Implementing Optogenetic-Controlled Bacterial Systems in Drosophila melanogaster for Alleviation of Heavy Metal Poisoning.

Drosophila melanogaster (fruit fly) is an animal model chassis in biological and genetic research owing to its short life cycle, ease of cultivation, and acceptability to genetic modification. While the D. melanogaster chassis offers valuable insights into drug efficacy, toxicity, and mechanisms, several obvious challenges such as dosage control and drug resistance still limit its utility in pharmacological studies. Our research combines optogenetic control with engineered gut bacteria to facilitate the precise delivery of therapeutic substances in D. melanogaster for biomedical research. We have shown that the engineered bacteria can be orally administered to D. melanogaster to get a stable density of approximately 28,000 CFUs/per fly, leading to no detectable negative effects on the growth of D. melanogaster. In a model of D. melanogaster exposure to heavy metal, these orally administered bacteria uniformly express target genes under green light control to produce MtnB protein for binding and detoxifying lead, which significantly reduces the level of oxidative stress in the intestinal tract of Pb-treated flies. This pioneering study lays the groundwork for using optogenetic-controlled bacteria in the model chassis D. melanogaster to advance biomedical applications.

Metallothionein-1 mitigates the advancement of osteoarthritis by regulating Th17/Treg balance.

Osteoarthritis (OA) is a chronic inflammatory joint disorder characterized by cartilage degradation and bone remodeling. This study investigated the regulatory role of metallothionein 1 (MT1) in modulating immune responses and the balance between regulatory T cells (Treg) and T helper 17 cells (Th17) in OA. Peripheral blood mononuclear cells (PBMCs) from healthy individuals and OA patients were assessed for cytokine expression linked to Treg/Th17 homeostasis. OA was induced in wild-type (WT) and Mt1 knockout (MT1KO) mice via surgical destabilization of the medial meniscus. Clinical scores, pathological features, inflammatory cytokines, and Treg/Th17 balance were evaluated. MT1KO mice showed significantly elevated Mt1, pro-inflammatory cytokines (IL-1, IL-6, TNF-α) and exacerbated OA progression, characterized by increased knee joint diameter, inflammatory infiltration, and cartilage destruction. Mechanistically, disrupted Treg/Th17 balance played a pivotal role in OA exacerbation, with MT1KO promoting Th17 differentiation and reducing Treg populations. Additionally, the compensatory elevation of anti-inflammatory interleukin-10 (IL-10) in OA patients hinted at a nuanced immune regulatory mechanism. The study illuminates intricate interactions involving MT1, Treg/Th17 cells, and pro-inflammatory cytokines in OA pathogenesis, suggesting MT1's potential as a pivotal regulatory factor and a therapeutic target for mitigating immune dysregulation in OA.

Investigation of the Relationship Between Heavy Metals (Cadmium, Arsenic, and Lead) and Metallothionein in Multiple Sclerosis.

BACKGROUND AND AIM: Multiple sclerosis (MS) is one of the most common neurological disorders. Metals are important for the maintenance and preservation of homeostasis and dysregulated metal homeostasis has an impact on neurodegeneration. Environmental factors are considered to contribute to MS risk and progression. Heavy metals such as arsenic (As), cadmium (Cd), and lead (Pb) are widely found in the environment and because of their toxic nature, they pose a great danger to human health. Metallothioneins (MTs) play important roles in metal homeostasis and detoxification of heavy metals. OBJECTIVE: The aim of this study was to investigate the relationship between levels of heavy metals (As, Cd, and Pb) and MT levels in MS patients and also to assess the oxidative stress status of patients. METHOD: Fifty subjects (20 healthy subjects and 30 MS patients) were included. Demographic characteristics of the patients, plasma MT levels, blood Cd, As, and Pb levels, as well as iron (Fe), copper (Cu), and zinc (Zn) levels, were determined. Malondialdehyde (MDA) levels were investigated as a marker of oxidative stress. RESULTS: MT levels were slightly higher in the MS group (p > 0.05). As Cd and Pb levels were significantly higher in the control subjects. MDA levels were significantly higher in MS patients. CONCLUSION: Our results support the relevance of MT and MDA levels in MS. Further clinical studies with larger cohorts will provide more insights into these factors.

Ex vivo modelling of cardiac injury identifies ferroptosis-related pathways as a potential therapeutic avenue for translational medicine.

BACKGROUND: Heart failure (HF) is a burgeoning health problem worldwide. Often arising as a result of cardiac injury, HF has become a major cause of mortality with limited availability of effective treatments. Ferroptotic pathways, triggering an iron-dependent form of cell death, are known to be potential key players in heart disease. This form of cell death does not exhibit typical characteristics of programmed cell death, and is mediated by impaired iron metabolism and lipid peroxidation signalling. OBJECTIVES: The aim of this study is to establish an ex-vivo model of myocardial injury in living myocardial slices (LMS) and to identify novel underlying mechanisms and potential therapeutic druggable target(s). METHODS AND RESULTS: In this study, we employed LMS as an ex vivo model of cardiac injury to investigate underlying mechanisms and potential therapeutic targets. Cryoinjury was induced in adult rat LMS, resulting in 30 % tissue damage. Cryoinjured LMS demonstrated impaired contractile function, cardiomyocyte hypertrophy, inflammation, and cardiac fibrosis, closely resembling in vivo cardiac injury characteristics. Proteomic analysis revealed an enrichment of factors associated with ferroptosis in the injured LMS, suggesting a potential causative role. To test this hypothesis, we pharmacologically inhibited ferroptotic pathways using ferrostatin (Fer-1) in the cryoinjured rat LMS, resulting in attenuation of structural changes and repression of pro-fibrotic processes. Furthermore, LMS generated from failing human hearts were used as a model of chronic heart failure. In this model, Fer-1 treatment was observed to reduce the expression of ferroptotic genes, enhances contractile function and improves tissue viability. Blocking ferroptosis-associated pathways in human cardiac fibroblasts (HCFs) resulted in a downregulation of fibroblast activation genes, a decrease in fibroblast migration capacity, and a reduction in reactive oxygen species production. RNA sequencing analysis of Fer-1-treated human LMS implicated metallothioneins as a potential underlying mechanism for the inhibition of these pathways. This effect is possibly mediated through the replenishment of glutathione reserves. CONCLUSIONS: Our findings highlight the potential of targeting ferroptosis-related pathways and metallothioneins as a promising strategy for the treatment of heart disease.

Exploring mercury detoxification in fish: The role of selenium from tuna byproduct diets for sustainable aquaculture.

Exposure to mercury (Hg) through fish consumption poses significant environmental and public health risks, given its status as one of the top ten hazardous chemicals. Aquaculture is expanding, driving a surge in demand for sustainable aquafeeds. Tuna byproducts, which are rich in protein, offer potential for aquafeed production, yet their use is challenged by the high content of heavy metals, particularly Hg. However, these byproducts also contain elevated levels of selenium (Se), which may counteract Hg adverse effects. This study examines the fate of dietary Hg and Se in an aquaculture model fish. Biomolecular speciation analyses through hyphenated analytical approaches were conducted on the water-soluble protein fraction of key organs of juvenile rainbow trout (Oncorhynchus mykiss) exposed to various combinations of Hg and Se species, including diets containing tuna byproducts, over a six-month period. The findings shed light on the dynamics of Hg and Se compounds in fish revealing potential Hg detoxification mechanisms through complexation with Hg-biomolecules, such as cysteine, glutathione, and metallothionein. Furthermore, the trophic transfer of selenoneine is demonstrated, revealing novel opportunities for sustainable aquafeed production. Understanding the interactions between Hg and Se in aquaculture systems is crucial for optimizing feed formulations and mitigating environmental risks. This research contributes to the broader goal of advancing sustainable practices in aquaculture while addressing food security challenges.

Thermodynamic origin of the affinity, selectivity and domain specificity of metallothionein for essential and toxic metal ions.

The small Cys-rich protein metallothionein (MT) binds several metal ions in clusters within its two domains. While the affinity of MT for both toxic and essential metals has been well studied, the thermodynamics of this binding has not. We have used isothermal titration calorimetry measurements to quantify the change in enthalpy (ΔH) and change in entropy (ΔS) when metal ions bind to the two ubiquitous isoforms of MT. The seven Zn2+ that bind sequentially at pH 7.4 do so in two populations with different coordination thermodynamics, an initial four that bind randomly with individual tetra-thiolate coordination and a subsequent three that bind with bridging thiolate coordination to assemble the metal clusters. The high affinity of MT for both populations is due to a very favourable binding entropy that far outweighs an unfavourable binding enthalpy. This originates from a net enthalpic penalty for Zn2+ displacement of protons from the Cys thiols and a favourable entropic contribution from the displaced protons. The thermodynamics of other metal ions binding to MT were determined by their displacement of Zn2+ from Zn7MT and subtraction of the Zn2+-binding thermodynamics. Toxic Cd2+, Pb2+ and Ag+, and essential Cu+, also bind to MT with a very favourable binding entropy but a net binding enthalpy that becomes increasingly favourable as the metal ion becomes a softer Lewis acid. These thermodynamics are the origin of the high affinity, selectivity and domain specificity of MT for these metal ions and the molecular basis for their in vivo binding competition.

Insight into brain metallothioneins from bidirectional Zn2+ signaling in synaptic dynamics.

The basal levels as the labile Zn2+ pools in the extracellular and intracellular compartments are in the range of ∼10 nM and ∼100 pM, respectively. The influx of extracellular Zn2+ is used for memory via cognitive activity and is regulated for synaptic plasticity, a cellular mechanism of memory. When Zn2+ influx into neurons excessively occurs, however, it becomes a critical trigger for cognitive decline and neurodegeneration, resulting in acute and chronic pathogenesis. Aging, a biological process, generally accelerates vulnerability to neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). The basal level of extracellular Zn2+ is age relatedly increased in the rat hippocampus, and the influx of extracellular Zn2+ contributes to accelerating vulnerability to the AD and PD pathogenesis in experimental animals with aging. Metallothioneins (MTs) are Zn2+-binding proteins for cellular Zn2+ homeostasis and involved in not only supplying functional Zn2+ required for cognitive activity, but also capturing excess (toxic) Zn2+ involved in cognitive decline and neurodegeneration. Therefore, it is estimated that regulation of MT synthesis is involved in both neuronal activity and neuroprotection. The present report provides recent knowledge regarding the protective/preventive potential of MT synthesis against not only normal aging but also the AD and PD pathogenesis in experimental animals, focused on MT function in bidirectional Zn2+ signaling in synaptic dynamics.

Dysregulation of metallothionein MT1 sub-types in TCF3::PBX1 pre-B-cell acute lymphoblastic leukemia.

The translocation between chromosomes 1 and 19 t(1;19) produces the TCF3::PBX1 fusion protein, which leads to childhood pre-B-cell acute lymphoblastic leukemia (ALL). The molecular mechanism of oncogenesis, however, remains obscure. This study aims to identify the genes specifically dysregulated in TCF3::PBX1 translocation. The publicly available expression microarray datasets on ALL were used for weighted gene co-expression network analysis (WGCNA) to identify modules associated with TCF3::PBX1. The available knockdown and ChIP-Seq datasets were used to assess the direct targets of TCF3::PBX1. The WGCNA revealed a module enriched in genes involved in the metal ion stress to be positively correlated with TCF3::PBX1, with metallothionein isoform MT1 subtypes MT1E, MT1F, MT1G, MT1H, and MT1X as the hub genes. Of the 145 positively correlated genes, 19 were downregulated upon TCF3::PBX1 knockdown. Eleven of these 19 genes including MT1G, showed TCF3::PBX1 occupancy at the promoter. The Metallothionein 1 family has been implicated in various cancers; however, their role in t(1;19) pre-B-cell ALL has not been previously demonstrated. Our analysis effectively accounts for the cellular and population-level heterogeneity and identifies a novel mechanism for the TCF3::PBX1 action.

Analysis of the role of the rice metallothionein gene OsMT2b in grain size regulation.

Seed size is one of the three main characteristics determining rice yield. Clarification of the mechanisms regulating seed size in rice has implications for improving rice yield. Although several genes have been reported to regulate seed size, most of the reports are fragmentary. The role of metallothioneins (MTs) in regulating seed size remains unknown. Here, we found that OsMT2b was expressed in both spikelets and developing seeds. OsMT2b-overexpression lines had large and heavy seeds, and RNAi (RNA interference) lines had small and light seeds. Scanning electron microscopy (SEM) observations revealed that OsMT2b regulated spikelet hull size by affecting cell expansion in the outer epidermis. Histological analysis indicated that OsMT2b affected the number of cells in the cross-section of spikelet hulls, which affected seed size. The fresh weight of seeds was consistently higher in OsMT2b-overexpression lines than in seeds of the wild-type (WT) and RNAi lines from 6 DAP (days after pollination) until maturity, indicating that OsMT2b affected seed filling. Reverse transcription-quantitative PCR (RT-qPCR) analyses revealed that OsMT2b regulates the expression of reactive oxygen species scavenging-related genes involved in seed size regulation. In conclusion, our results indicated that OsMT2b positively regulates seed size, which provides a novel approach for regulating seed size with genetic engineering technology.

Cadmium exposure and its association with oxidative stress, MT1A methylation, and idiopathic male infertility in Egypt: A case-control study.

Idiopathic male infertility, a significant health concern, lacks a clear etiology. Cadmium (Cd), a widespread environmental pollutant known to impact male reproductive health negatively, can accumulate in mussels, a common food source in Egypt. This study investigated the link between ecological Cd exposure, oxidative stress, MT1A methylation, and idiopathic male infertility in two regions of Alexandria. Thirty-three infertile men and 33 fertile controls were included. Cd levels were measured in mussels from the study sites and in participants' blood and semen. Biomarkers reflecting Cd exposure and its effects were assessed. Mussel Cd levels exceeded regulatory limits. Infertile men revealed significantly higher blood and semen Cd levels, reduced semen quality, increased oxidative stress, and elevated MT1A methylation compared to controls. MT1A methylation was inversely correlated with sperm count and is the strongest predictor of idiopathic male infertility, demonstrating the lowest p-value and considerable effect size. This study suggests that environmental Cd exposure, potentially through mussel consumption, may contribute to idiopathic male infertility in Egypt by increasing oxidative stress, inducing epigenetic modifications, and impairing semen quality. These findings underscore the need for further research into the mechanisms underlying Cd-induced male infertility and the development of preventative strategies.

Polyamines: Rising stars against metal and metalloid toxicity.

Globally, metal/metalloid(s) soil contamination is a persistent issue that affects the atmosphere, soil, water and plant health in today's industrialised world. However, an overabundance of these transition ions promotes the excessive buildup of reactive oxygen species (ROS) and ion imbalance, which harms agricultural productivity. Plants employ several strategies to overcome their negative effects, including hyperaccumulation, tolerance, exclusion, and chelation with organic molecules. Polyamines (PAs) are the organic compounds that act as chelating agents and modulate various physiological, biochemical, and molecular processes under metal/metalloid(s) stress. Their catabolic products, including H2O2 and gamma amino butyric acid (GABA), are also crucial signalling molecules in abiotic stress situations, particularly under metal/metalloid(s) stress. In this review, we explained how PAs regulate genes and enzymes, particularly under metal/metalloid(s) stress with a specific focus on arsenic (As), boron (B), cadmium (Cd), chromium (Cr), and zinc (Zn). The PAs regulate various plant stress responses by crosstalking with other plant hormones, upregulating phytochelatin, and metallothionein synthesis, modulating stomatal closure and antioxidant capacity. This review presents valuable insights into how PAs use a variety of tactics to reduce the harmful effects of metal/metalloid(s) through multifaceted strategies.

Blocking Metallothionein-2 Expression by Copper-Doped Carbon Dots Induces Cellular Antioxidant System Collapse for Antitumor Therapy.

The insufficient antioxidant reserves in tumor cells play a critical role in reactive oxygen species (ROS)-mediated therapeutics. Metallothionein-2 (MT-2), an intracellular cysteine-rich protein renowned for its potent antioxidant properties, is intricately involved in tumor development and correlates with a poor prognosis. Consequently, MT-2 emerges as a promising target for tumor therapy. Herein, we present the development of copper-doped carbon dots (Cu-CDs) to target MT-2 to compromise the delicate antioxidant reserves in tumor cells. These Cu-CDs with high tumor accumulation and prolonged body retention can effectively suppress tumor growth by inducing oxidative stress. Transcriptome sequencing unveils a significant decrease in MT-2 expression within the in vivo tumor samples. Further mechanical investigations demonstrate that the antitumor effect of Cu-CDs is intricately linked to apolipoprotein E (ApoE)-mediated downregulation of MT-2 expression and the collapse of the antioxidant system. The robust antitumor efficacy of Cu-CDs provides invaluable insights into developing MT-2-targeted nanomedicine for cancer therapies.

Antioxidant-related enzymes and peptides as biomarkers of metallic nanoparticles (eco)toxicity in the aquatic environment.

Increased awareness of the impact of human activities on the environment has emerged in recent decades. One significant global environmental and human health issue is the development of materials that could potentially have negative effects. These materials can accumulate in the environment, infiltrate organisms, and move up the food chain, causing toxic effects at various levels. Therefore, it is crucial to assess materials comprising nano-scale particles due to the rapid expansion of nanotechnology. The aquatic environment, particularly vulnerable to waste pollution, demands attention. This review provides an overview of the behavior and fate of metallic nanoparticles (NPs) in the aquatic environment. It focuses on recent studies investigating the toxicity of different metallic NPs on aquatic organisms, with a specific emphasis on thiol-biomarkers of oxidative stress such as glutathione, thiol- and related-enzymes, and metallothionein. Additionally, the selection of suitable measurement methods for monitoring thiol-biomarkers in NPs' ecotoxicity assessments is discussed. The review also describes the analytical techniques employed for determining levels of oxidative stress biomarkers.

Dexamethasone reduces cisplatin-induced hair cell damage by inducing cisplatin resistance through metallothionein-2.

PURPOSE: Hair cell damage is a common side effect caused by the anticancer drug cisplatin (CDDP), which reduces patient quality of life. One CDDP resistance mechanism that occurs in recurrent cancers is heavy metal detoxification by metallothionein-2 (mt2). Here, we show that in zebrafish larvae, dexamethasone (DEX) reduces CDDP-induced hair cell damage by enhancing mt2 expression. METHODS: Transgenic zebrafish (cldn: gfp; atoh1: rfp) that express green and red fluorescent proteins in neuromasts and hair cells, respectively, were used. The zebrafish were pretreated with DEX at 52 h post-fertilization (hpf) for 8 h, followed by CDDP treatment for 12 h. The lateral line hair cells of CDDP-treated zebrafish at 72 hpf were observed by fluorescence microscopy. RESULTS: Reporting odds ratio (ROR) analysis using an adverse event database indicated an association between a decrease in CDDP-induced ototoxicity and DEX as an antiemetic treatment for cancer chemotherapy. Pretreatment with DEX protected 72 hpf zebrafish hair cells from CDDP-induced damage. The expression of mt2 mRNA was significantly increased by the combination of 10 µM DEX with CDDP. Gene editing of mt2 reversed the protective effect of DEX against CDDP-induced damage in hair cells. CONCLUSION: DEX protects hair cells from CDDP-induced damage through increased mt2 expression, which is a resistance mechanism for platinum-based anticancer drugs.

Functional analysis of root-preferential oil palm metallothionein promoter in tobacco.

Root-specific or preferential promoters are essential to genetically modify plants with beneficial root traits. We have characterised the promoter from an oil palm metallothionein gene (EgMT) and performed a serial 5' deletion analysis to identify the region(s) essential for transgenes expression in roots. Stable functional characterisation of tobacco transgenic lines using the T1 generation showed that a deletion construct, designated as RSP-2D (1107 bp), directed strong GUS expression at all stages of root development, particularly in mature roots. Other constructs, RSP-2A (2481 bp) and RSP-2C (1639 bp), drove GUS expression in roots with an intensity lower than RSP-2D. The promoter activity was also detectable in seed pods and immature seeds, albeit at lower levels than CaMV35S. The promoter activity may also be induced by wounding as intact GUS staining was observed at the flower- and leaf-cutting sites of T1 samples carrying either RSP-2C or RSP-2D constructs. The promoter sequence contains cis-acting elements that may act as negative regulators and be responsible for root specificity. The results further indicated that the 5' UTR and ATATT sequences are essential for strong promoter activity. This study highlights the potential of RSP-2D promoter as a tool for modifying root traits through genetic engineering.

Short-term impact of vitamin K2 supplementation on biochemical parameters and lipoprotein fractions.

This study explored the short-term effects of vitamin K2 (VK2) supplementation on biochemical parameters (vitamin D, vitamin E, vitamin A, alkaline phosphatase, calcium, phosphorus (P), magnesium, metallothionein, triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and lipoprotein fractions (albumin, HDL, very low-density lipoprotein (VLDL), LDL, and chylomicrons). A short-term experiment (24 h, six probands) was performed to track changes in VK2 levels after a single-dose intake (360 µg/day). Liquid chromatography-tandem mass spectrometry was used to monitor vitamin K levels (menaquinone-4 (MK-4), menaquinone-7 (MK-7), and vitamin K1 [VK1]) with a limit of detection of 1.9 pg/mL for VK1 and 3.8 pg/mL for the two forms of VK2. Results showed that MK-7 levels significantly increased within 2-6 h post-administration and then gradually declined. MK-4 levels were initially low, showing a slight increase, whereas VK1 levels rose initially and then decreased. Biochemical analyses indicated no significant changes in sodium, chloride, potassium, calcium, magnesium, albumin, or total protein levels. A transient increase in P was observed, peaking at 12 h before returning to baseline. Agarose gel electrophoresis of lipoprotein fractions revealed distinct chylomicron bands and variations in VLDL and HDL mobility, influenced by dietary lipids and VK2 supplementation. These findings suggest effective absorption and metabolism of MK-7 with potential implications for bone metabolism and cardiovascular health.

Protection of cultured vascular endothelial cells against cadmium cytotoxicity by simultaneous treatment or pretreatment with manganese.

Cadmium is a heavy metal that pollutes the environment and foods and is a risk factor for vascular disorders. We have previously demonstrated that pretreatment of vascular endothelial cells with zinc and copper protects the cells against cadmium cytotoxicity. In contrast, cadmium cytotoxicity was potentiated in cells following exposure to lead, thereby indicating that in vascular endothelial cells, cadmium cytotoxicity can be differentially modified by the co-occurrence of other heavy metals. In this study, we revealed that simultaneous treatment or pretreatment with manganese protects vascular endothelial cells against cadmium cytotoxicity. Intracellular accumulation of cadmium was observed to be reduced by simultaneous treatment with manganese, although not by pretreatment. The mRNA expression of metal transporters that regulate the uptake of both cadmium and manganese (ZIP8, ZIP14, and DMT1) remained unaffected by either simultaneous treatment or pretreatment with manganese, and simultaneous treatment with manganese suppressed the cadmium-induced expression of metallothionein but pretreatment with manganese did not exhibit such suppressive effect. Thus, the protection of vascular endothelial cells against cadmium cytotoxicity conferred by simultaneous treatment with manganese is assumed to be partially attributed to a reduction in the intracellular accumulation of cadmium, whereas the effects of pretreatment with manganese are independent of both the reduced intracellular accumulation of cadmium and the induction of metallothionein. These observations accordingly indicate that the protective effects of manganese are mediated via alternative (as yet unidentified) mechanisms.

Effect of CUP1 copy number and pH on copper resistance of Saccharomyces cerevisiae enological strains.

The widespread use of copper-based pesticides in winemaking can affect wine fermentation. Therefore, it is crucial to assess the resistance levels of Saccharomyces cerevisiae wine strains in enological growth conditions. In the context of winemaking, grape juice is a complex environment capable of chelating copper and is characterized by a distinctly acidic pH. In this work, the effects of copper concentration on the growth of 10 S. cerevisiae strains, isolated from an enological environment, and one commercial starter were tested in YNB minimal medium and synthetic must, mimicking enological conditions. In minimal medium, resistance to copper varied among yeasts (50-600 µM), revealing the presence of three resistance levels (high, intermediate, and low). Representative strains of the three groups were tested at a pH range from 5.2 to 3.0 at the copper concentration that showed a 20-25 % growth reduction. At pH range 5.2-4.5, a growth reduction was observed, while, conversely, a strain-specific recovery was observed at pH range 3.2-3.0. In synthetic must, the strains showed higher copper resistance levels than in minimal medium (50-4000 µM). In both synthetic must and minimal medium, a significant logarithmic correlation was found between copper resistance and CUP1 gene copy number. The copy number tended to better explain resistance in minimal medium compared to synthetic must. The results shed light on the role of CUP1 copy number within an enological environment.

Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia.

OBJECTIVE: The metabolic reprogramming of acute myeloid leukemia (AML) cells is a compensatory adaptation to meet energy requirements for rapid proliferation. This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells. METHODS: SKM-1 cells (an AML cell line) were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, a glutaminase inhibitor) or cytarabine. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis and reactive oxygen species (ROS) by flow cytometry. Western blotting was conducted to examine the levels of apoptotic proteins, including cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, the human long noncoding RNA (lncRNA) microarray was used to analyze gene expression after glutamine deprivation, and results were confirmed with quantitative RT-PCR (qRT-PCR). The expression of metallothionein 2A (MT2A) was suppressed using siRNA. Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry, respectively, in cells with MT2A knockdown. RESULTS: Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells. The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation. MT2A knockdown increased apoptosis, while proliferation was not affected in SKM-1 cells. In addition, metformin inhibited cell growth and induced apoptosis in SKM-1 cells. Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine. Furthermore, the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells. CONCLUSION: Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

LATS1 inhibitor and zinc supplement synergistically ameliorates contrast-induced acute kidney injury: Induction of Metallothionein-1 and suppression of tubular ferroptosis.

Contrast-induced acute kidney injury (CI-AKI) is a prevalent cause of renal dysfunction among hospitalized patients, yet the precise pathogenesis and effective therapeutic strategies remain elusive. In this study, we investigated the role of tubular ferroptosis in both experimental CI-AKI models and in primary tubular epithelial cells (PTECs) treated with ioversol. Using whole exome sequencing, we identified metallothioneins (MTs) as being among the most significantly downregulated genes following ioversol exposure. Our findings reveal that overexpression of Mt1 mitigates, whereas suppression of Mt-1 exacerbates, ioversol-induced tubular ferroptosis. Interestingly, the level of MTF1 (metal regulatory transcription factor 1), a principal regulator of Mt1, was found to increase in response to ioversol treatment. We further elucidated that ioversol activates LATS1 (Large tumor suppressor homolog 1), a kinase that promotes the phosphorylation and nuclear translocation of MTF1, thereby inhibiting its transcriptional activity for Mt1. Both genetic and pharmacological inhibition of LATS1 reversed the ioversol-induced suppression of Mt-1. From a therapeutic perspective, the LATS1 inhibitor TDI-011536, in combination with zinc acetate, was administered to a rodent model of CI-AKI. Our data indicate that this combination synergistically upregulates Mt1 expression and provides protection against contrast media-induced tubular ferroptosis. In summary, our study demonstrates that the reduction of Mt-1 contributes to tubular ferroptosis associated with CI-AKI. We show that contrast media activate LATS1, which in turn suppresses the transcriptional activity of MTF1 for Mt1. Herein, the combination of zinc acetate and a LATS1 inhibitor emerges as a potential therapeutic approach for the treatment of CI-AKI.