Search Results (94)

Hydralazine is widely used to treat hypertension during pregnancy and has epigenetic effects in cancer therapy. Cryoplatable human hepatocytes showed concentration-dependent increase in DNA damage response (linear trend p = 0.0069) following 24 h hydralazine treatment. DNA repair-deficient UV5 Chinese hamster ovary (CHO) cell lines expressing human CYP1A2 and either NAT2*4 (reference allele) or NAT2*5 (variant allele) were treated with hydralazine for 24 h. CHO cells expressing NAT2*4 showed a higher acetylation rate than those with NAT2*5 (p < 0.001), whereas CHO cell viability did not differ significantly following hydralazine treatment (p > 0.05). Hydralazine caused a concentration-dependent increase in DNA damage response in the un-transfected UV5 CHO cell line, as well as in each of the UV5 CHO cell lines transfected with human CYP1A2 and/or NAT2 alleles. CHO cells with CYP1A2 only showed higher DNA damage response from hydralazine compared to cells with CYP1A2/NAT2*4 or CYP1A2/NAT2*5 (p < 0.05 and p < 0.0001, respectively), and higher in CYP1A2/NAT2*4 versus CYP1A2/NAT2*5 cells (p = 0.0011). Apurinic/apyrimidinic (AP) sites in CHO cells expressing only CYP1A2 were significantly higher than in the un-transfected UV5 CHO cell line (p < 0.01) and higher in CHO cells expressing CYP1A2/NAT2*4 compared to CYP1A2/NAT2*5, but the difference was not significant (p > 0.05). In contrast, ROS levels were reduced following hydralazine treatment in CHO cells with CYP1A2/NAT2*4 and CYP1A2/NAT2*5 (p < 0.001 and p < 0.05, respectively). The results of the current study document DNA damage responses associated with hydralazine in human hepatocytes and CHO cells. The DNA damage response was increased following N-hydroxylation by CYP1A2, which competes with N-acetylation by NAT2. Full article

Driven by the large-scale deployment of renewable electricity, water electrolysis has emerged as a leading pathway for high-efficiency hydrogen production. Under practical operating conditions, gas evolution and gas–liquid two-phase flow inside electrolyzers substantially reshape electrode interfacial states and the in-cell mass transfer environment and have been reported to cause performance losses on the order of 10–30% under unfavorable conditions. This review summarizes the evolution of electrode-generated bubbles during nucleation, growth, detachment, and coalescence, and consolidates the fundamental features of two-phase hydrodynamics and phase-distribution patterns in electrolyzer channels. Progress and limitations of major two-phase modeling approaches are then assessed with respect to their capability to resolve the relevant interfacial and transport processes. The impacts of gas evolution and two-phase flow on electrochemical performance, stability, and durability are subsequently discussed. Finally, recent advances in two-phase-flow management—through flow-field organization and structural design, as well as the introduction of external physical fields—are reviewed, together with experimental and diagnostic methods used to quantify bubble behavior and phase distributions. This review aims to provide a coherent understanding of the governing behaviors, research tools, and performance implications of gas evolution and two-phase flow in water electrolysis, and to inform electrode/transport-layer design, flow-field management, and the development of predictive numerical models. Full article

Enzymes are indispensable in fields such as biotechnology, medicine, and industrial manufacturing due to their high catalytic specificity and efficiency under mild conditions. However, their natural versions often suffer from limitations, including low activity toward non-natural substrates, poor stability under extreme conditions, and narrow substrate spectra. Directed evolution, a key protein engineering strategy that optimizes protein function via genetic diversity introduction and directed selection, has become the primary solution to these limitations. Among its mature methodological systems, in vivo evolution platforms (advanced by synthetic biology) are particularly efficient, as they integrate in-cell mutation, translation, selection, and replication into an automated process, significantly improving experimental efficiency. This review will focus on two core strategies that enhance these platforms: in vivo targeted gene hypermutation and heterologous polymerase-mediated targeted hypermutation. These techniques enable the rapid optimization of enzymes to acquire novel functions, as well as the comprehensive engineering of microbial strains to enhance their performance and stress tolerance. Analyzing these strategies provides a robust technical framework for enzyme engineering and promises to drive future innovations across multiple fields. Full article

Transmetalation, the exchange of metal ions between coordination complexes and biomolecules, has emerged as a powerful design lever in cancer metallopharmacology. Using thiosemicarbazones (TSCs) as a unifying case study, we show how redox-inert carrier states such as zinc(II) or gallium(III) can convert in situ into redox-active copper(II) or iron(III/II) complexes within acidic, metal-rich lysosomes. This conditional activation localizes reactive oxygen species (ROS) generation and iron deprivation to tumor cells. We critically compare redox-active and redox-inert states, delineating how steric and electronic tuning, backbone rigidity, and sulfur-to-selenium substitution govern exchange hierarchies and kinetics. We further map downstream consequences for metal trafficking, lysosomal membrane permeabilization, apoptosis, and ferroptosis. Beyond TSCs, iron(III)-targeted transmetalation from titanium(IV)-chelator “chemical transferrin mimetics” illustrates a generalizable Trojan horse paradigm. We conclude with translational lessons, including mitigation of hemoprotein oxidation via steric shielding, stealth zinc(II) prodrugs, and dual-chelator architectures and outline biomarker, formulation, and imaging strategies that de-risk clinical development. Collectively, these insights establish transmetalation as a central therapeutic principle. We also highlight open challenges such as quantifying in-cell exchange kinetics, predicting speciation under non-equilibrium conditions, and rationally combining these agents with existing therapies. Full article

We pose the question of whether life has become unlivable for the young in America amidst the current political climate, which has systematically deregulated our social structures that safeguard against oppressive and unjust practices. What leads the young to become demoralized to the point of wanting to end their lives? Drawing on several established psychosocial models for suicide, including those of Durkheim, Joiner, and Butler, we highlight how groups of youth as disparate as youth of color, LGBTQ+ youth, and young men experience unique sociopolitical stressors that contribute to increased suicidality. We argue that despite differences in their contexts, they experience shared pathways to suicide. At a time when U.S. funding cuts threaten to dismantle the progress made in recent years to address structural racism and sexism, we also make a case for the importance of mental health clinicians’ engagement in advocacy work that recognizes the sociopolitical influences on mental health and highlight universal school-based social emotional learning (USB SEL) as one beneficial intervention to target mental health outcomes across disparate youth groups. Full article

The assessment of a ligand’s activity is typically established by measuring its binding affinity in a biochemical assay, often expressed as K_a or K_d values. Further validation of its biological activity is achieved through cellular assays. There is frequently an inconsistency between the activity values obtained from those assays, which could delay research progress as well as drug development. Factors such as the permeability, solubility, specificity, and stability of active compounds are usually held responsible for this discrepancy. Even when these values are known, inconsistencies in activity measurements remain challenging to explain. This is not surprising since intracellular physicochemical conditions are undoubtedly different from the simplified conditions used in most in vitro biochemical assays. It is therefore reasonable to assume that these differences would be minimized if biochemical measurements were performed under conditions that more accurately mimic the intracellular environment. These physicochemical conditions can alter the K_d values. While the cellular environment has been extensively studied for decades, more recent efforts have focused on obtaining equilibrium and kinetic data directly from in-cell environments. Clarifying molecular crowding, salt composition, and lipophilic parameters inside the cell and thus their effect on molecular equilibrium is a crucial step toward replicating the intracellular environment. Full article

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

We report two conjugates of gem-diethyl pyrroline nitroxide radicals with D-mannosamine as potential metabolic organic radical contrast agents, mORCAs, circumventing the need for biorthogonal reactions. In-cell EPR spectroscopy, using Jurkat cells and analogous conjugate, based on a pyrrolidine nitroxide radical, shows an efficient incorporation of highly immobilized nitroxides, with a correlation time of τ_cor = 20 ns. In vivo MRI experiments in mice show that the paramagnetic nitroxide radical shortens the T₁ and T₂ relaxation times of protons in water located in the kidney and brain by only up to ~10% after 3 d. Ex vivo EPR spectroscopic analyses indicate that the contrast agents in mouse tissues are primarily localized in the kidney, lung, liver, heart, and blood, which primarily contain immobilized nitroxide radicals with τ_cor = 4–9 ns. The spin concentrations in tissues remain low (1–3 nmol g⁻¹) at 24 h after the third mORCA injection, approximately one to two orders of magnitude lower than those of ORCAFluor and BASP-ORCA (measured at ~24 h post-injection). These low spin concentrations explain the small proton T₁ and T₂ relaxation changes observed in in vivo MRI. Full article

We describe a method tailored to the in-cell selection of protease inhibitors. In this method, a target protease is co-expressed with a selective substrate, the product of which kills host cells. Therefore, the method can be applied to identify potential inhibitors based on cell host survival when inhibition of the target protease occurs. The TEV protease was chosen for this proof-of-concept experiment. The genetically encoded selective substrate is a single polypeptide chain composed of three parts: (1) a ccdB protein, which can cause host cell death when it accumulates inside the cell; (2) a protease cleavage sequence that can be changed according to the target protease, in this case the TEV substrate ENLYFQ↓G (↓-predicted cleavage site); and (3) the ssrA sequence (AANDENYALAA), which drives the polypeptide to degradation by the ClpX/ClpP complex inside host E. coli cells. In our experiment, co-expression of the active TEV protease and this selective substrate (ccdB-ENLYFQG-ssrA) caused the death of a significant host cell population, while control assays with an inactive mutant TEV Asp81Asn did not. Details of the methodology used are given, providing the basis for the application of similar systems for other proteases of interest. Full article

Dengue virus (DV) infection poses a severe life-threatening risk in certain cases. This is mainly due to endothelial dysregulation, which causes plasma leakage and hemorrhage. However, the etiology of DV-induced endothelial dysregulation remains incompletely understood. To identify the potential mechanisms of endothelial dysregulation caused by DV, the effects of conditioned media from Dengue virus (CMDV) on the mechanics and transcriptional profile of the endothelial cells were examined using permeability assays, atomic force microscopy, In-Cell Western blot and in silico transcriptomics. Exposure of HMEC-1 cells to the CMDV increased endothelial permeability and cellular stiffness. It also induced the expression of the key proteins associated with endothelial-to-mesenchymal transition (EndMT). These data support the notion that the DV promotes endothelial dysfunction by triggering transcriptional programs that compromise the endothelial barrier function. Understanding the molecular mechanisms underlying DV-induced endothelial dysregulation is crucial for developing targeted therapeutic strategies to mitigate the severe outcomes associated with dengue infection. 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

In-cell touch and display integrated panels, along with their integrated readout systems, are widely adopted in mobile devices for their cost-effectiveness and compact design. This paper proposes an analog Gaussian-shaped filter and a current mode compensation technique for dot-matrix Touch Screen Panel (TSP) readout systems. Specifically, this article presents a noise management strategy for both intrinsic and external noise, offering simulation guidelines for determining intrinsic circuit noise levels in relation to scan time and enhancing external noise immunity through the Gaussian-shaped filter response. The system achieved an intrinsic SNR of 66 dB with a 200 kHz TSP driving frequency and a 160 μs scan time, while the 4-bit quantized Gaussian coefficients filter provided 33 dB noise suppression for out-of-band noise. The compensation error in the dot-matrix capacitance compensation was measured at 1.24 pF, which corresponds to a 0.078% deviation. The simulated power consumption of the proposed readout system is 24 mW, with a layout area of 1.017 mm² for the 10-channel readout front-end. 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

Fragment-based drug discovery is a powerful approach in drug discovery, applicable to a wide range of targets. This method enables the discovery of potent compounds that can modulate target functions, starting from fragment compounds that bind weakly to the targets. While biochemical, biophysical, and cell-based assays are commonly used to identify fragments, ¹⁹F-NMR spectroscopy has emerged as a powerful tool for exploring interactions between biomolecules and ligands. Because fluorine atoms are not naturally present in biological systems, ¹⁹F-NMR serves as a sensitive method for fragment screening against diverse targets. Herein, we reviewed the applications of ¹⁹F-NMR in fragment screening, highlighting its effectiveness in identifying fragments that bind weakly to various targets such as proteins and RNA. The accumulated evidence suggests that ¹⁹F-NMR will continue to be a crucial tool in drug discovery. Full article