Search Results (39)

Laboratory data from in-cell tests at and near open circuit potentials (OCV) and ex-situ H₂O₂ vapor exposure tests are used to develop a fluoride emission rate (FER) model for a state-of-the-art 12-µm thin, low equivalent weight, long-chain perfluorosulfonic acid (PFSA) ionomer membrane that is mechanically reinforced with expanded PTFE and chemically stabilized with 2 mol% cerium as an anti-oxidant. The anode FER at OCV linearly correlates with O₂ crossover from the cathode and the high yield of H₂O₂ at anode potentials, as observed in rotating ring disk electrode (RRDE) studies. The cathode FER may be linked to the energetic formation of reactive hydroxyl radicals (·OH) from the decomposition of H₂O₂ produced as an intermediate in the two-electron ORR pathway at high cathode potentials. Both anode and cathode FERs are significantly enhanced at low relative humidity and high temperatures. The modeled FER is strongly influenced by the gradients in water activity and cerium concentration that develops in operating fuel cells. Membrane stability maps are constructed to illustrate the relationship between the cell voltage, temperature, and relative humidity for FER thresholds that define H₂ crossover failure by chemical degradation over a specified lifetime. Full article

Background/Objectives: The low permeability of the blood-brain barrier (BBB) represents a significant challenge to effective systemic chemotherapy for primary and metastatic brain cancers. Kinin receptors play a crucial role in modulating BBB permeability, and their agonist analogs have been explored in preclinical animal models to enhance drug delivery to the brain. In this study, we investigated whether des-Arg⁹-bradykinin (DBK), a physiological agonist of kinin B₁ receptor (B1R), acts as a brain drug delivery adjuvant by promoting the transient opening of the BBB. Methods: Human brain microvascular endothelial cells (HBMECs) were treated with DBK in the culture medium and in conditioned media from glioblastoma cell lines, namely T98G (CMT98G) and U87MG (CMU87). Immunofluorescence, RT-qPCR, in-cell Western assay, and proximity ligation assay (PLA) were performed to analyze BBB components, kinin receptors and TLR4, a receptor associated with the kinin pathway and inflammation. The effect of DBK on enhancing paracellular molecule transport was evaluated using Evans blue dye (EB) quantification in a cell culture insert assay and in an in vivo model, where mice with and without brain tumors were treated with DBK. To assess the functional impact of the transient BBB opening induced by DBK, the chemotherapeutic drug doxorubicin (DOX) was administered. Results: Treatment with DBK facilitates the presence of EB in the brain parenchyma by transiently disrupting the BBB, as further evidenced by the increased paracellular passage of the dye in an in vitro assay. B1R activation by DBK induces transient BBB opening lasting less than 48 h, enhancing the bioavailability of the DOX within the brain parenchyma and glioma tumor mass. The interaction between B1R and TLR4 is disrupted by the secreted factors released by glioblastoma cells, as conditioned media from T98G and U87 reduce TLR4 staining in endothelial cells without affecting B1R expression. Conclusions: These results further support the potential of B1R activation as a strategy to enhance targeted drug delivery to the brain. Full article

The term incel is a neologism combining “involuntary” and “celibate”, describing men who experience involuntary absence from sexual and romantic relationships. Incels frequently espouse conspiratorial and misogynistic ideologies, often engaging in verbal aggression. The present study aimed at qualitatively exploring the emotional experiences and beliefs articulated within an online incel community, contributing to the formation of its collective identity. A thread (453 comments) sampled from an Italian “incel” forum was analyzed by two independent raters, using thematic analysis. Four themes emerged: (a) “non-persons (i.e., women) are not like us” revealed perceived gender inequality and misogynistic beliefs, (b) “without experiences in adolescence you are ruined” reflected the belief that early romantic and sexual experiences are crucial for later relationship satisfaction, (c) “I have no life” captured expressions of profound distress and life dissatisfaction, and (d) “only ours is true suffering” highlighted a sense of unique victimhood. Incel group identity was shaped by gender role stress, primarily stemming from the perceived failure to meet socially constructed expectations of hegemonic masculinity. Suffering emerged as the dominant emotional experience, seemingly exacerbated by feelings of hopelessness regarding romantic and sexual prospects. The perceived subordinate status of men and male suffering were exploited to rationalize and perpetuate misogyny. The observed incels’ openness in discussing experiences of suffering and trauma may present an opportunity for the development of preventive interventions aimed at increasing help-seeking behavior in this population. Full article

Based on the established neuroprotective properties of indole-based compounds and their significant potential as multi-targeted therapeutic agents, a series of synthetic indole–phenolic compounds was evaluated as multifunctional neuroprotectors. Each compound demonstrated metal-chelating properties, particularly in sequestering copper ions, with quantitative analysis revealing approximately 40% chelating activity across all the compounds. In cellular models, these hybrid compounds exhibited strong antioxidant and cytoprotective effects, countering reactive oxygen species (ROS) generated by the Aβ(25–35) peptide and its oxidative byproduct, hydrogen peroxide, as demonstrated by quantitative analysis showing on average a 25% increase in cell viability and a reduction in ROS levels to basal states. Further analysis using thioflavin T fluorescence assays, circular dichroism, and computational studies indicated that the synthesized derivatives effectively promoted the self-disaggregation of the Aβ(25–35) fragment. Taken together, these findings suggest a unique profile of neuroprotective actions for indole–phenolic derivatives, combining chelating, antioxidant, and anti-aggregation properties, which position them as promising compounds for the development of multifunctional agents in Alzheimer’s disease therapy. The methods used provide reliable in vitro data, although further in vivo validation and assessment of blood–brain barrier penetration are needed to confirm therapeutic efficacy and safety. Full article

Background and objective: Cannabinoids are commonly used as adjuvant cancer drugs to overcome numerous adverse side effects for patients. The aim of this study was to identify the target genes that show a synergistic anti-tumor role in combination with the cannabinoid WIN55212-2 in vitro and in vivo. Methods: A human kinome RNAi library was used to screen the targeted gene that silencing plus WIN55212-2 treatment synergistically inhibited cancer cell growth in an INCELL Analyzer 2000. Cell viability, cell phase arrest and apoptosis were evaluated by MTT and flow cytometry assay. In vivo combined anti-tumor effects and regulatory mechanisms were detected in immunocompromised and immunocompetent mice. Results: Using RNAi screening, we identified the tyrosine receptor kinase AXL as a potential gene whose silencing plus WIN55212-2 treatment synergistically inhibited the proliferation of cancer cells in an INCELL Analyzer 2000. Subsequently, we demonstrated that inhibition of AXL by TP-0903 potentiated the inhibitory role of WIN55212-2 on cellular viability, colony formation and 3D tumor sphere in HCT-8 cells. Meanwhile, TP-0903 plus WIN55212-2 treatment promoted the apoptosis of HCT-8 cells. We then investigated the synergistic anti-tumor effect of TP-0903 and WIN55212-2 using colon cancer cell xenografts in immunocompromised and immunocompetent mice. The in vivo study demonstrated that combined administration of TP-0903 plus WIN55212-2 effectively reduced tumor volume and microvessel density and promoted apoptotic cells of tumor tissues in HCT-8 exogenous mice compared to either TP-0903 or WIN55212-2 treatment alone. Moreover, in addition to tumor suppression, the combination therapy of TP-0903 and WIN55212-2 induced the infiltration of cytotoxic CD8⁺ T cells and significantly reduced mTOR and STAT3 activation in tumor tissues of C57BL/6J mice bearing MC-38 cells. Conclusions: This study demonstrated that targeting AXL could sensitize cannabinoids to cancer therapy by interfering with tumor cells and tumor-infiltrating CD8⁺ T cells. Full article

Background: Intracellular tracking is commonly used in trafficking research. Until today, the respective techniques have remained complex, and complicated, mostly transgenic target protein changes are necessary, often requiring expensive equipment and expert knowledge. Methods: We present a novel method, which we term “cell-sonar”, that enables the user to track expression changes of specific protein markers that serve as points of interaction. Our study provides comparable analyses of expression changes of these marker proteins by in-cell Western analyses in two otherwise isogenic cell lines that only differ in the overexpression of the tracked target protein. Using the overexpressed human adult muscle-type nicotinic acetylcholine receptor as an example, we demonstrate that cell-sonar can cover multiple intracellular compartments such as the endoplasmic reticulum, the pathway between it and the Golgi apparatus, and the endocytic pathway. Results: We provide evidence for receptor maturation in the Golgi and storage in recycling endosomes, rather than the fate of increased insertion into the plasma membrane. Additionally, we demonstrate with the implementation of nicotine that the receptor’s destiny is exasperated up to secondary degradation. Conclusions: Cell-sonar is an affordable, easy-to-implement, and cheap method that can be adapted to a broad variety of proteins and cellular pathways of interest to researchers. Full article

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1—critical to the cell cycle—selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (I_Na) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak I_Na density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor’s opposite effects on I_Na. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase—in concert with the AKT/GSK3 pathway—in regulating the Nav1.2 channelosome. Full article

Hantaviruses are zoonotic agents responsible for causing Hantavirus Cardiopulmonary Syndrome (HCPS) in the Americas, with Brazil ranking first in number of confirmed HCPS cases in South America. In this study, we simulate the monthly spread of highly lethal hantavirus in natural hosts by conjugating a Kermack–McCormick SIR model with a cellular automata model (CA), therefore simultaneously evaluating both in-cell and between-cell infection dynamics in host populations, using recently compiled data on main host species abundances and confirmed deaths by hantavirus infection. For both host species, our models predict an increase in the area of infection, with 22 municipalities where no cases have been confirmed to date expected to have at least one case in the next decade, and a reduction in infection in 11 municipalities. Our findings support existing research and reveal new areas where hantavirus is likely to spread within recognized epicenters. Highlighting spatial-temporal trends and potential expansion, we emphasize the increased risk due to pervasive habitat fragmentation and agricultural expansion. Consistent prevention efforts and One Health actions are crucial, especially in newly identified high-risk municipalities. Full article

Epithelial–mesenchymal transition (EMT) refers to the transformation of polar epithelial cells into motile mesenchymal cells under specific physiological or pathological conditions, thus promoting the metastasis of cancer cells. Epithelial cadherin (E-cadherin) is a protein that plays an important role in the acquisition of tumor cell motility and serves as a key EMT epithelial marker. In the present study, AW01178, a small-molecule compound with potential therapeutic efficacy, was identified via in-cell Western high-throughput screening technology using E-cadherin as the target. The compound induced the upregulation of E-cadherin at both mRNA and protein levels and inhibited the EMT of breast cancer cells in vitro as well as metastasis in vivo. Mechanistically, AW01178 is a novel benzacetamide histone deacetylase inhibitor (HDACi) mainly targeting class I histone deacetylases. AW01178 promoted the transcription and expression of E-cadherin through enhancing the acetylation level of histone H3 in the E-cadherin promoter region, thereby inhibiting the metastasis of breast cancer cells. The collective findings support the potential utility of the novel HDACi compound identified in this study, AW01178, as a therapeutic drug for breast cancer and highlight its value for the future development of HDACi structures as anticancer drugs. Full article

This article investigates conceptions of morality within the framework of ressentimentful victimhood in the manosphere, while also exploring avenues for resistance among young individuals encountering the “hatred pipeline”. In Study 1, we use the emotional mechanism of ressentiment to examine how incels construct narratives of victimhood rooted in the notion of sexual entitlement that remains owed and unfulfilled, alongside its “black pill” variant emphasising moral and epistemic superiority. Through a linguistic corpus analysis and content examination of 4chan and Incel.is blog posts, we find evidence of ressentiment morality permeating the language and communication within the incel community, characterised by blame directed at women, and the pervasive themes of victimhood, powerlessness, and injustice. In Study 2, we delve into young individuals’ reflections on incel morality and victimhood narratives as they engage with online networks of toxic masculinity in the manosphere. Drawing from semi-structured interviews with young participants who have accessed the manosphere, we explore their perceptions of risks, attribution of blame, and experiences of empathy towards individuals navigating the “hatred pipeline”. Our analysis underscores the significance of ressentiment in elucidating alternative conceptions of morality and victimhood, while shedding light on the potential for acceptance or resistance within online environments characterised by hatred. Full article

Honey bees play a critical role as pollinators. However, their reproduction success and survival face severe threats due to the deterioration of their living environment. Notably, environmental conditions during their preimaginal stage inside brood cells can influence their immune capabilities and overall health after emergence. During the in-cell developmental stage, workers are in close contact with cocoons, which can become a source of stress due to accumulated metals. To investigate this potential threat, experiments were conducted to examine the impact of cocoons in brood cells used to rear different generations on the metal content and detoxification gene expression levels in Apis cerana cerana. Our findings indicated significant differences in the layers, weight, base thickness, and metal contents like Cr, Cd, Pb, Mn, Ni, and As of cocoons in multi-generation brood cells compared to single-generation brood cells. These increases led to significant elevations in metal levels and upregulations of the four CYP450 detoxification genes in both six-day-old larvae and newly emerged workers. In conclusion, this study highlights the negative impact of cocoons in multi-generation brood cells on bee health and provides evidence supporting the development of rational apiculture management strategies for ecosystem stability. Full article

The global scholarly attention has shifted toward the phenomenon of inceldom (involuntary celibacy) due to violent incidents involving self-identified incels. There is a growing number of platforms promoting the proliferation of these ideologies, and cases of violence are becoming increasingly severe. This research constitutes one of the limited empirical investigations within an Italian context. This study aims to examine the mental well-being and its associations with self-esteem and temporal perspectives among individuals identifying as incels. Fifty-eighth male subjects aged between 18 and 45 years old participated in the study. Participants, recruited through online communication channels, completed three questionnaires focused on assessing mental well-being, self-esteem, and temporal orientation. The results reveal that incel subjects exhibit low self-esteem and are inclined toward a hedonistic present-focused perspective aimed at immediate gratification rather than future planning. Of note are the data related to the future temporal perspective, which does not show any predictive value on the well-being of incel subjects. Their ability to plan for the long term, defer immediate gratification, and control behavior through the anticipation and evaluation of possible consequences appears diminished. This study discusses the implications of developing targeted intervention programs, given that the incel phenomenon is becoming increasingly widespread. It is, therefore, crucial not to underestimate the potential threat that inceldom could pose in the future. Full article

For the quantification of insulin activity, United States Pharmacopeia (USP) general chapter <121> continues to require the rabbit blood sugar test. For new insulin or insulin analogue compounds, those quantitative data are expected for stability or comparability studies. At Sanofi, many rabbits were used to fulfil the authority’s requirements to obtain quantitative insulin bioactivity data until the in vivo test was replaced. In order to demonstrate comparability between the in vivo and in vitro test systems, this study was designed to demonstrate equivalency. The measurement of insulin lispro and insulin glargine drug substance and drug product batches, including stress samples (diluted or after temperature stress of 30 min at 80 °C), revealed a clear correlation between the in vitro and in vivo test results. The recovery of quantitative in vitro in-cell Western (ICW) results compared to the in vivo test results was within the predefined acceptance limits of 80% to 125%. Thus, the in vitro ICW cell-based bioassay leads to results that are equivalent to the rabbit blood sugar test per USP <121>, and it is highly suitable for insulin activity quantification. For future development compounds, the in vitro in-cell Western cell-based assay can replace the rabbit blood sugar test required by USP <121>. Full article

Cancers utilize sugar residues such as sialic acids (Sia) to improve their ability to survive. Sia presents a variety of functional group alterations, including O-acetylation on the C6 hydroxylated tail. Previously, sialylation has been reported to suppress EGFR activation and increase cancer cell sensitivity to Tyrosine Kinase Inhibitors (TKIs). In this study, we report on the effect of deacetylated Sia on the activity of three novel EGFR-targeting Cucurbitacin-inspired estrone analogs (CIEAs), MMA 294, MMA 321, and MMA 320, in lung and colon cancer cells. Acetylation was modulated by the removal of Sialate O-Acetyltransferase, also known as CAS1 Domain-containing protein (CASD1) gene via CRISPR-Cas9 gene editing. Using a variety of cell-based approaches including MTT cell viability assay, flow cytometry, immunofluorescence assay and in-cell ELISA we observed that deacetylated Sia-expressing knockout cells (1.24–6.49 μM) were highly sensitive to all CIEAs compared with the control cells (8.82–20.97 μM). Apoptosis and varied stage cell cycle arrest (G0/G1 and G2/M) were elucidated as mechanistic modes of action of the CIEAs. Further studies implicated overexpression of CIEAs’ cognate protein target, phosphorylated EGFR, in the chemosensitivity of the deacetylated Sia-expressing knockout cells. This observation correlated with significantly decreased levels of key downstream proteins (phosphorylated ERK and mTOR) of the EGFR pathway in knockout cells compared with controls when treated with CIEAs. Collectively, our findings indicate that Sia deacetylation renders lung and colon cancer cells susceptible to EGFR therapeutics and provide insights for future therapeutic interventions. Full article

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling. Full article