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Background: Lepidium meyenii Walp. (LmW), known as maca, has been shown to increase physical performance. However, the effect size (ES) of LmW on the different manifestations of physical performance has not yet been described. Objectives: To examine and qualitatively describe the studies published up to 2024 that employed LmW supplementation to increase physical performance in animal and human experimentation. In addition, the ES associated with the different interventions was calculated. Methods: The research followed PRISMA® guidelines for systematic reviews and meta-analyses, using Web of Science, Scopus, SPORTDiscus, PubMed, and MEDLINE databases until 2024. Randomized controlled studies with a pre- and post-test design, conducted both in vitro and in vivo in animals and humans, were included. Methodological quality assessment was performed using the CAMARADES tool for animal studies and the Newcastle Ottawa Scale for human studies. The main variables were the forced swimming test (FST), the rota-rod test (RRT), the grip strength test (GST), blood lactic acid (BLA), and lactic acid (LA). The analysis was conducted with a pooled standardized mean difference (SMD) through Hedges' g test (95% CI). Results: Twenty-one studies were included in the systematic review and sixteen in the meta-analysis. They revealed a large effect for all outcomes (SMD: FST = 2.26, RRT = 6.26, GST = 5.23, LA = −1.01, and BLA = −1.70). Conclusions: The phytochemical compounds of LmW, mainly macamides, increase physical performance, showing a greater effect at higher doses (dose–response effect). [ABSTRACT FROM AUTHOR]

Abstract Background Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. Methods To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC’s therapeutic effects on chondrocytes. Results The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. Conclusions This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC’s comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC’s therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Abstract The present study aimed to compare the effect of photobiomodulation with different energy densities on the angiogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and stem cells from human exfoliated deciduous teeth (SHED). Photobiomodulation therapy with a 660 nm diode laser (2.4 J/cm2 and 3.9 J/cm2) on two consecutive days post-culture was applied to two types of stem cells (hPDLSCs and SHED). The Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) test was undertaken to investigate Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin I (ANG-I) genes on days 1, 3, 5, 7, and 10 after the first session of laser application. The 4′,6-diamidino-2-phenylindole (DAPI) staining and Methyl Thiazolyl Tetrazolium (MTT) test were conducted on days 1, 3, and 5 after the first session of laser application, to assess the cell viability. The Two-way ANOVA with Tukey post hoc test was used to analyze the outcomes of the MTT and RT-qPCR tests. The results of the MTT and DAPI convergently illustrated that the groups receiving photobiomodulation with 2.4 J/cm2 had higher cell viability compared to 3.9 J/cm2. All experimental groups showed an upregulation of VEGF-A and ANG-I gene expression from day 1 to 5, followed by a downregulation from day 5 to 10. The groups with cultured hPDLSCs and SHED receiving photobiomodulation using 2.4 J/cm2 had the most amounts of VEGF-A and ANG-I gene expression on day 5, respectively. In conclusion, the 660 nm mediated photobiomodulation therapy of cultured SHED and hPDLSCs with 2.4 J/cm2 energy density may be associated with higher angiogenic differentiation (the expression of VEGF-A and ANG-I) as well as higher cell viability compared to the photobiomodulation therapy with 3.9 J/cm2.

Background: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. Methods: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. Results: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. Conclusions: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders. [ABSTRACT FROM AUTHOR]

Skin grows old due to intrinsic factors, such as age and associated hormonal changes, and external factors, like solar radiation and chemical substances to which we are exposed. With age, skin shows thinning, laxity, pallor, increased dryness, and the appearance of wrinkles, attributed to a decrease in collagen and elastin produced by fibroblasts. Several theories explain skin aging, including the free radical formation, neuroendocrine, and mitochondrial decline theories. Based on a scientific understanding of skin behavior, different in vitro methods are used to evaluate the effects of new ingredients in cosmetics. Commonly used methods include anti-collagenase, anti-elastase, and anti-hyaluronidase activity alongside techniques utilizing skin cells or 3D models. Although these methods are recognized and widely used, they lack standardization. In this review, a literature search has been conducted to examine the characteristics and variations of these methods across the laboratories. A key issue identified in this review is that many papers provide insufficient detail regarding their protocols. Moreover, the number of studies using cells is less significant than the ones determining enzyme inhibition. Our findings revealed that, in many cases, there is limited information available, underscoring the urgent need to initiate a comprehensive standardization process for the methodologies used to demonstrate anti-aging activity. [ABSTRACT FROM AUTHOR]

The present study aimed to compare the effect of photobiomodulation with different energy densities on the angiogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and stem cells from human exfoliated deciduous teeth (SHED). Photobiomodulation therapy with a 660 nm diode laser (2.4 J/cm2 and 3.9 J/cm2) on two consecutive days post-culture was applied to two types of stem cells (hPDLSCs and SHED). The Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) test was undertaken to investigate Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin I (ANG-I) genes on days 1, 3, 5, 7, and 10 after the first session of laser application. The 4′,6-diamidino-2-phenylindole (DAPI) staining and Methyl Thiazolyl Tetrazolium (MTT) test were conducted on days 1, 3, and 5 after the first session of laser application, to assess the cell viability. The Two-way ANOVA with Tukey post hoc test was used to analyze the outcomes of the MTT and RT-qPCR tests. The results of the MTT and DAPI convergently illustrated that the groups receiving photobiomodulation with 2.4 J/cm2 had higher cell viability compared to 3.9 J/cm2. All experimental groups showed an upregulation of VEGF-A and ANG-I gene expression from day 1 to 5, followed by a downregulation from day 5 to 10. The groups with cultured hPDLSCs and SHED receiving photobiomodulation using 2.4 J/cm2 had the most amounts of VEGF-A and ANG-I gene expression on day 5, respectively. In conclusion, the 660 nm mediated photobiomodulation therapy of cultured SHED and hPDLSCs with 2.4 J/cm2 energy density may be associated with higher angiogenic differentiation (the expression of VEGF-A and ANG-I) as well as higher cell viability compared to the photobiomodulation therapy with 3.9 J/cm2. [ABSTRACT FROM AUTHOR]

Background/Aim. The clinical success of prosthetic rehabilitation that commences after the attained implant osseointegration is dependent on the influence of several factors, of which screw loosening is a frequent one, and it is highly related to inadequate tightening (torquing) using torque wrenches. Although the wrenches are initially calibrated by the manufacturer, it is of great importance to evaluate their function after usage for a certain period of time. The aim of this study was to evaluate the accuracy and repeatability of the performance of implant torque wrenches in delivering necessary torque values before and after one year of usage. Methods. Two types of wrenches were used in the study: the beam-type and the toggle-type. Four various brands of beam-type wrench were marked as Beam 1 - Beam 4, and three various brands of toggle-type wrench were marked as Toggle 1 - Toggle 3, according to their design. Torque values delivered by wrenches were measured and analyzed using the One-Sample t-test, Independent-Samples t-test, and Mann-Whitney U test. The Bland-Altman bias test was used as an index of accuracy, whereas Forkman's comparison of datasets coefficients of variation (CV) served as an index of repeatability. Results. All wrenches except new Beam 2 and Beam 3 showed differences between the average measured torque value and target torque value. Differences were found in the average measured values between all used and new wrenches. Higher bias was observed in Toggle 1, Toggle 2, and Toggle 3 brands, whereas lower bias was recorded between used and new Beam 1 and Beam 3 wrenches. When comparing the CV for used and new wrenches, Beam 1, Beam 4, Toggle 1, and Toggle 2 revealed differences, whereas the CV for Beam 2, Beam 3, and Toggle 3 did not differ significantly. Conclusion. Compared to toggle-type, the beam-type wrenches offer greater accuracy in achieving the target torque value. The torque deteriorates in all wrenches after aging/usage and is more prominent in toggle-type devices. [ABSTRACT FROM AUTHOR]

Lepidium meyenii Walp (LmW) or Maca, including its bioactive components such as macamides, among others, has demonstrated antioxidant effects. However, the effect size (ES) of LmW on oxidative stress has not been qualitatively described and calculated. The primary objective of this systematic review and meta-analysis was to review and qualitatively describe the studies published up to 2023 that supplemented LmW to control cellular oxidative stress; the secondary objective was to calculate the ES of the different interventions. The search was designed following the PRISMA® guidelines for systematic reviews and meta-analyses and performed in the Web of Science, Scopus, SPORTDiscus, PubMed, and MEDLINE until 2023. The selection of studies included randomized controlled trials, with tests and post-tests, both in vitro and in vivo in animals and humans. The methodological quality and risk of bias were evaluated with the CAMARADES tool. The main variables were reduced glutathione, glutathione peroxidase, superoxide dismutase, and malondialdehyde. The analysis was conducted with a pooled standardized mean difference (SMD) through Hedges' g test (95% CI). Eleven studies were included in the systematic review and eight in the meta-analysis. They revealed a small effect for reduced glutathione (SMD = 0.89), a large effect for glutathione peroxidase (SMD = 0.96), a moderate effect for superoxide dismutase (SMD = 0.68), and a moderate effect for malondialdehyde (SMD = −0.53). According to the results, the phytochemical compounds of LmW effectively controlled cellular oxidative stress, mainly macamides. It was also determined that a higher dose of LmW generated a greater antioxidant effect. However, information concerning humans is scarce. [ABSTRACT FROM AUTHOR]

The endometrium plays a fundamental role in the reproductive system yet many etiologies of infertility-related endometrial diseases such as endometriosis, adenomyosis, Asherman’s syndrome or endometrial cancer remain unknown. There are currently no treatments that minimize the effects of this devastating disorder. Appropriate model systems that closely mimic the architecture and function of the endometrium in healthy and pathological states are needed to understand the underlying molecular pathways and develop novel or more effective treatments. This review summarizes the key milestones of in vitro culture models of the human endometrium throughout history, as well as the applications of advanced bioengineering techniques in the modelling of both healthy and pathological endometrium. Opportunities for future approaches are also discussed.

Objective To investigate the effect of miR-181c-5p on vasculogenic mimicry (VM) in ovarian cancer stem-like cells (OCS-LCs) and its mechanism. Methods OVCAR3 cells were induced into OCS-LCs by serum-free suspension culture. OVCAR3 cells were divided into groups A, B, and C and were transfected with NC-miR-181c-5p, siRNA-miR-181c-5p, and pRNA-miR-181c-5p, respectively. The sphere-forming experiment was used to assess the sphere formation ability of groups A, B, and C. RT-qPCR was used to measure the relative expression level of miR-181c-5p in cells of groups A, B, and C, and Western blot was used to measure the relative protein expression levels of Oct-4, Nanog, HIF-1α, and VEGF in cells of groups A, B, and C. CCK-8 assay was used to measure the viability of cells in groups A, B, and C, and the three-dimensional culture experiment was used to measure the angiogenesis rate of groups A, B, and C. Results OVCAR3 cells were successfully induced into OCS-LCs. RT-qPCR showed that group B had a significantly lower relative expression level of miR-181c-5p than group A, and group C had a significantly higher relative expression level than group A (t=2.25,8.68,P

Background: Plumbago zeylanicum, commonly known as `Chitramula’ and `Chitrak’ and belonging to the family Plumbaginaceae, is a rambling perennial under shrub grown in most parts of India. The present work deals with the pharmacognostical and preliminary phytochemical studies on the roots of Plumbago zeylanica Linn. Objectives: This study aimed to look at the preliminary phytochemical screening and pharmacognostic characteristics of P. zeylanicum roots. Material and Methods: Macroscopy, microscopy, phytochemical screening, physicochemical parameters, and the production of thin-layer chromatograms were used to evaluate the pharmacognostic properties of the Plumbago zeylanicum root. Results: Macroscopic examination revealed brown roots with an unpleasant flavor. Transverse cuts displayed visible features like medullary rays, arteries, secondary xylem, and starch grains. Powder microscopy further revealed secondary xylem elements, trichomes, fibers, and starch grains. Physicochemical properties of Plumbago zeylanicum were determined, including thin-layer chromatography fingerprinting, ash levels, extractive values, and fluorescence analysis, indicating diverse phytoconstituents in the root extract. Conclusion: The extracts contain steroids, alkaloids, saponins, glycosides, terpenoids, flavonoids, and carbohydrates, suggesting potential medicinal applications. The method is a superior alternative to time-consuming and complex steam distillation-based isolation techniques.

Bioactive peptides derived from native proteins modulate physiological processes in the metabolic pathways. Given that multiple protocols in the literature mimic the digestion of dietary components, gathering studies that use such models directed at protein digestion processes is critical. This systematic review aimed to gather evidence that adopted adequate experimental models to simulate human protein digestion. The databases searched were PubMed, Web of Science, ScienceDirect, Embase, Virtual Health Library, and Scopus. A total of 1985 articles were found, resulting in 20 eligible in vitro studies. The Office of Health Assessment and Translation was used to evaluate methodological quality. Seven studies used plant-based protein sources, twelve used animal protein sources, and one used both. The duration of the oral phase varied, although 60% of the studies employed a protein digestion period of 120 min. Amylase, pepsin, and pancreatin enzymes were utilized in 40% of the studies, with pH levels of 7, 3, and 7, respectively, during the oral, gastric, and intestinal phases. The INFOGEST harmonized static model was adopted by 65% of the studies; INFOGEST is the most effective model for simulating gastrointestinal protein processes in humans and can be used to answer several research questions because it describes experimental conditions close to the human physiological situation. [ABSTRACT FROM AUTHOR]

Copyright of Cumhuriyet Dental Journal is the property of Cumhuriyet University, Faculty of Dentistry and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Acta Odontologica Latinoamericana: AOL is the property of Acta Odontologica Latinoamericana and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Vascular injury such as central venous stenosis (CVS) is a common complication in hemodialysis patients with central venous catheters (CVCs), yet the impact of the microstructure and partial physic characteristics of catheter surface on the chronic injury of central vein has not been elucidated. In this study, the microscopic morphology of tips and bodies of six different brands of polyurethane CVCs was observed and their roughness was assessed. Subsequently, an in vitro model was established to measure the coefficients of friction (COF) between CVCs (tips and bodies) and the vena cava intima of Japanese rabbits under the same condition in a linear reciprocating mode, and changes in the intima of vessels after friction were observed. The study found that there was a significant variation in surface roughness among different brands of CVCs (tips P

Background and Objectives: Limited diabetes drug availability and frequent adverse reactions to oral medications highlight the need for improved drug delivery. This study suggests optimizing drug efficiency through process modifications, focusing on anti-diabetic medications like metformin hydrochloride. Matrix-type formulations are utilized to overcome current drug targeting and dosage management limitations. Methods: This study aims to improve anti-diabetic drugs, focusing on metformin hydrochloride, via process modifications in drug delivery. We developed a sustainable drug release process with matrix-type formulations, comparing hydroxyl propyl methylcellulose (HPMC) and guar gum as rate-controlling polymers. The optimized formulation was then compared with Glyciphage SR 500 to identify similarities and differences in drug release profiles. Results: We utilized matrix-type formulations to administer metformin hydrochloride. It develops a sustainable drug release process with HPMC and guar gum as rate-controlling polymers. Next, we conducted comparative drug release studies, systematically optimized the formulation, and compared it to Glyciphage SR 500 using appropriate analytical methods. Results: Comparative dissolution data, including a high similarity factor (f2) of 75.33 with Glyciphage SR 500, emphasizes the promising resemblance between metformin hydrochloride tablet (MT5) and the market sample, warranting further stability studies for potential upscaling. Conclusion: The study's novelty lies in successfully formulating sustained-release MT5 using HPMC K100M and guar gum via wet granulation, demonstrating a potential reduction in dosing frequency and adverse effects. MT5's optimal physical and chemical parameters and sustained drug release exceeded 99% at 12 hours.

Zinc oxide nanoparticles (ZnO NPs) are metal oxide nanomaterials, which are important for several applications: antibacterial, anthelmintic, antiprotozoal and antitumoral, among others. These applications are mainly related to the ability to spontaneously produce and induce the production of reactive oxygen species that are important components for the destruction of pathogens and tumor cells. While trying to potentiate ZnO NPs, studies have associated these NPs with silver oxide (AgO) or silver (Ag) NPs. It has already been reported that this combination (Ag-ZnO/AgO NPs) is able to enhance the microbicidal potential. Although possessing much potential for several purposes, it is important to evaluate whether this association also poses the risk of toxicity to cells and experimental models. Therefore, this work aimed to evaluate the toxicity of various Ag-ZnO/AgO NP nanocomposites, in vitro and in vivo. Accordingly, ZnO nanocrystals and nanocomposites with various concentrations of AgO (ZnO:5Ag, ZnO:9Ag or ZnO:11Ag) were used in different cytotoxicity models: Galleria mellonella (G. mellonella), cell lines (VERO and RAW 264.7) and C57BL/6 mice. In the G. mellonella model, four concentrations were used in a single dose, with subsequent evaluation of mortality. In the case of cells, serial concentrations starting at 125 µg/mL were used, with subsequent cytotoxicity assessment. Based on the safe doses obtained in G. mellonella and cell models, the best doses were used in mice, with subsequent evaluations of weight, biochemistry as also renal and liver histopathology. It was observed that the toxicity, although low, of the nanocomposites was dependent upon the concentration of AgO used in association with ZnO NPs, both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

Introduction: Microorganism infiltration through the im-plant-abutment interface causes oral health problems such as periimplantitis, leading to implant loss. Materials and Methods: A feasible new method to quantify the Streptococcus mutans (S. mutans) infiltration through the implant-abutment interface gap is introduced in the present work. Internal hexagon (IH; n = 10), external hexagon (EH; n = 10), Morse taper (MT; n = 10), and a control for each group (n = 1) were tested. Bacteria suspension was prepared at 1.5x108 CFU/mL (CFU: colony forming units), and the implants were individually submerged up to the connection level, allowing the bacteria to contact it. The abutment was removed, and bacteria count was performed. Results: The implant sets were tested under normal bacterial growth and early and late biofilm growth conditions. Colony-forming units per mL were obtained, and the results were compared among groups. Differences in bacterial count between the MT and EH (p

Objective: To evaluate the influence of opacity and the layering technique on the fluorescence of different composite resins. Materials and Methods: Two opacities (enamel and dentin) and the layering technique (enamel + dentin) of the composite resins: Filtek® Z350 and Palfique LX5 were evaluated in vitro. Composite resin discs were fabricated using a preformed matrix of 10 mm diameter and 0.5 mm thick for the single opacity groups and 10 mm thick for the layering technique groups, using 2 layers of 0.5 mm thickness of each opacity (n = 5). Specimens were analyzed using the Raman spectroscopy method. Data were analyzed using the Kruskall-wallis and Mann-Whitney U tests. Results: When evaluating the intensity of fluorescence, no statistically significant difference was found when comparing the layering technique and enamel opacity (p2> 0.05) and an increase in the dentin opacity value for both brands of composite resin. Regarding wavelength, no statistically significant difference was found when comparing the layering technique with enamel opacity and dentin opacity for both Filtek® Z350 and Palfique LX5® composite resins (p2 > 0.05). Conclusions: The fluorescence intensity of the layering technique is similar to enamel opacity for both composite resins. Likewise, the wavelength of the layering technique is similar to the enamel opacity and dentin opacity for both brands.

Abstract Objectives This in vitro study assessed the potential of tooth discoloration by aerosols generated from three heated tobacco products (HTPs) with different specifications: in‐direct heating tobacco system platform 1.0a (IT1.0a), in‐direct heating tobacco system platform 2.0a (IT2.0a), and direct heating tobacco system platform 3.0a (DT3.0a). In addition, three flavor types (regular, menthol, and berry menthol) were selected for each HTP to characterize the effect of flavor types on tooth discoloration. Material and Methods Six bovine tooth samples were exposed directly to aerosols generated from one pack of each HTP: 350 puffs for IT1.0a, 325 puffs for IT2.0a, and 220 puffs for DT3.0a. Six bovine tooth samples were also exposed to air (350 puffs) and smoke generated from one pack of cigarettes (160 puffs) as negative and positive controls, respectively. The color of each tooth sample was measured before and after exposure. The overall color changes were assessed using overall color differences (ΔE) calculated according to the Commission International de I'Eclairage color system. A one‐way analysis of variance followed by Tukey's post hoc test was used to compare ΔE among bovine tooth samples exposed to air, cigarette smoke, and aerosols generated from each HTP. Results ΔE values for tooth samples exposed to air and aerosols generated from the three HTPs (IT1.0a, IT2.0a, and DT3.0a) were significantly lower than ΔE value for tooth samples exposed to cigarette smoke. ΔE values obtained with DT3.0a were significantly higher than those obtained with air‐exposed control samples. However, ΔE values obtained with IT1.0a and IT2.0a were not significantly different from that obtained with air‐exposed control samples. No HTPs showed significant differences in ΔE values among the three flavor types. Conclusions This study showed that HTP aerosols reduce tooth discoloration potential compared with cigarette smoke, regardless of flavor types, and the tooth discoloration potential of the product may depend on product specifications.

Skin grows old due to intrinsic factors, such as age and associated hormonal changes, and external factors, like solar radiation and chemical substances to which we are exposed. With age, skin shows thinning, laxity, pallor, increased dryness, and the appearance of wrinkles, attributed to a decrease in collagen and elastin produced by fibroblasts. Several theories explain skin aging, including the free radical formation, neuroendocrine, and mitochondrial decline theories. Based on a scientific understanding of skin behavior, different in vitro methods are used to evaluate the effects of new ingredients in cosmetics. Commonly used methods include anti-collagenase, anti-elastase, and anti-hyaluronidase activity alongside techniques utilizing skin cells or 3D models. Although these methods are recognized and widely used, they lack standardization. In this review, a literature search has been conducted to examine the characteristics and variations of these methods across the laboratories. A key issue identified in this review is that many papers provide insufficient detail regarding their protocols. Moreover, the number of studies using cells is less significant than the ones determining enzyme inhibition. Our findings revealed that, in many cases, there is limited information available, underscoring the urgent need to initiate a comprehensive standardization process for the methodologies used to demonstrate anti-aging activity.

ABSTRACT Objective To evaluate the in vitro and in vivo toxicities of polyethylene glycol-coated gold nanoparticles synthesized using a one-step process. Methods Gold nanoparticles were prepared via a co-precipitation method using polyethylene glycol, and the synthesis product was characterized. For the in vitro evaluation, a flow cytometry analysis with Annexin V and iodide propidium staining was used to assess cytotoxicity in MG-63 cells labeled with 10, 50, and 100µg/mL of nanoparticle concentration. For the in vivo evaluation, nanoparticles were administered intraperitoneally at a dose of 10mg/kg dose in 10-week-old mice. Toxicity was assessed 24 hours and 7 days after administration via histopathological analysis of various tissues, as well as through renal, hepatic, and hematopoietic evaluations. Results Synthesized nanoparticles exhibited different hydrodynamic sizes depending on the medium: 51.27±1.62nm in water and 268.12±28.45nm (0 hour) in culture medium. They demonstrated a maximum absorbance at 520nm and a zeta potential of -8.419mV. Cellular viability exceeded 90%, with less than 3% early apoptosis, 6% late apoptosis, and 1% necrosis across all labeling conditions, indicating minimal cytotoxicity differences. Histopathological analysis highlighted the accumulation of nanoparticles in the mesentery; however, no lesions or visible agglomeration was observed in the remaining tissues. Renal, hepatic, and hematopoietic analyses showed no significant differences at any time point. Conclusion Polyethylene glycol-coated gold nanoparticles exhibit extremely low toxicity and high biocompatibility, showing promise for future studies.

Bone density at the implant site is correlated to the success of osseointegration. The objective of this in vitro study was to evaluate the efficacy of osseodensification burs in increasing bone density using a solid polyurethane foam block model. The osseodensification burs kit was used to perform 48 osteotomies on a rigid polyurethane foam test ground. Burs were utilized on a TMM2 implant motor for data collection. The osteotomies were divided into two study groups (A and C) in which implant sites, extended 12 and 14 mm deep, respectively, were prepared using the drills to a compaction rotation; two control groups, B and D, represented the osteotomies for which the drills were used in cutting direction. A 3.8 × 12 mm conical implant was inserted into each site; for each implant, data were collected on the peak torque (Cp), mean torque (Cm), and integral depth curve (I). The implants underwent resonance frequency analysis (RFA) to assess the implant stability quotient (ISQ). Correlation analysis was performed between I, Cm, Cp and ISQ. One-way analysis of variance (ANOVA) was used to identify statistically significant differences between groups. Group C, representing osteotomies prepared at 14 mm with osseodensification burs, showed a significantly higher value for each parameter. Implants at sites obtained with osteocondensation drills and prepared at greater depth for autologous particle grafting showed significant increases in each implant stability parameter. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the in vitro and in vivo toxicities of polyethylene glycol-coated gold nanoparticles synthesized using a one-step process. Methods: Gold nanoparticles were prepared via a co-precipitation method using polyethylene glycol, and the synthesis product was characterized. For the in vitro evaluation, a flow cytometry analysis with Annexin V and iodide propidium staining was used to assess cytotoxicity in MG-63 cells labeled with 10, 50, and 100µg/mL of nanoparticle concentration. For the in vivo evaluation, nanoparticles were administered intraperitoneally at a dose of 10mg/kg dose in 10-week-old mice. Toxicity was assessed 24 hours and 7 days after administration via histopathological analysis of various tissues, as well as through renal, hepatic, and hematopoietic evaluations. Results: Synthesized nanoparticles exhibited different hydrodynamic sizes depending on the medium: 51.27±1.62nm in water and 268.12±28.45nm (0 hour) in culture medium. They demonstrated a maximum absorbance at 520nm and a zeta potential of -8.419mV. Cellular viability exceeded 90%, with less than 3% early apoptosis, 6% late apoptosis, and 1% necrosis across all labeling conditions, indicating minimal cytotoxicity differences. Histopathological analysis highlighted the accumulation of nanoparticles in the mesentery; however, no lesions or visible agglomeration was observed in the remaining tissues. Renal, hepatic, and hematopoietic analyses showed no significant differences at any time point. Conclusion: Polyethylene glycol-coated gold nanoparticles exhibit extremely low toxicity and high biocompatibility, showing promise for future studies. [ABSTRACT FROM AUTHOR]

Objectives: This in vitro study assessed the potential of tooth discoloration by aerosols generated from three heated tobacco products (HTPs) with different specifications: in‐direct heating tobacco system platform 1.0a (IT1.0a), in‐direct heating tobacco system platform 2.0a (IT2.0a), and direct heating tobacco system platform 3.0a (DT3.0a). In addition, three flavor types (regular, menthol, and berry menthol) were selected for each HTP to characterize the effect of flavor types on tooth discoloration. Material and Methods: Six bovine tooth samples were exposed directly to aerosols generated from one pack of each HTP: 350 puffs for IT1.0a, 325 puffs for IT2.0a, and 220 puffs for DT3.0a. Six bovine tooth samples were also exposed to air (350 puffs) and smoke generated from one pack of cigarettes (160 puffs) as negative and positive controls, respectively. The color of each tooth sample was measured before and after exposure. The overall color changes were assessed using overall color differences (ΔE) calculated according to the Commission International de I'Eclairage color system. A one‐way analysis of variance followed by Tukey's post hoc test was used to compare ΔE among bovine tooth samples exposed to air, cigarette smoke, and aerosols generated from each HTP. Results: ΔE values for tooth samples exposed to air and aerosols generated from the three HTPs (IT1.0a, IT2.0a, and DT3.0a) were significantly lower than ΔE value for tooth samples exposed to cigarette smoke. ΔE values obtained with DT3.0a were significantly higher than those obtained with air‐exposed control samples. However, ΔE values obtained with IT1.0a and IT2.0a were not significantly different from that obtained with air‐exposed control samples. No HTPs showed significant differences in ΔE values among the three flavor types. Conclusions: This study showed that HTP aerosols reduce tooth discoloration potential compared with cigarette smoke, regardless of flavor types, and the tooth discoloration potential of the product may depend on product specifications. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is a representative malignant brain tumor characterized by a dismal prognosis, with survival rates of less than 2 years and high recurrence rates. Despite surgical resection and several alternative treatments, GBM remains a refractory disease due to its aggressive invasiveness and resistance to anticancer therapy. In this report, we explore the role of fibronectin type III domain containing 3B (FNDC3B) and its potential as a prognostic and therapeutic biomarker in GBM. GBM exhibited a significantly higher cancer-to-normal ratio compared to other organs, and patients with high FNDC3B expression had a poor prognosis (p < 0.01). In vitro studies revealed that silencing FNDC3B significantly reduced the expression of Survivin, an apoptosis inhibitor, and also reduced cell migration, invasion, extracellular matrix adhesion ability, and stem cell properties in GBM cells. Furthermore, we identified that FNDC3B regulates PTEN/PI3K/Akt signaling in GBM cells using MetaCore integrated pathway bioinformatics analysis and a proteome profiler phospho-kinase array with sequential western blot analysis. Collectively, our findings suggest FNDC3B as a potential biomarker for predicting GBM patient survival and for the development of treatment strategies for GBM. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

CGG expansions between 55 and 200 in the 5'-untranslated region of the fragile-X mental retardation gene (FMR1) increase the risk of developing the late-onset debilitating neuromuscular disease Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). While the science behind this mutation, as a paradigm for RNA-mediated nucleotide triplet repeat expansion diseases, has progressed rapidly, no treatment has proven effective at delaying the onset or decreasing morbidity, especially at later stages of the disease. Here, we demonstrated the beneficial effect of the phytochemical sulforaphane (SFN), exerted through NRF2-dependent and independent manner, on pathways relevant to brain function, bioenergetics, unfolded protein response, proteosome, antioxidant defenses, and iron metabolism in fibroblasts from FXTAS-affected subjects at all disease stages. This study paves the way for future clinical studies with SFN in the treatment of FXTAS, substantiated by the established use of this agent in clinical trials of diseases with NRF2 dysregulation and in which age is the leading risk factor.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection.

Objective To investigate the effect of lenvatinib on the autophagy and migration of cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC) tissue. Methods Primary CAFs from HCC patients were isolated and cultured and were then treated with an equal volume of DMSO (DMSO group) or 46 nmol/L lenvatinib (lenvatinib group) for 72 h. Transcriptomic sequencing and the omics analysis of drug targets were performed for the two groups, and GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for differentially expressed genes and peptides. Transmission electron microscopy was used to observe the effect of lenvatinib on autophagy of CAFs, and Transwell assay and wound hea-ling assay were used to observe the effect of lenvatinib on the migration ability of CAFs. Results The differentially expressed genes and peptides between the DMSO group and the lenvatinib group were mainly enriched in the functions and pathways such as cell migration and autophagy. Transmission electron microscopy showed that the lenvatinib group had a significantly higher number of autophagosomes than the DMSO group (t=5.376,P

In the postnatal brain, neurogenesis occurs only within a few regions, such as the hippocampal sub-granular zone (SGZ). Postnatal neurogenesis is tightly regulated by factors that balance stem cell renewal with differentiation, and it gives rise to neurons that participate in learning and memory formation. The Kv1.1 channel, a voltage-gated potassium channel, was previously shown to suppress postnatal neurogenesis in the SGZ in a cell-autonomous manner. In this study, we have clarified the physiological and molecular mechanisms underlying Kv1.1-dependent postnatal neurogenesis. First, we discovered that the membrane potential of neural progenitor cells is highly dynamic during development. We further established a multinomial logistic regression model for cell-type classification based on the biophysical characteristics and corresponding cell markers. We found that the loss of Kv1.1 channel activity causes significant depolarization of type 2b neural progenitor cells. This depolarization is associated with increased tropomyosin receptor kinase B (TrkB) signaling and proliferation of neural progenitor cells; suppressing TrkB signaling reduces the extent of postnatal neurogenesis. Thus, our study defines the role of the Kv1.1 potassium channel in regulating the proliferation of postnatal neural progenitor cells in mouse hippocampus.

Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.

Lepidium meyenii Walp (LmW) or Maca, including its bioactive components such as macamides, among others, has demonstrated antioxidant effects. However, the effect size (ES) of LmW on oxidative stress has not been qualitatively described and calculated. The primary objective of this systematic review and meta-analysis was to review and qualitatively describe the studies published up to 2023 that supplemented LmW to control cellular oxidative stress; the secondary objective was to calculate the ES of the different interventions. The search was designed following the PRISMA® guidelines for systematic reviews and meta-analyses and performed in the Web of Science, Scopus, SPORTDiscus, PubMed, and MEDLINE until 2023. The selection of studies included randomized controlled trials, with tests and post-tests, both in vitro and in vivo in animals and humans. The methodological quality and risk of bias were evaluated with the CAMARADES tool. The main variables were reduced glutathione, glutathione peroxidase, superoxide dismutase, and malondialdehyde. The analysis was conducted with a pooled standardized mean difference (SMD) through Hedges’ g test (95% CI). Eleven studies were included in the systematic review and eight in the meta-analysis. They revealed a small effect for reduced glutathione (SMD = 0.89), a large effect for glutathione peroxidase (SMD = 0.96), a moderate effect for superoxide dismutase (SMD = 0.68), and a moderate effect for malondialdehyde (SMD = −0.53). According to the results, the phytochemical compounds of LmW effectively controlled cellular oxidative stress, mainly macamides. It was also determined that a higher dose of LmW generated a greater antioxidant effect. However, information concerning humans is scarce.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a β cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other β cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the β cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.

Oxylipins are metabolites of polyunsaturated fatty acids that mediate cardiovascular health by attenuation of inflammation, vascular tone, hemostasis, and thrombosis. Very low-density lipoproteins (VLDL) contain oxylipins, but it is unknown whether the liver regulates their concentrations. In this study, we used a perfused liver model to observe the effect of inflammatory lipopolysaccharide (LPS) challenge and soluble epoxide hydrolase inhibition (sEHi) on VLDL oxylipins. A compartmental model of deuterium-labeled linoleic acid and palmitic acid incorporation into VLDL was also developed to assess the dependence of VLDL oxylipins on fatty acid incorporation rates. LPS decreased the total fatty acid VLDL content by 30% [6%,47%], and decreased final concentration of several oxylipins by a similar amount (13-HOTrE, 35% [4%,55%], -1.3 nM; 9(10)-EpODE, 29% [3%,49%], -2.0 nM; 15(16)-EpODE, 29% [2%,49%], -1.6 nM; AA-derived diols, 32% [5%,52%], -2.4 nM; 19(20)-DiHDPA, 31% [7%,50%], -1.0 nM). However, the EPA-derived epoxide, 17(18)-EpETE, was decreased by 75% [49%,88%], (-0.52 nM) with LPS, double the suppression of other oxylipins. sEHi increased final concentration of DHA epoxide, 16(17)-EpDPE, by 99% [35%,193%], (2.0 nM). Final VLDL-oxylipin concentrations with LPS treatment were not correlated with linoleic acid kinetics, suggesting they were independently regulated under inflammatory conditions. We conclude that the liver regulates oxylipin incorporation into VLDL, and the oxylipin content is altered by LPS challenge and by inhibition of the epoxide hydrolase pathway. This provides evidence for delivery of systemic oxylipin signals by VLDL transport.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

Circulating TFH (cTFH ) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cTFH 1, cTFH 2, cTFH 17, and cTFR cells, respectively. This CD4+ T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cTFH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-β-estradiol (E2) directly increased the percentage of different cTFH subsets. While E2 and progesterone (P4) increased the proportion of differentiated TFH cells derived from naïve CD4+ T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/TFH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10+ TFR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cTFH and B cell subsets.

ObjectiveJoint injury rapidly induces expression of primary response genes (PRGs), which activate a cascade of secondary genes that destroy joint tissues and initiate post-traumatic osteoarthritis (PTOA). Bromodomain-containing-protein-4 (Brd4) and cyclin-dependent-kinase-9 (CDK9) cooperatively control the rate-limiting step of PRG transactivation, including pro-inflammatory genes. This study investigated whether Brd4 and CDK9 inhibitors suppress inflammation and prevent PTOA development in vitro and in a mouse PTOA model.MethodsThe effects of Brd4 and CDK9 inhibitors (JQ1 and Flavopiridol) on PRG and associated secondary damage were rigorously tested in different settings. Short-term effects of inflammatory stimuli (IL-1β, IL-6, TNF) on human chondrocyte PRG expression were assessed by RT-PCR and microarray after 5-h. We quantified glycosaminoglycan release from IL-1β-treated bovine cartilage explants after 3-6 days, and osteoarthritic changes in mice after ACL-rupture using RT-PCR (2-24hrs), in vivo imaging of MMP activity (24hrs), AFM-nanoindentation (3-7days), and histology (3days-4wks).ResultsFlavopiridol and JQ1 inhibitors act synergistically, and a combination of both almost completely prevented the activation of most IL-1β-induced PRGs in vitro by microarray analysis, and prevented IL-1β-induced glycosaminoglycan release from cartilage explants. Mice given the drug combination showed reduced IL-1β and IL-6 expression, less in vivo MMP activity, and lower synovitis (1.5 vs 4.9) and OARSI scores (2.8 vs 6.0) than untreated mice with ACL-rupture.ConclusionsJQ1 and Flavopiridol work synergistically to reduce injury response after joint trauma, suggesting that targeting Brd4 and/or CDK9 could be a viable strategy for PTOA prevention and treatment of early OA.

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.

Formation of Aβ oligomers and fibrils plays a central role in the pathogenesis of Alzheimer's disease. There are two major forms of Aβ in the brain: Aβ42 and Aβ40. Aβ42 is the major component of the amyloid plaques, but the overall abundance of Aβ40 is several times that of Aβ42. In vitro experiments show that Aβ42 and Aβ40 affect each other's aggregation. In mouse models of Alzheimer's disease, overexpression of Aβ40 has been shown to reduce the plaque pathology, suggesting that Aβ42 and Aβ40 also interact in vivo. Here we address the question of whether Aβ42 and Aβ40 interact with each other in the formation of oligomers using electron paramagnetic resonance (EPR) spectroscopy. When the Aβ42 oligomers were formed using only spin-labeled Aβ42, the dipolar interaction between spin labels that are within 20 Å range broadened the EPR spectrum and reduced its amplitude. Oligomers formed with a mixture of spin-labeled Aβ42 and wild-type Aβ42 gave an EPR spectrum with higher amplitude due to weakened spin-spin interactions, suggesting molecular mixing of labeled and wild-type Aβ42. When spin-labeled Aβ42 and wild-type Aβ40 were mixed to form oligomers, the resulting EPR spectrum also showed reduced amplitude, suggesting that wild-type Aβ40 can also form oligomers with spin-labeled Aβ42. Therefore, our results suggest that Aβ42 and Aβ40 form mixed oligomers with direct molecular interactions. Our results point to the importance of investigating Aβ42-Aβ40 interactions in the brain for a complete understanding of Alzheimer's pathogenesis and therapeutic interventions.

Background/Aim. One of the disadvantages of the cement-retained fixed implant-supported restorations is the residual cement, which is found on the superstructure after the cementation procedure and has been identified as a risk factor for the occurrence of peri-implantitis. The aim of the study was to examine the influence of cementation techniques on the amount of residual cement at different levels of demarcation of the abutment in relation to the gingiva in the process of cementing restorations on implants. Methods. The research was conducted in in vitro conditions on casts obtained after implant placement. The abutments are milled at the level of the gingiva, 1.5 mm subgingivally and 3 mm subgingivally. Zirconium dioxide ceramic restorations were cemented using a standard cementation technique, a cementation technique using Teflon tape, and a precementation method using a silicone replica of the abutment and a 3D printed replica. The amount of residual cement was measured by photograph analysis. Adobe Photoshop was used for software analysis of photographs and determination of cement surface. Statistical data processing was performed in the SPSS program, and the Kruskal-Wallis test was used for data analysis. Results. A comparative analysis of the effectiveness of cementation techniques at the gingival margin level revealed a statistically significant difference in the amount of residual cement in relation to the cementation technique (p < 0.001). Analyzing the cementation technique effectiveness at the level of the finish line, 1.5 mm subgingivally, it was established that there was a statistically significant difference in the amount of residual cement compared to the cementation technique (p = 0.001). Comparing the effectiveness of cementation techniques at the 3 mm subgingival finish line level, it was established that there was a statistically significant difference in the amount of residual cement compared to the cementation technique (p < 0.001). Conclusion. Subgingival localization significantly affects the amount of residual cement in fixed prosthetic restorations on implants. Applying precementation techniques significantly reduces the amount of residual cement. [ABSTRACT FROM AUTHOR]

Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca++ . Over several days, induced by Ca++ , the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca++ -induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.

BackgroundWe previously identified two acute kidney injury (AKI) sub-phenotypes (AKI-SP1 and AKI-SP2) with different risk of poor clinical outcomes and response to vasopressor therapy. Plasma biomarkers of endothelial dysfunction (tumor necrosis factor receptor-1, angiopoietin-1 and 2) differentiated the AKI sub-phenotypes. However, it is unknown whether these biomarkers are simply markers or causal mediators in the development of AKI sub-phenotypes.MethodsWe tested for associations between single-nucleotide polymorphisms within the Angiopoietin-1, Angiopoietin-2, and Tumor Necrosis Factor Receptor 1A genes and AKI- SP2 in 421 critically ill subjects of European ancestry. Top performing single-nucleotide polymorphisms (FDR  T, near ANGPT2 was associated with reduced risk of AKI-SP2 (odds ratio, 0.45; 95% CI, 0.31-0.66; adjusted FDR = 0.003) and decreased plasma angiopoietin-2 (p = 0.002). Causal inference analysis showed that for each minor allele (T) the risk of developing AKI-SP2 decreases by 16%. Plasma angiopoietin-2 mediated 41.5% of the rs2920656 related risk for AKI-SP2. Human kidney microvascular endothelial cells carrying the T allele of rs2920656 produced numerically lower levels of angiopoietin-2 although this was not statistically significant (p = 0.07). Finally, analyses demonstrated that angiopoietin-2 is minimally renally cleared in critically ill subjects.ConclusionGenetic mediation analysis provides supportive evidence that angiopoietin-2 plays a causal role in risk for AKI-SP2.

Mesoporous silica has attracted significant attention in the drug delivery area; however, impurities can be a source of toxicity. The current study used commercial microparticles produced at large scale in a well-controlled environment. Micrometer sized mesoporous silica particles were acquired through a commercial vendor and pore structures were characterized by SEM. The three silica particle formulations had a diameter of 15 micrometers and three different pore sizes of 10 nm, 30 nm, and 100 nm. The fourth formulation had particle size of 20-40 micrometers with 50 nm pores. Before in vivo tests, an in vitro cytotoxicity test was conducted with silicic acid, derived from the sol-gel particles, on EA.hy926 cells. Low concentration (2.5 µg/mL) of silicic acid showed no cytotoxicity; however, high concentration (25 µg/mL) was cytotoxic. In vivo intravitreal injection demonstrated that 15 um silica particles with 10 nm pore were safe in both rabbit and guinea pig eyes and the particles lasted in the vitreous for longer than two months. Formulations of with larger pores demonstrated variable localized vitreous cloudiness around the sol-gel particle depot and mild inflammatory cells in the aqueous humor. The incidence of reaction trended higher with larger pores (10 nm: 0%, 30 nm: 29%, 50 nm: 71%, 100 nm: 100%, p

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor 'M1'-type to protumor 'M2'-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolves as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell-cell dynamics at high spatial and temporal resolution. Using our recently developed microphysiological 'tumor-on-a-chip' (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells, whereas the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polarized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of C-X-C motif chemokines (CXCL9), CXCL10 and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of matrix metalloproteinase 7 and angiopoietin 2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment.

Cytoskeletal transport in cells is driven by enzymes whose activity shows sensitive, typically Arrhenius, dependence on temperature. Often, the duration and outcome of cargo transport is determined by the relative success of kinesin vs. dynein motors, which can simultaneously bind to individual cargos and move in opposite direction on microtubules. The question of how kinesin and dynein activity remain coupled over the large temperature ranges experienced by some cells is one of clear biological relevance. We report a break in the Arrhenius behavior of both kinesin-1 and kinesin-3 enzymatic activity at 4.7 °C and 10.5 °C, respectively. Further, we report that this transition temperature significantly changes as a function of chemical background: addition of 200 mM TMAO increases transition temperatures by ∼6 °C in all cases. Our results show that Arrhenius trend breaks are common to all cytoskeletal motors and open a broad question of how such activity transitions are regulated in vivo. STATEMENT OF SIGNIFICANCE: Many cytoskeletal motors studied to date follow Arrhenius kinetics, at least from room temperature up to mammalian body temperature. However the thermal dynamic range is typically finite, and breaks in Arrhenius trends are commonly observed at biologically relevant temperatures. Here we report that the thermal dynamic range of kinesins is also limited and moreover that the location of the Arrhenius break for kinesins can shift significantly based on chemical backgrounds. This implies that the balance of multiple motor cargo transport along the cytoskeleton is far more tunable as a function of temperature than previously appreciated.