO mice were co-cultured with M2 compared to the control. However, TGF-β1 expression was significantly increased when PD-1 KO B cells were co-cultured with M2 compared to the control. IL-35 expression in activated B cells was increased upon stimulation with PD-L1. However, TGF-β1 expression in activated B cells was increased regardless of the PD-L1 availability. This study demonstrates that pro-resolving macrophage-induced IL-35⁺ but not TGF-β1⁺ regulatory B cell activation requires the PD-L1/PD-1 pathway. Full article

In this piece, we approach graffiti from the perspective of the ‘circuits of valorization’ that qualify as well as quantify it. We understand a valorization circuit as an assemblage of cultural, legal, economic and geographic dynamics surrounding a given artefact, which eventually confer a certain ‘value’ to it. Here, we look at examples of global graffiti, with attention to how cities and administrations juggle with its controversial valorization, implementing various policies to rein it in, but also to exploit it. Typically, graffiti appears and lives in ill-defined, metamorphic urban spaces: as an urban artefact, graffiti occupies loose, interstitial places and rhymes with an aesthetic of defacement and infestation. The ‘in place/out of place’ dialectic is thus central for claims to legitimacy, legality, and, ultimately, also the ‘quality’ of graffiti. Through the lens of radical legal pluralism, we argue that graffiti can insert a distinctive dynamism into the lawscape, rather than be a sheer inert object of urban policies. Graffiti itself actively participates, not simply in populating the lawscape, but in its actual crafting. Full article

Background/Objectives: Pancreatic cancer is the seventh most lethal type of cancer in the world, and its treatment, which is largely inefficient, is based on surgery and/or non-specific chemotherapy. Its malignant features are characterized by complex cell signaling pathways, which can be used as targets for new drugs. Methods: In this study, glucal-based compounds were synthetized, with substitution based on fluorine, nitrogen and aromatic ring addition. The compounds were tested in the pancreatic cell culture Mia-PaCa-2 and cell viability was assessed, with further IC50 calculation, stability and selectivity. Molecular docking was performed to evaluate the probable molecular target for 5b and in silico physicochemical properties were determined. Results: One molecule, named 5b, with two fluorine atoms inserted in the aromatic ring, exerted potent inhibitory activity on cell growth (IC50 = 1.39 µM), which was selective for pancreatic cells. Through molecular docking studies, the compound was found to be positioned in the active site of JAK3, indicating inhibition of such protein, which has a role in tumoral cell growth. Moreover, 5b was stable for 24 months and had physicochemical properties to permeate cell membranes, good oral absorption, and low potential to cause toxicity. Conclusions: These data suggest that 5b can be druggable and can be considered as a prototype for a new course of treatment in pancreatic cancer. Full article

Porcine deltacoronavirus (PDCoV) is a newly discovered enteropathogenic coronavirus primarily responsible for diarrhea and mortality in piglets, with the potential to infect humans, thereby posing a significant threat to both human health and the global pig industry. Currently, there is no commercially available live-attenuated vaccine for PDCoV. In this study, an isolated virulent PDCoV strain, DHeB1, was continuously passaged in LLC-PK1 cells for up to 110 passages. The virus growth kinetics in cell culture and complete genome sequences of various passages (F11, F40, F70, F90, and F110) were determined. The results indicated significant increases in virus titers at passages F40 and F90. Sequence analysis revealed that only a few single-nucleotide mutations (some of which resulted in amino acid changes) and one nucleotide insertion were observed throughout successive passages. Notably, the eight and seven amino acid mutations that emerged in F40 and F70, respectively, remained stable in subsequent passages and were predominantly located in the S glycoprotein. The pathogenicity of F11, F40, F70, and F90 was assessed in 5-day-old piglets, revealing markedly reduced clinical symptoms, histopathological lesions, and intestinal PDCoV antigen distributions in piglets inoculated with F70 or F90. Importantly, F90 exhibited little to no virulence in piglets. The immunogenicity of F70, F90, and F110 was further evaluated in weaned piglets, with results indicating that the neutralizing antibody titers induced by F70 and F90 were comparable and significantly higher than those induced by F110. Collectively, these findings suggest that the PDCoV strain DHeB1 has been attenuated and can be used to develop a live-attenuated vaccine against PDCoV. Full article

Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes—glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells. Full article

The “metaverse city” is defined as an immersive, interactive, and experiential digital environment that replicates or reinvents elements of physical cities, inserting them into an alternative reality. This concept involves transposing the urban, social, and cultural aspects of real cities into the metaverse, thus creating new ways of interacting with and experiencing urban space. Thus, it is not necessarily a digital replica of a physical city; however, it is invariably distinguished by its immersive nature, offering users a sensory and interactive experience. This concept goes beyond the mere digital replication of a city, evolving into a multifaceted space that integrates urban, social, cultural, and technological elements. It is shaped by digital interactions mediated by social actors (users), whose relationships in the metaverse are influenced by the power dynamics occurring in the virtual environment, much like in physical cities. The metaverse city is not merely an extension of the physical city; rather, it is a digital construct that enables alternative ways of living and relating to urban space. Consequently, it is a dynamic and ever-evolving construct, contingent on the active engagement of multiple social actors and their interactions for its consolidation. without the strategic involvement of diverse social actors, the identity and practices that characterize this contemporary urban entity—made possible by emerging technologies—risk losing their viability. Full article