Glutathione-activated biotin-targeted dual-modal imaging probe with improved PDT/PTT synergistic therapy.

BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer. RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation. SIGNIFICANCE: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.

Improved Preservation of Rat Small Intestine Transplantation Graft by Introduction of Mesenchymal Stem Cell-Secreted Fractions.

Segmental grafts from living donors have advantages over grafts from deceased donors when used for small intestine transplantation. However, storage time for small intestine grafts can be extremely short and optimal graft preservation conditions for short-term storage remain undetermined. Secreted factors from mesenchymal stem cells (MSCs) that allow direct activation of preserved small intestine grafts. Freshly excised Luc-Tg LEW rat tissues were incubated in preservation solutions containing MSC-conditioned medium (MSC-CM). Preserved Luc-Tg rat-derived grafts were then transplanted to wild-type recipients, after which survival, injury score, and tight junction protein expression were examined. Luminance for each graft was determined using in vivo imaging. The findings indicated that 30-100 and 3-10 kDa fractions of MSC-CM have superior activating effects for small intestine preservation. Expression of the tight-junction proteins claudin-3, and zonula occludens-1 preserved for 24 h in University of Wisconsin (UW) solution containing MSC-CM with 50-100 kDa, as shown by immunostaining, also indicated effectiveness. Reflecting the improved graft preservation, MSC-CM preloading of grafts increased survival rate from 0% to 87%. This is the first report of successful transplantation of small intestine grafts preserved for more than 24 h using a rodent model to evaluate graft preservation conditions that mimic clinical conditions.

Loss of ß-catenin reveals a role for glutathione in regulating oxidative stress during cholestatic liver disease.

BACKGROUND: Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/ß-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury. METHODS: α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of ß-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses. RESULTS: We found that the presence of ß-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated ß-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality. CONCLUSIONS: Loss of ß-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Tentative identification of phytochelatins, their derivatives, and Cd-phytochelatin complexes in Ocimum basilicum L. roots by Paper Spray Mass Spectrometry.

An unprecedented and direct PS-MS (paper spray ionization mass spectrometry) method was proposed for the detection of native peptides, that is, glutathiones (GSHs), homoglutathiones (hGSHs), and phytochelatins (PCs), in basil (Ocimum basilicum L.) roots before and after cadmium exposure. The roots were submitted to cold maceration followed by sonication with formic acid as the extractor solvent for sample preparation. PS-MS was used to analyze such extracts in the positive mode, and the results allowed for the detection of several GSHs, hGSHs, and PCs. Some of these PCs were not distinguished in the control samples, that is, basil roots not exposed to cadmium. Other PCs were noticed in both types of roots, uncontaminated and cadmium-contaminated, but the intensities were higher in the former samples. Moreover, long-time exposure to cadmium stimulated the formation of some of these PCs and their cadmium complexes. The results, therefore, provided some crucial insights into the defense mechanism of plants against an external stress condition due to exposure to a toxic heavy metal. The present study represents a promising alternative to investigate other crucial physiological processes in plants submitted to assorted stress conditions.

Allicin inhibits the growth of HONE-1 and HNE1 human nasopharyngeal carcinoma cells by inducing ferroptosis.

Allicin (AL) is one of garlic-derived organosulfides and has a variety of pharmacological effects. Studies have reported that AL has notable inhibitory effects on liver cancer, gastric cancer, breast cancer, and other cancers. However, there are no relevant reports about its role in human nasopharyngeal carcinoma. Ferroptosis is an iron-dependent form of non-apoptotic regulated cell death. Increasing evidence indicates that induction of ferroptosis can inhibit the proliferation, migration, invasion, and survival of various cancer cells, which act as a tumor suppressor in cancer. In this study, we confirmed that AL can inhibit cell proliferation, migration, invasion, and survival in human nasopharyngeal carcinoma cells. Our finding shows that AL can induce the ferroptosis axis by decreasing the level of GSH and GPX4 and promoting the induction of toxic LPO and ROS. AL-mediated cytotoxicity in human nasopharyngeal carcinoma cells is dependent on ferroptosis. Therefore, AL has good anti-cancer properties and is expected to be a potential drug for the treatment of nasopharyngeal carcinoma.

Myricetin mitigates motor disturbance and decreases neuronal ferroptosis in a rat model of Parkinson's disease.

Ferroptosis is an iron-dependent cell death form characterized by reactive oxygen species (ROS) overgeneration and lipid peroxidation. Myricetin, a flavonoid that exists in numerous plants, exhibits potent antioxidant capacity. Given that iron accumulation and ROS-provoked dopaminergic neuron death are the two main pathological hallmarks of Parkinson's disease (PD), we aimed to investigate whether myricetin decreases neuronal death through suppressing ferroptosis. The PD models were established by intraperitoneally injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into rats and by treating SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP+), respectively. Ferroptosis was identified by assessing the levels of Fe2+, ROS, malondialdehyde (MDA), and glutathione (GSH). The results demonstrated that myricetin treatment effectively mitigated MPTP-triggered motor impairment, dopamine neuronal death, and α-synuclein (α-Syn) accumulation in PD models. Myricetin also alleviated MPTP-induced ferroptosis, as evidenced by decreased levels of Fe2+, ROS, and MDA and increased levels of GSH in the substantia nigra (SN) and serum in PD models. All these changes were reversed by erastin, a ferroptosis activator. In vitro, myricetin treatment restored SH-SY5Y cell viability and alleviated MPP+-induced SH-SY5Y cell ferroptosis. Mechanistically, myricetin accelerated nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and subsequent glutathione peroxidase 4 (Gpx4) expression in MPP+-treated SH-SY5Y cells, two critical inhibitors of ferroptosis. Collectively, these data demonstrate that myricetin may be a potential agent for decreasing dopaminergic neuron death by inhibiting ferroptosis in PD.

Durian fruit pulp extract enhances intracellular glutathione levels, mitigating oxidative stress and inflammation for neuroprotection.

Durian (Durio zibethinus L.) fruit pulp is a rich source of γ-glutamylcysteine (γ-EC), a direct precursor to the antioxidant glutathione (GSH). This study elucidated the in vitro neuroprotective potential of unripe durian fruit pulp extract (UDE) against H2O2-induced neurotoxicity in SH-SY5Y cells and neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells. Treatments with γ-EC, GSH standards, or UDE exhibited no cytotoxicity in SH-SY5Y and BV-2 cells, except at high concentrations. A 4-h pretreatment with 100 µM γ-EC or UDE containing 100 µM γ-EC significantly increased SH-SY5Y cell viability post H2O2 induction. Moreover, a similar pretreatment reduced LPS-stimulated production of proinflammatory cytokines in BV-2 cells. The neuroprotective effect of UDE is primarily attributed to γ-EC provision and the promotion of GSH synthesis, which in turn elevates intracellular GSH levels and reduces proinflammatory cytokines. This study identifies γ-EC in UDE as a potential neuroprotective biomarker boosting intracellular GSH levels, providing insights into UDE's therapeutic potential.

Alteration of salivary LPO, MDA, LDH, glutathione, GPx, SOD and vitamins in oral submucous fibrosis: A three-level meta-analysis study.

This study aims to investigate the alteration of salivary biomarker profiling in the development of oral submucous fibrosis (OSMF) and to explore the influence of saliva in the diagnosis of OSMF. A systematic search of published articles using the PRISMA guidelines was conducted to identify relevant studies on OSMF and saliva. All eligible studies, including case-control, cross-sectional studies, cohort, and pilot studies, contained the evaluation of salivary biomarker profiling in patients with OSMF. Salivary biomarker data from 28 selected articles were categorized into nine groups, and their mean values were determined. A three-step meta-analysis was performed by grouping salivary biomarker profiling into more heterogeneous categories based on OSMF classification, considering functional, histological, and clinical grading. The salivary biomarker profiling analysis revealed significant alterations in all markers, indicating their efficacy in OSMF diagnosis. Subgroup analyses highlighted significant associations in oxidative stress and protein with increased mean values, particularly emphasizing lipid peroxidase (LPO), malondialdehyde (MDA), and lactate dehydrogenase (LDH). Conversely, decreased mean values were observed in glutathione, glutathione peroxidase (GPx), superoxide dismutase (SOD), and vitamins. Notably, OSMF grading analysis demonstrated a significant difference in weighted effect sizes for histological grading, particularly in stage IV. The study underscores the alteration of specific salivary biomarkers, particularly those associated with LPO, MDA, LDH, glutathione, GPx, SOD, and vitamins, in diagnosing and grading OSMF.

Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

Chlorogenic acid mitigates potassium dichromate-induced acute hepato-nephrotoxicity by attenuating the NF-κB signalling pathway.

BACKGROUND: Hexavalent chromium (CrVI) is known to be a potentially hepatotoxic and nephrotoxic contaminant in humans and other animals, whose toxicity is associated with oxidative stress and inflammation. The aim of this study was to evaluate the potential protective effect of chlorogenic acid (CGA), which has known anti-inflammatory and antioxidant effects, on potassium dichromate (PDC)-induced acute hepatotoxicity and nephrotoxicity in rats. METHODS AND RESULTS: Thirty-six Wistar albino rats were treated with CGA (10, 20, or 40 mg/kg, intraperitoneally) and/or PDC (15 mg/kg/day, intraperitoneally) as a single dose. Serum, liver, and kidney tissues were examined biochemically, histopathologically, and immunohistochemically. Compared to the control group, a significant increase in interleukin-6 (IL-6) levels and a significant decrease in serum and renal reduced glutathione (GSH) levels, liver catalase (CAT), tumour necrosis factor-alpha (TNF-α), and interleukin 1ß (IL-1ß) levels were observed in the PDC group. The administration of PDC led to histopathological and immunohistochemical changes in rat liver and kidney tissues. With the administration of CGA, especially at the 10 mg/kg dosage, the above-mentioned parameters approached normal levels. CONCLUSIONS: CGA had antioxidant and anti-inflammatory effects that alleviated PDC-induced acute hepato- and nephrotoxicity.

Metabolic Reprogramming Is an Initial Step in Pancreatic Carcinogenesis That Can Be Targeted to Inhibit Acinar-to-Ductal Metaplasia.

Metabolic reprogramming is a hallmark of cancer and is crucial for cancer progression, making it an attractive therapeutic target. Understanding the role of metabolic reprogramming in cancer initiation could help identify prevention strategies. To address this, we investigated metabolism during acinar-to-ductal metaplasia (ADM), the first step of pancreatic carcinogenesis. Glycolytic markers were elevated in ADM lesions compared with normal tissue from human samples. Comprehensive metabolic assessment in three mouse models with pancreas-specific activation of KRAS, PI3K, or MEK1 using Seahorse measurements, nuclear magnetic resonance metabolome analysis, mass spectrometry, isotope tracing, and RNA sequencing analysis revealed a switch from oxidative phosphorylation to glycolysis in ADM. Blocking the metabolic switch attenuated ADM formation. Furthermore, mitochondrial metabolism was required for de novo synthesis of serine and glutathione (GSH) but not for ATP production. MYC mediated the increase in GSH intermediates in ADM, and inhibition of GSH synthesis suppressed ADM development. This study thus identifies metabolic changes and vulnerabilities in the early stages of pancreatic carcinogenesis. Significance: Metabolic reprogramming from oxidative phosphorylation to glycolysis mediated by MYC plays a crucial role in the development of pancreatic cancer, revealing a mechanism driving tumorigenesis and potential therapeutic targets. See related commentary by Storz, p. 2225.

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro.

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Effects of Nitrosyl Iron Complexes with Thiol, Phosphate, and Thiosulfate Ligands on Hemoglobin.

Nitrosyl iron complexes are remarkably multifactorial pharmacological agents. These compounds have been proven to be particularly effective in treating cardiovascular and oncological diseases. We evaluated and compared the antioxidant activity of tetranitrosyl iron complexes (TNICs) with thiosulfate ligands and dinitrosyl iron complexes (DNICs) with glutathione (DNIC-GS) or phosphate (DNIC-PO4-) ligands in hemoglobin-containing systems. The studied effects included the production of free radical intermediates during hemoglobin (Hb) oxidation by tert-butyl hydroperoxide, oxidative modification of Hb, and antioxidant properties of nitrosyl iron complexes. Measuring luminol chemiluminescence revealed that the antioxidant effect of TNICs was higher compared to DNIC-PO4-. DNIC-GS either did not exhibit antioxidant activity or exerted prooxidant effects at certain concentrations, which might have resulted from thiyl radical formation. TNICs and DNIC-PO4- efficiently protected the Hb heme group from decomposition by organic hydroperoxides. DNIC-GS did not exert any protective effects on the heme group; however, it abolished oxoferrylHb generation. TNICs inhibited the formation of Hb multimeric forms more efficiently than DNICs. Thus, TNICs had more pronounced antioxidant activity than DNICs in Hb-containing systems.

The Impact of Vitamin D and L-Cysteine Co-Supplementation on Upregulating Glutathione and Vitamin D-Metabolizing Genes and in the Treatment of Circulating 25-Hydroxy Vitamin D Deficiency.

Vitamin D receptors are expressed in many organs and tissues, which suggests that vitamin D (VD) affects physiological functions beyond its role in maintaining bone health. Deficiency or inadequacy of 25(OH)VD is widespread globally. Population studies demonstrate that a positive association exists between a high incidence of VD deficiency and a high incidence of chronic diseases, including dementia, diabetes, and heart disease. However, many subjects have difficulty achieving the required circulating levels of 25(OH)VD even after high-dose VD supplementation, and randomized controlled clinical trials have reported limited therapeutic success post-VD supplementation. Thus, there is a discordance between the benefits of VD supplementation and the prevention of chronic diseases in those with VD deficiency. Why this dissociation exists is currently under debate and is of significant public interest. This review discusses the downregulation of VD-metabolizing genes needed to convert consumed VD into 25(OH)VD to enable its metabolic action exhibited by subjects with metabolic syndrome, obesity, and other chronic diseases. Research findings indicate a positive correlation between the levels of 25(OH)VD and glutathione (GSH) in both healthy and diabetic individuals. Cell culture and animal experiments reveal a novel mechanism through which the status of GSH can positively impact the expression of VD metabolism genes. This review highlights that for better success, VD deficiency needs to be corrected at multiple levels: (i) VD supplements and/or VD-rich foods need to be consumed to provide adequate VD, and (ii) the body needs to be able to upregulate VD-metabolizing genes to convert VD into 25(OH)VD and then to 1,25(OH)2VD to enhance its metabolic action. This review outlines the association between 25(OH)VD deficiency/inadequacy and decreased GSH levels, highlighting the positive impact of combined VD+LC supplementation on upregulating GSH, VD-metabolizing genes, and VDR. These effects have the potential to enhance 25(OH)VD levels and its therapeutic efficacy.

Prodromic Inflammatory-Oxidative Stress in Peritoneal Leukocytes of Triple-Transgenic Mice for Alzheimer's Disease.

Inflammatory-oxidative stress is known to be pivotal in the pathobiology of Alzheimer's disease (AD), but the involvement of this stress at the peripheral level in the disease's onset has been scarcely studied. This study investigated the pro-inflammatory profile and oxidative stress parameters in peritoneal leukocytes from female triple-transgenic mice for AD (3xTgAD) and non-transgenic mice (NTg). Peritoneal leukocytes were obtained at 2, 4, 6, 12, and 15 months of age. The concentrations of TNFα, INFγ, IL-1ß, IL-2, IL-6, IL-17, and IL-10 released in cultures without stimuli and mitogen concanavalin A and lipopolysaccharide presence were measured. The concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), lipid peroxidation, and Hsp70 were also analyzed in the peritoneal cells. Our results showed that although there was a lower release of pro-inflammatory cytokines by 3xTgAD mice, this response was uncontrolled and overstimulated, especially at a prodromal stage at 2 months of age. In addition, there were lower concentrations of GSH in leukocytes from 3xTgAD and higher amounts of lipid peroxides at 2 and 4 months, as well as, at 6 months, a lower concentration of Hsp70. In conclusion, 3xTgAD mice show a worse pro-inflammatory response and higher oxidative stress than NTg mice during the prodromal stages, potentially supporting the idea that Alzheimer's disease could be a consequence of peripheral alteration in the leukocyte inflammation-oxidation state.

A diverse proteome is present and enzymatically active in metabolite extracts.

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Glutathione determines chronic myeloid leukemia vulnerability to an inhibitor of CMPK and TMPK.

Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.

Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 µM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

Prepubertal Repeated Berberine Supplementation Enhances Cerebrocerebellar Functions by Modulating Neurochemical and Behavioural Changes in Wistar Rats.

Antioxidant-rich supplementation plays an essential role in the function of mammals' central nervous system. However, no research has documented the effect of berberine (BER) supplementation on the cerebrocerebellar function of prepubertal rats. The present study was designed to investigate the impact of BER supplementation on neurochemical and behavioural changes in prepubertal male rats. Five groups (90 ± 5 g, n = 7 each) of experimental rats were orally treated with corn oil or different doses of BER (25, 50, 100, and 200 mg/kg bw) from the 28th at 68 post-natal days. On the 69 days of life, animals underwent behavioural assessment in the open field, hanging wire, and negative geotaxis tests. The result revealed that BER administration improved locomotive and motor behaviour by increasing distance travelled, line crossings, average speed, time mobile, and absolute turn angle in open field test and decrease in time to re-orient on an incline plane, a decrease in immobility time relative to the untreated control. Furthermore, BER supplementation increased (p < 0.05) antioxidant enzyme activities such as SOD, CAT, GPx, GSH, and TSH and prevented increases (p < 0.05) in oxidative and inflammatory levels as indicated by decreases in RONS, LPO, XO, carbonyl protein, NO, MPO, and TNF-α compared to the untreated control. BER-treated animals a lessened number of dark-stained Nissl cells compared to the untreated control rats. Our findings revealed that BER minimised neuronal degeneration and lesions, improved animal behaviour, and suppressed oxidative and inflammatory mediators, which may probably occur through its agonistic effect on PPAR-α, PPAR-δ, and PPAR-γ - essential proteins known to resolve inflammation and modulate redox signalling towards antioxidant function.