p53 inhibitor or antioxidants reduce the severity of ethmoid plate deformities in zebrafish Type 3 Treacher Collins syndrome model.

Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.

Fate characteristics and risk quantification of cyflumetofen from tomato cultivation to processing based on large-scale applications.

Elucidating the fate characteristics of cyflumetofen and its main metabolite 2-TFMBA in tomato from cultivation to processing is crucial for safeguarding the environment and humans from hazardous effects. Cyflumetofen and 2-TFMBA could exist stably in tomato matrices for at least 343 days under frozen and dark conditions according to UHPLC-MS/MS, with a limit of quantitation of 0.001 mg/kg and retention time within 2.12 min. The occurrence, dissipation, and concentration variation of cyflumetofen were reflected by original depositions of 0.02-0.44 mg/kg, half-lives of 1.7-7.2 days, and terminal magnitudes of 0.005-0.30 mg/kg, respectively, with various influencing factors, e.g., climate conditions and tomato cultivars. Additionally, 13.5-59.3% of cyflumetofen was metabolized to 2-TFMBA, showing significant toxicological effects ranging from cultivation to processing. When the concentration decreased by 0.06 mg/kg, cyflumetofen was effectively removed by peeling, while washing was the recommended method for removing 2-TFMBA with a processing factor of 0.70. The comparative dietary risks of sum cyflumetofen were assessed for all life cycle populations using deterministic and probabilistic models. The risk quotients decreased to 1.3-4.8 times during the preparation of home canning tomato paste. Despite the low exposure risk, the potential health hazards of sum cyflumetofen should be considered, given its ubiquity and cumulative effects.

Selenium deficiency exacerbated Bisphenol A-induced intestinal toxicity in chickens: Apoptosis and cell cycle arrest mediated by ROS/P53.

Bisphenol A (BPA) is a phenolic organic synthetic compound that is used as the raw material of polycarbonate plastics, and its safety issues have recently attracted wide attention. Selenium (Se) deficiency has gradually developed into a global disease affecting intestinal function via oxidative stress and apoptosis. However, the toxic effects and potential mechanisms of BPA exposure and Se deficiency in the chicken intestines have not been studied. In this study, BPA exposure and/or Se deficiency models were established in vivo and in vitro to investigate the effects of Se deficiency and BPA on chicken jejunum. The results showed that BPA exposure and/or Se deficiency increased jejunum oxidative stress and DNA damage, activated P53 pathway, led to mitochondrial dysfunction, and induced apoptosis and cell cycle arrest. Using protein-protein molecular docking, we found a strong binding ability between P53 and peroxisome proliferator-activated receptor γ coactivator-1, thereby regulating mitochondrial dysfunctional apoptosis. In addition, we used N-acetyl-L-cysteine and pifithrin-α for in vitro intervention and found that N-acetyl-L-cysteine and pifithrin-α intervention reversed the aforementioned adverse effects. This study clarified the potential mechanism by which Se deficiency exacerbates BPA induced intestinal injury in chickens through reactive oxygen species/P53, which provides a new idea for the study of environmental combined toxicity of Se deficiency, and insights into animal intestinal health from a new perspective.

Protocol for preparing dynamic covalent macrocycles for co-delivering genes and drugs to cancer cell lines.

Combination therapy using effective drug molecules and functional genes such as small interfering RNA (siRNA) has been suggested as a powerful strategy against multiple drug resistance. Here, we present a protocol for preparing a delivery system by developing dynamic covalent macrocycles using a dithiol monomer to co-deliver doxorubicin and siRNA. We describe steps for preparing the dithiol monomer, followed by co-delivery to form nanoparticles. We then detail procedures for cell uptake and assessing enhanced anti-cancer efficacy in vitro. For complete details on the use and execution of this protocol, please refer to Lyu et al.1.

SIRT1720 promotes survival of corneal epithelial cells via the P53 pathway.

PURPOSE: To investigate the protective role of SRT1720 (SIRT1 activator) against the oxidative stress caused by H2O2 in the corneal cell line. METHODS: Human corneal (2.040 pRSV-T) cell lines were cultured and treated with SRT1720 (as SIRT1 activator) and nicotinamide (NAM, a SIRT1 inhibitor), and incubated with H2O2. The expression level of SIRT1, p53, and acetyl-p53 was measured by western blot. Propidium iodine/annexin V-FITC staining, and flow cytometry was used to evaluate apoptosis. The trypan blue assay was used to assess the morphological modifications that occurred after the treatment, and Pifithrin-α (PFT-α) was used to inhibit the p53 pathway. RESULTS: The investigation revealed that under oxidative stress, SRT1720 caused a reduction in acetyl-p53 expression and increased SIRT1 expression. It was also found that under oxidative stress, SRT1720 suppressed apoptosis. In comparison, NAM promoted cell apoptosis under oxidative stress. NAM's destructive effect was eliminated by PFT-α, a suppressor of the p53 pathway. PFT-α reduced the morphological changes in 2.040 pRSV-T cell lines compared to NAM treatment and inhibited apoptosis. CONCLUSIONS: The protective effects of the SIRT1 activator (SRT1720) indicate that H2O2 induces oxidative stress-associated cell damage. The results also encouraged us to consider using SRT1720 to improve corneal safety and reduce the adverse effects of oxidative damage.

Preserving DNA in Biodegradable Organosilica Encapsulates.

A core-shell strategy was developed to protect synthetic DNA in organosilica particles encompassing dithiol linkages allowing for a DNA loading of 1.1 wt %. DNA stability tests involving bleach as an oxidant showed that following the procedure DNA was sandwiched between core particles of ca. 450 nm size and a protective outer layer, separating the DNA from the environment. Rapid aging tests at 60 °C and 50% relative humidity revealed that the DNA protected within this material was significantly more stable than nonprotected DNA, with an expected ambient temperature half-life of over 60 years. Still, and due to the presence of the dithiol linkages in the backbone of the organosilica material, the particles degraded in the presence of reducing agents (TCEP and glutathione) and disintegrated within several days in a simulated compost environment, which was employed to test the biodegradability of the material. This is in contrast to DNA encapsulated following state of the art procedures in pure SiO2 particles, which do not biodegrade in the investigated timeframes and conditions. The results show that synthetic DNA protected within dithiol comprising organosilica particles presents a strategy to store digital data at a high storage capacity for long time frames in a fully biodegradable format.

Dithiol Based on l-Cysteine and Cysteamine as a Disulfide-Reducing Agent.

We report the synthesis, chemical properties, and disulfide bond-reducing performance of a dithiol called NACMEAA, conceived as a hybrid of two biologically relevant thiols: cysteine and cysteamine. NACMEAA is conveniently prepared from inexpensive l-cystine in an efficient manner. As a nonvolatile, highly soluble, and neutral compound at physiological pH with the first thiol pKa value of 8.0, NACMEAA is reactive and user-friendly. We also demonstrate that NACMEAA reduces disulfide bonds in GSSG and lysozyme.

Ionic Liquid-Mediated One-Pot 3-Acylimino-3H-1,2-dithiole Synthesis from Thiocarboxylic Acids and Alkynylnitriles via In Situ Generation of Disulfide Intermediates.

A practical and straightforward strategy for the synthesis of 3-acylimino-3H-1,2-dithiol derivatives via a metal-free annulation reaction of alkynylnitriles with thiocarboxylic acids mediated by ionic liquids [BMIM]Br has been reported. This operationally simple protocol offers an easy and rapid access to a library of dithiol derivatives in moderate to good yields. The mechanistic studies show a benzoyldithio anion addition to alkynylnitriles followed by an annulation reaction through the involvement of a disulfide moiety as the key intermediate.

PFT-α inhibits gallid alpha herpesvirus 1 replication by repressing host nucleotide metabolism and ATP synthesis.

Therapeutics targeting virus-host interactions have been considered promising strategies for treating herpesvirus infection. Our previous study on avian infectious laryngotracheitis virus (ILTV), an avian herpesvirus economically important to the poultry industry worldwide, identified the small molecule Pifithrin-α (PFT-α) as a potential therapeutic agent. However, the underlying mechanisms of its antiviral function remain largely unknown. Using the ILTV-permissive chicken cell line LMH as the model, we found that PFT-α effectively suppressed the transcription and genome replication of ILTV and greatly reduced the level of infectious virions. Genome-wide transcriptome analysis revealed extensive repression of the metabolic processes of infected cells by PFT-α administration. Further metabolome assays of ILTV-infected cells using liquid chromatography coupled with mass spectrometry suggest host nucleotide metabolism and ATP synthesis as the key targets of PFT-α treatment during its repression of ILTV replication, which was experimentally supported by the reduced transcription of many key enzymes essential to nucleotide metabolism and ATP synthesis. The present study provides insights into the mechanisms by which PFT-α inhibits ILTV infection, which may increase the probability of successful clinical application of this molecule.

Pifithrin-α ameliorates glycerol induced rhabdomyolysis and acute kidney injury by reducing p53 activation.

OBJECTIVES: Rhabdomyolysis is a series of symptoms caused by the dissolution of striped muscle, and acute kidney injury (AKI) is a potential complication of severe rhabdomyolysis. The underlying causes of AKI are remarkably complex and diverse. Here, we aim to investigate whether pifithrin-α protected against rhabdomyolysis-induced AKI and to determine the involved mechanisms. METHODS: Intramuscular injection in the right thigh caudal muscle of C57BL/6J mice with 7.5 ml/kg saline (Group A) or of the same volume 50% glycerol was used to induce rhabdomyolysis and subsequent AKI (Group B). Pifithrin-α was injected intraperitoneally 4 h before (Group C) or 4 h after (Group D) the glycerol injection. Serum creatine kinase, blood urea nitrogen, and creatinine were determined, and the renal cortex was histologically analyzed. Renal expression levels of interested mRNAs and proteins were determined and compared, too. RESULTS: Intramuscular injection of glycerol induced rhabdomyolysis and subsequent AKI in mice (Groups B-D). Renal function reduction and histologic injury of renal tubular epithelial cells were associated with increased p53 activation, oxidative stress, and inflammation. Notably, compared with pifithrin-α rescue therapy (Group D), pretreatment of pifithrin-α (Group C) protected the mice from severe injury more effectively. CONCLUSIONS: Our present study suggests that p53 may be a therapeutic target of AKI caused by glycerol, and the inhibition of p53 can block glycerol-mediated AKI by using pharmacological agents instead of genetic inhibitory approaches, which further supports that p53 played a pivotal role in renal tubular injury when challenged with glycerol.

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

Urea transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation.

Urea transporter B (UT-B, encoded by the SLC14A1 gene) is a membrane channel protein involved in urea transmembrane transport. Compared with normal tissues, UT-B expression is significantly decreased in most tumours, especially melanoma. However, the UT-B role in tumorigenesis and development is still unclear. Herein, we investigated the effects of UT-B overexpression on polyamine metabolism and the urea cycle in murine melanoma B16 cells, to explore the roles of mitochondrial dysfunction and p53 activation in cell growth and polyamines metabolism. UT-B overexpression in B16 cells decreased cell growth, increased apoptosis, and significantly altered metabolic pathways related to the urea cycle, which were characterized by reduced production of urea and polyamines and increased production of nitric oxide. Subsequently, we observed that activation of the p53 pathway may be the main cause of the above phenomena. The p53 inhibitor pifithrin-α partially restored the production of polyamines, but the mitochondrial morphology and function were still impaired. Further treatment of UT-B-overexpressing B16 cells with reactive oxygen species scavenging agent N-acetyl-l-cysteine and coenzyme Q10 restored cell viability and mitochondrial function and increased polyamine production. In conclusion, UT-B overexpression caused mitochondrial dysfunction and increased oxidative stress in B16 cells, and then activated p53 expression, which may be one of the mechanisms leading to the decrease in intracellular polyamines.

Design and Development of a Bioorthogonal, Visualizable and Mitochondria-Targeted Hydrogen Sulfide (H2 S) Delivery System.

Hydrogen sulfide (H2 S) is an important endogenous gasotransmitter, but the targeted delivery and real-time feedback of exogenous H2 S are still challenging. With the aid of density functional theory (DFT) calculations, we designed a new 1,3-dithiolium-4-olate (DTO) compound, which can react with a strained alkyne via the 1,3-dipolar cycloaddition and the retro-Diels-Alder reaction to generate carbonyl sulfide (COS) as the precursor of H2 S, and a thiophene derivative with turn-on fluorescence. Moreover, the diphenylamino substituent in DTO greatly increases the mitochondrial targeting of this H2 S delivery system. Such a bioorthogonal click-and-release reaction has integrated three functions in one system for the first time: (1) in situ controllable H2 S release, (2) concomitant fluorescence response, and (3) mitochondria-targeted delivery. In addition, we investigated the mitochondrial membrane potential loss alleviation by using this system in H9c2 cells under oxidative stress.

Spherical silver oxide nanoparticles for fabrication of electrochemical sensor for efficient 4-Nitrotoluene detection and assessment of their antimicrobial activity.

This research article reports an economic and affordable microwave-assisted synthesis of spherical silver oxide nanoparticles (Ag2O NPs) (80-90 nm) for an efficient electrochemical sensing of a hazardous organic pollutant 4-nitrotoluene (4-NT). Such well-characterized Ag2O NPs were utilized to modify gold (Au) electrode in order to fabricate Ag2O-NPs/Au sensor to detect 4-NT using cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques. Ag2O-NPs/Au sensor exhibited a distinguished electrical response as a function of varying 4-NT concentration in neutral medium samples. Ag2O-NPs/Au sensor demonstrated an ultrahigh sensitivity as 15.33 µA (µM)-1 cm-2, a low detection limit of 62.3 nM, and linear response in detection ranges of 0.6-5.9 µM and 37-175 µM. The sensing performance of fabricated Ag2O-NPs/Au sensor is reproducible, reusable, selective in presence of other chemical species, and validated using real samples. The Ag2O/Au sensor had much rapid and easy fabrication process and offered much lower LOD for 4-NT detection than many previously reported sensors. Along with efficient electrochemical activity, the spherical Ag2O NPs also exhibit remarkable antimicrobial activity against harmful gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacteria. Herein projected efficient Ag2O-NPs/Au electrochemical sensor for 4-NT is affordable with the capability of scaling up to perform point-of-care 4-NT testing needed for successful environmental monitoring and water quality assurance.

Full Characterization of the UV Hydrodebromination Products of the Current-Use Brominated Flame Retardants Hexabromobenzene, Pentabromotoluene, and Pentabromoethylbenzene.

UV transformation was studied with three structurally closely related current-use brominated flame retardants (cuBFRs), i.e., hexabromobenzene (HBB), pentabromotoluene (PBT), and pentabromoethylbenzene (PBEB). Irradiation in toluene and benzotrifluoride (BTF) showed pseudo-first-order kinetics. Repeated high-performance liquid chromatographic (HPLC) fractionation, available reference standards, dedicated syntheses, gas chromatography with mass spectrometry (GC/MS), GC separation on two different phases including retention time rules based on dipole interactions, and proton magnetic resonance spectroscopy (1H NMR) evaluation enabled a full structural characterization of all 22 transformation products formed by hydrodebromination. In addition to pentabromobenzene (only transformation product with five bromine), tetra- and tribrominated transformation products were predominantly formed in the case of all three cuBFRs. Hydrodebromination was favored by bromine removal from positions with a high Br density. Br → H exchange was about 3 times faster in positions flanked by two vicinal Br atoms. This favored pathway explained why hydrodebromination sharply dropped at the level of tribrominated cuBFRs because readily degradable precursors were no more available at this point. Hence, a full degradation of tribrominated and lower-brominated transformation products may only be achieved in combination with a different process such as microbial transformation.

A New Inhibitor of ADAM17 Composed of a Zinc-Binding Dithiol Moiety and a Specificity Pocket-Binding Appendage.

A disintegrin and metalloproteinase 17 (ADAM17) is a zinc-dependent enzyme that catalyzes the cleavage of the extracellular domains of various transmembrane proteins. ADAM17 is regarded as a promising drug target for the suppression of various diseases, including cancer metastasis. We synthesized a new ADAM17 inhibitor, SN-4, composed of a zinc-binding dithiol moiety and an appendage that specifically binds to a pocket of ADAM17. We show that SN-4 inhibits the ability of ADAM17 to cleave tumor necrosis factor α (TNF-α) in vitro. This activity was reduced by the addition of zinc, indicating the importance of the zinc chelating dithiol moiety. Inhibition of TNF-α cleavage by SN-4 in cells was also observed, and with an IC50 of 3.22 µM, SN-4 showed slightly higher activity than the well-studied ADAM17 inhibitor marimastat. Furthermore, SN-4 was shown to inhibit cleavage of CD44 by ADAM17, but not by ADAM10, and to suppress cell invasion. Molecular docking showed good fitting of the specificity pocket-binding group and one SH of SN-4 and hinted at possible means of structural optimization. This study provides clues for the development of potent and selective ADAM17 inhibitors.

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors.

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.

Autophagic Activation and Decrease of Plasma Membrane Cholesterol Contribute to Anticancer Activities in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.