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Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc−, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia‐induced changes in neuronal responsiveness from wild‐type and xCT KO (xCT−/−) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild‐type and xCT−/− slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re‐oxygenation, an effect we termed 'post‐hypoxic long‐term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post‐hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post‐hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired‐pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta‐burst stimulation‐induced LTP was equivalent between genotypes. Importantly, post‐hypoxic LTP was generated in wild‐type slices pretreated with system xc− inhibitor, S‐4‐carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc− interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post‐ischaemic recovery of the penumbra. What is the central question of this study?Does glutamate antiporter system xc− influence recovery in the ischemic penumbra?What is the main finding and its importance?Our data reveal that mild hypoxia elicits a persistent potentiation of synaptic transmission in hippocampus when system xc− is genetically deleted or pharmacologically inhibited. This 'post‐hypoxic long‐term potentiation (LTP)' engages similar mechanims as classical NMDAR‐dependent LTP induced by theta‐burst stimulation (TBS). Because this neuroplasticity potentially fosters recovery in penumbral tissue, these findings portend system xc− antagonism as a post‐stroke therapeutic intervention. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is a disabling and highly prevalent condition affecting millions worldwide. Recently discovered, disulfidptosis represents a novel form of cell death induced by the excessive accumulation of cystine. Despite its significance, a systematic exploration of disulfidptosis-related genes (DRGs) in OA is lacking. Methods: This study utilized three OA-related datasets and DRGs. Differentially expressed (DE)-DRGs were derived by intersecting the differentially expressed genes (DEGs) from GSE114007 with DRGs. Feature genes underwent screening through three machine learning algorithms. High diagnostic value genes were identified using the receiver operating characteristic curve. Hub genes were confirmed through expression validation. These hub genes were then employed to construct a nomogram and conduct enrichment, immune, and correlation analyses. An additional validation of hub genes was performed through in vitro cell experiments. Results: SLC3A2 and PDLIM1 were designated as hub genes, displaying excellent diagnostic performance. PDLIM1 exhibited low expression in early chondrocyte differentiation, rising significantly in the late stage, while SLC3A2 showed high overall expression, declining in the late differentiation stage. Cellular experiments corroborated the correlation of SLC3A2 and PDLIM1 with chondrocyte inflammation. Conclusions: Two hub genes, SLC3A2 and PDLIM1, were identified in relation to disulfidptosis, providing potential directions for diagnosing and treating OA. [ABSTRACT FROM AUTHOR]

SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy. [ABSTRACT FROM AUTHOR]

Background: Lentil is an important legume crop and plays an important role in human nutrition and soil fertility improvement. It contains 57-60% carbohydrate, 24-26% protein, 3.2% fiberand only 1.3% fat with the full complement of amino acids. It is a potential crop for about 10 million hectares of rice-fallow areas of eastern India. This study aimed to standardized sulphur and boron requirements of lentil in Eastern India. Methods: The experiment was conducted in a glass house during 2018-19 and 2019-20 in a factorial completely randomized design with three replications and 16 treatment combinations of sulphur and boron. The treatment combinations were product of 4 levels each of sulphur and boron as 0, 15, 30 and 45 kg ha-1 (0, 0.067, 0.134 and 0.201 g pot-1) and 0, 1, 2 and 3 kg ha-1 (0, 0.004, 0.009 and 0.013 g pot-1), respectively. Lentil grain yield was observed at harvest and sulphur-containing amino acids, as well as seed nutrient content, were analyzed followingstandard operating procedures. Result: Lentil grain yield was enhanced by 1.85 times with a combined application of 45 kg S and 2 kg B ha-1 with a recommended dose of NPK. The sulphur-containing amino acids namely methionine, cystine and cysteine content showed significant improvement with increasing levels of sulphur application however were not significantly affected with boron levels. The protein content in lentil seed increased considerably with application of sulphur levels as compared to boron levels and it was highest with 30 kg S ha-1 + 2kg B ha-1 along with recommended dose of NPK fertilizers. [ABSTRACT FROM AUTHOR]

It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans. Synopsis: Metabolic utilization of cysteine requires reduction of cystine taken up from extracellular sources. Here, TXNDC17 -encoded TRP14 is identified as the main enzyme catalyzing this step, regulating protein cysteinylation and cysteine homeostasis in concert with the transsulfuration pathway. Thioredoxin-related protein TRP14 is the rate-limiting enzyme in mammalian cytosolic cystine reduction. Genetic ablation of TRP14 redirects cysteine biosynthesis towards the transsulfuration pathway as alternative, methionine-based source of cysteine. TRP14 also reduces cysteinylated proteins and potentially modulates protein function, such as reactivation of cysteinylated peroxiredoxin 2 through its decysteinylation. Txndc17 -deficient mice are protected from acute pancreatitis pathophysiology through activation of Nrf2-mediated antioxidant pathways and primed upregulation of transsulfuration. Interplay between TRP14 and transsulfuration in cysteine metabolism appears conserved across species, as knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. A thioredoxin-related protein controls cysteine homeostasis in parallel to the transsulfuration pathway, and can also modulate protein function via protein decysteinylation. [ABSTRACT FROM AUTHOR]

Disintegrating cystine and calcium oxalate monohydrate stones present a formidable challenge owing to their hardness and distinct composition. This study aimed to establish optimal laser settings for these hard stones lithotripsy. Cystine and calcium oxalate monohydrate stones were extracted from two patients. Two experiments were conducted in vitro by utilizing a 272 μm laser fiber with variable settings to disintegrate the cystine and calcium oxalate monohydrate stones. In the first experiment, energy was adjustable while frequency was constant, whereas the second experiment involved constant energy with adjustable frequency on each type of stone and each experiment was repeated three times to ensure robustness and reliability. Our findings indicated that for cystine stones, use of higher total power with high energy and low frequency proved to be effective. Conversely, for calcium oxalate monohydrate stones, settings involving higher total power with low energy and high frequency demonstrated superior efficacy and safety. Holmium (Ho: YAG) laser settings with higher total power, high energy, and low frequency effectively disintegrate cystine stones despite increased heat, which was measured by a thermometer with a thermocouple. For calcium oxalate monohydrate stones, higher total power, high frequency, and low energy settings are recommended and safe. [ABSTRACT FROM AUTHOR]

Corrosion has been identified as the primary mechanism causing pipeline failures, leading to significant economic losses and environmental problems. One of the effective and economical methods to prevent metal corrosion is to add corrosion inhibitors. Although environmentally friendly corrosion inhibitors are beneficial to the ecological environment, their lower corrosion inhibition efficiency compared to traditional corrosion inhibitors has limited their application. Therefore, this paper aims to develop an environmentally friendly compound corrosion inhibitor that can meet the practical industrial requirements. The corrosion inhibition effect of two complexes of cystine, namely cystine + sodium molybdate (Cys-Cys + MS) and cystine + zinc gluconate (Cys-Cys + ZG), on pipeline steel in 1 M HCl was investigated. And the synergistic corrosion inhibition mechanism of these two composite corrosion inhibitors was discussed. The results indicated that the corrosion inhibition performance of Cys-Cys + MS and Cys-Cys + ZG complexes was significantly better than that single inhibitors at higher concentration. Furthermore, it was observed that the corrosion inhibition performance of Cys-Cys + ZG was superior to that of Cys-Cys + MS. The maximum corrosion inhibition efficiency of the two compound corrosion inhibitors was achieved at the concentration of (2 + 4) mM. [ABSTRACT FROM AUTHOR]

Background & aimsHepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown.MethodsHepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development.ResultsHepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH.ConclusionsDespite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset.Impact and implicationsLiver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer.

Cystinosis is a rare autosomal recessive disease characterised by an accumulation of cystine in the lysosomes. It is caused by pathogenic variants of the cystinosin gene (CTNS), which interrupts the transport of cystine from the lysosomes into the cytosol. Intra-lysosomal cystine accumulation leads to subsequent cellular dysfunction. Cystinosis has an incidence of 0.5–1/100,000 live births. There are three forms of cystinosis: nephropathic cystinosis, juvenile cystinosis, and ocular cystinosis, with nephropathic cystinosis being the most prevalent disease subtype. Renal impairment is the most common manifestation of disease. Extrarenal manifestations of cystinosis include hypothyroidism, diabetes, and hypogonadism. The current treatment for cystinosis is cysteamine, a cystine-depleting agent. This is not a curative treatment and only aims to slow the progression of disease. A total of 90% of cystinosis patients progress to kidney failure within the first 20 years of life. Kidney transplantation is the only option available to patients once the disease has progressed to this stage. This review highlights the pathogenesis and clinical manifestations of cystinosis, as well as potential future treatment options. [ABSTRACT FROM AUTHOR]

Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns−/− zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns−/− zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation. NEW & NOTEWORTHY: In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns−/− zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns−/− zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation. [ABSTRACT FROM AUTHOR]

Background: Crystallization experiments of renal-calculi-forming compounds (calcium oxalate, calcium phosphates, uric acid) are normally performed by monitoring these processes during periods of time similar to the residence of urine inside the kidney. Nevertheless, cystine requires high supersaturation for its crystallization, and most experiments last for longer periods. It must be considered that at high supersaturation, the inhibitors of crystalline development have poor effects. Methods: The induction time of crystallization (ti) of cystine in experimental conditions similar to those of the formation of cystine renal calculi and the effect of different cystine-binding thiol agents was determined through turbidimetric measurements. We also studied the macro- and microstructure of 30 cystine kidney stones through stereoscopic microscopy and scanning electron microscopy. Results: Under the studied conditions, the ti in absence of crystallization inhibitors was 15 min, and the presence of 9 mM of penicillamine, tiopronin, or N-acetylcysteine totally inhibited crystallization, as their effects relate to the formation of complexes with cystine, although N-acetylcysteine also delayed cystine crystalline development and modified cystine crystal morphology. Cystine stones have traditionally been classified as smooth and rough. The study of their structure shows that all of them begin their formation from a few crystals that generate a compact radial structure. Their subsequent growth, depending on the renal cavity where they are located, gives rise to the rough structure in the form of large blocks of cystine crystals or the smooth structure with small crystals. Conclusions: To prevent the development of cystine renal stones, the formation of small crystals must be avoided by reducing urinary cystine supersaturation, with N-acetylcysteine being the most effective among the studied cystine-binding thiol agents. Also, the removal of cystine crystals through increased water intake and physical activity can be a very important preventive measure. [ABSTRACT FROM AUTHOR]

While cysteine (CysSH) is known to be exported into the extracellular space, its biological significance is not well understood. The present study examined the movement of extracellular CysSH using stable isotope-labeled cystine (CysSSCys), which is transported into cells and reduced to CysSH. Exposure of HepG2 cells to 100 µM stable isotope-labeled CysSSCys resulted in 70 µM labeled CysSH in cell medium 1 h after CysSSCys exposure. When the cell medium was collected and incubated with either hydrogen peroxide (H2O2) or atmospheric electrophiles, such as 1,2-naphthoquinone, 1,4-naphthoquinone and 1,4-benzoquinone, CysSH in the cell medium was almost completely consumed. In contrast, extracellular levels of CysSH were unaltered during exposure of HepG2 cells to H2O2 for up to 2 h, suggesting redox cycling of CysSSCys/CysSH in the cell system. Experiments with and without changing cell medium containing CysSH from HepG2 cells revealed that oxidative and electrophilic modifications of cellular proteins, caused by exposure to H2O2 and 1,2-naphthoquinone, were significantly repressed by CysSH in the medium. We also examined participation of enzymes and/or antioxidants in intracellular reduction of CysSSCys to CysSH. These results provide new findings that extracellular CysSH derived from CysSSCys plays a role in the regulation of oxidative and electrophilic stress. [ABSTRACT FROM AUTHOR]

Cancer cells exhibit high levels of oxidative stress and consequently require a high amount of cysteine for glutathione synthesis. Solute Carrier Family 7 Member 11 (SLC7A11), or xCT, mediates the cellular uptake of cystine in exchange for intracellular glutamate; imported extracellular cystine is reduced to cysteine in the cytosol through a NADPH-consuming reduction reaction. SLC7A11/xCT expression is under the control of stress-inducing conditions and of several transcription factors, such as NRF2 and ATF4. Formyl-peptide receptor 2 (FPR2) belongs to the FPR family, which transduces chemotactic signals mediating either inflammatory or anti-inflammatory responses according to the nature of its ligands and/or FPR2 binding with other FPR isoforms. The repertoire of FPR2 agonists with anti-inflammatory activities comprises WKYMVm peptide and Annexin A1 (ANXA1), and the downstream effects of the intracellular signaling cascades triggered by FPR2 include NADPH oxidase (NOX)-dependent generation of reactive oxygen species. Herein, we demonstrate that stimulation of CaLu-6 cells with either WKYMVm or ANXA1: (i) induces the redox-regulated activation of SLC7A11/xCT; (ii) promotes the synthesis of glutathione; (iii) prevents lipid peroxidation; and (iv) favors NRF2 nuclear translocation and activation. In conclusion, our overall results demonstrate that FPR2 agonists and NOX modulate SLC7A11/xCT expression and activity, thereby identifying a novel regulative pathway of the cystine/glutamate antiport that represents a new potential therapeutical target for the treatment of human cancers. [ABSTRACT FROM AUTHOR]

Many cancer cells undergo metabolic reprogramming that results in heightened oxidative stress. Solute carrier family 7 member 11 (SLC7A11), a cystine transporter, is often overexpressed in cancer cells and plays a critical role in promoting glutathione synthesis and suppressing ferroptosis. However, cystine import by SLC7A11 imposes a significant cost to SLC7A11-high cancer cells, because the reduction of cystine to cysteine requires a large amount of glucose-derived NADPH, rendering such cells highly dependent on glucose. Glucose starvation in SLC7A11-high cancer cells depletes NADPH and induces aberrant accumulation of disulfides and disulfide bonding in actin cytoskeleton proteins, resulting in actin network collapse and disulfidptosis, a form of regulated cell death caused by disulfide stress. Targeting disulfidptosis may offer a new therapeutic strategy for treating SLC7A11-high cancers. The cystine transporter solute carrier family 7 member 11 (SLC7A11) (also known as xCT) promotes glutathione synthesis and counters oxidative stress–induced cell death, including ferroptosis, by importing cystine. Also, SLC7A11 plays a crucial role in tumor development. However, recent studies have uncovered an unexpected role of SLC7A11 in promoting disulfidptosis, a novel form of regulated cell death induced by disulfide stress. In this review, we examine the opposing roles of SLC7A11 in regulating redox homeostasis and cell survival/death, summarize current knowledge on disulfidptosis, and explore its potential in disease treatment. A deeper understanding of disulfidptosis will offer new insights into fundamental cellular homeostasis and facilitate the development of innovative therapies for disease treatment. [ABSTRACT FROM AUTHOR]

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by cystine buildup in various tissues, including the kidneys. Renal involvement is the primary manifestation, leading to end-stage renal disease (ESRD) if left untreated. Kidney transplantation (KT) in patients with cystinosis has significantly improved their prognosis for the disease outcome. Detailed reports on preoperative and Long-term postoperative management in these patients remain sparse. This report discusses the outcomes of two young adult patients of Middle Eastern descent with cystinosis who underwent KT. The first patient, diagnosed with infantile nephropathic cystinosis treated by cystine-depleting therapy, was operated by KT at the age of 18. The second patient, diagnosed with juvenile cystinosis, underwent transplantation at the age of 35 after being treated with hemodialysis. Our report describes detailed pre- and postoperative managements, including laboratory results, and pharmacological interventions. Both cases highlighted the varying clinical manifestations and disease severity between infantile and juvenile cystinosis. Pre-transplant conditions included renal dysfunction, growth retardation, secondary hyperparathyroidism, anemia, and extrarenal manifestations. Following KT, both patients experienced regained renal function, resolution of extrarenal complications, and normalization of laboratory parameters. Furthermore, both patients showed excellent postoperative outcomes with no acute rejection or allograft-related complications. KT is the treatment of choice for cystinosis patients with ESRD. Long-term follow-up post-transplantation is crucial to maintain good graft function. Further studies may elucidate optimal pre- and postoperative management protocols for this rare condition. [ABSTRACT FROM AUTHOR]

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy. [ABSTRACT FROM AUTHOR]

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of β-turn nucleation on cyclotide oxidative folding. Two types of β-turn mimics were grafted into kalata B1, individually replacing each of the four β- turns in the folded cyclotide. Insertion of D-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded fourtimes faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the Möbius subfamily of cyclotides. Grafting D-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a β-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides. [ABSTRACT FROM AUTHOR]

Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns-/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns-/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34+ cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis.

Objective: This retrospective hospital-based study is aimed at investigating the six-year prevalence and clinical characteristics of pediatric cystinuria in Ahvaz, southwest Iran. Method: The demographic and clinical data of children referred to nephrology wards with kidney stone disease (KSD) were obtained from the medical records. Data were statistically analyzed and compared for two groups of children with non-cystine kidney stones and those with cystinuria. Results: From a total of 415 patients with KSD, 45 cases (10.8%) had cystinuria and 370 (89.2%) had non-cystine kidney stones. Overall, the incidence rate of KSD was higher in boys (53.7%) than girls (46.3%, p=0.03). However, no significant difference in age and gender was observed between the two groups (p>0.05). Parental consanguinity (OR [95%]:4.1 [2.15_7.8], p=0.0001) and bilateral renal involvement (OR [95%]: 2.38 [1.25_4.53], p=0.01) were more frequent in children with cystinuria. The number and size of cystine stones was significantly higher than non-cystine stones (p=0.0001). Hypercalciuria (60.72%) and hyperuricosuria (40.1%) were the most frequent metabolic disorders and were significantly more frequent in patients with non-cystine stones (p<0.01). The rate of complete recovery in cystinuria patients was significantly lower than in patients with non-cystine stones (51% v. 83.2%, p=0.0001). Conclusion: Parental consanguinity may increase the risk of cystinuria development. Also, the notable prevalence of symptomatic cystinuria at younger ages highlights the importance of metabolic assessment in early childhood. Bilateral renal involvement, the large number/size of cystine stones and their recurrent nature may make cystinuria patients more vulnerable to renal parenchymal damage and more resistant to treatment. [ABSTRACT FROM AUTHOR]

Glutathione (GSH), an oxidative stress biomarker plays a major role in different pathological processes and lead to a vast number of diseases including cancer. Biologically, cystine uptake is a fundamental factor in maintaining GSH levels in cells; however, the interaction between cystine and GSH in biological systems is least explored in developing sensors for GSH. In the present work, we have explored this interaction as the fundamental factor in "turn‐on" sensing of GSH from body fluids using a fluorescent gold nanocluster platform. The experimental results revealed that the probe displayed appreciable performance for GSH over a linear range from 0.2 mM to 1.3 mM with a limit of detection (LoD) of 0.43 μM. The probe also revealed sensitive as well as selective response towards GSH even in the presence of possible co‐existing interferants and provided satisfactory recovery percentage of 94.17 % to 108.12 % from real serum samples. These findings of the present study can be extended in future applications of sensing GSH from clinical samples. [ABSTRACT FROM AUTHOR]

The primary aim of the present in vitro study is to analyze the chemical content of the bubbles occurring during the fragmentation of cystine stones with both the high-power and low-power holmium:YAG (Ho:YAG) lasers. The secondary aim is to discuss their clinical importance. Three types of human renal calculi calcium oxalate monohydrate (COM), cystine, and uric acid were fragmented with both low-power and high-power Ho:YAG lasers in separate experimental setups at room temperature, during which time it was observed whether gas was produced. After laser lithotripsy, a cloudy white gas was obtained, after the fragmentation of cystine stones only. A qualitative gas content analysis was performed with a gas chromatography–mass spectrometry (GC–MS) device. The fragments in the aqueous cystine calculi setup were dried and taken to the laboratory to be examined by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM–EDX) and X-ray diffraction analysis. No gas production was observed after fragmentation in the COM and uric acid stones. Free cystine, sulfur, thiophene, and hydrogen sulfide gas were produced by both low-power and high-power Ho:YAG laser lithotripsy of the cystine stones. In the SEM–EDX mapping analysis, a free cystine molecule containing 42.8% sulfur (S), 21% oxygen (O), 14.9% carbon (C), and 21% nitrogen (N) atoms was detected in the cystine stone experimental setup. The evidence obtained, which shows that hydrogen sulfide emerges in the gaseous environment during Ho:YAG laser fragmentation of cystine stones, indicates that caution is required to prevent the risk of in vivo production and toxicity. [ABSTRACT FROM AUTHOR]

Currently, over 88 million people are estimated to have adopted a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and can be converted to taurine and glutathione. We found earlier that intermolecular cystine bridges are essential for the barrier function of the intestinal mucus layer. Therefore, we now investigate the effect of low dietary cystine on the intestine. Mice (8/group) received a high fat diet with a normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels or bile acid conjugation after 2 weeks of low cystine feeding. In the colon, dietary cystine restriction results in an increase in goblet cell numbers, and a borderline significant increase mucus layer thickness. Gut microbiome composition and expression of stem cell markers did not change on the low cystine diet. Remarkably, stem cell markers, as well as the proliferation marker Ki67, were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of increased epithelial proliferation. In conclusion, 2 weeks of cystine restriction did not result in apparent systemic effects, but the low cystine diet increased the proliferative capacity specifically of the small intestine and induced the number of goblet cells in the colon. [ABSTRACT FROM AUTHOR]

Thermophilic β-1,4-endoglucanases (Cel5A) have garnered significant interest due to their potential applications in various industries, particularly in biofuel production and biorefineries. However, despite inherent stability, thermophilic Cel5A still face challenges in terms of further enhancing their catalytic efficiency and thermostability. In this study, a novel B-factor analysis method was used to predict beneficial amino acid substitutions within a 4 Å radius of the catalytic site in the tunnel of thermophilic Cel5A from Acidomyces richmondensis (ArCel5A). A combined strategy involving site-saturation mutagenesis and high-throughput screening was employed to identify the variants with the highest endoglucanase activity. Genomic sequencing revealed a mutation at position 299 in the starting strain T. reesei A2H, where the nucleotide sequence changed from TAC to TGC, resulting in a corresponding amino acid substitution from Tyrosine(Y) to Cystine(C). The endoglucanase activity of the mutant ArCel5A reached 3251 IU/mL, representing an 85.2% increase compared to wild-type ArCel5A at the fermentation time of 94 h. Significantly, the ArCel5A-Y299C mutant showed superior thermostability, retaining 93.8% of its initial activity after 30 min at 70 °C, and 91.5% after 10 min at 80 °C. Various computational simulation methods confirmed that the Y299C mutation enhanced the stability of the catalytic pocket, thereby improving the overall stability and catalytic efficiency of ArCel5A. This study offers an effective strategy for mining target sites for rational mutagenesis based on highly conserved sequences, which simultaneously improves both the thermostability and catalytic efficiency of thermophilic Cel5A. [ABSTRACT FROM AUTHOR]

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporters conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.

Ferroptosis is an important mediator of pathophysiological cell death and an emerging target for cancer therapy. Whether ferroptosis sensitivity is governed by a single regulatory mechanism is unclear. Here, based on the integration of 24 published chemical genetic screens combined with targeted follow-up experimentation, we find that the genetic regulation of ferroptosis sensitivity is highly variable and context-dependent. For example, the lipid metabolic gene acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) appears far more essential for ferroptosis triggered by direct inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4) than by cystine deprivation. Despite this, distinct pro-ferroptotic stimuli converge upon a common lethal effector mechanism: accumulation of lipid peroxides at the plasma membrane. These results indicate that distinct genetic mechanisms regulate ferroptosis sensitivity, with implications for the initiation and analysis of this process in vivo.

Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.

The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns-/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies.Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded. [ABSTRACT FROM AUTHOR]

To evaluate if videos during laser lithotripsy increase accuracy and confidence of stone identification by urologists compared to still pictures. We obtained representative pictures and videos of 4 major stone types from 8 different patients during ureteroscopy with holmium laser lithotripsy. A REDCap survey was created and emailed to members of the Endourological Society. The survey included a picture followed by the corresponding video of each stone undergoing laser lithotripsy and additional clinical information. Each picture and video included multiple-choice questions about stone composition and response confidence level. Accuracy, confidence levels, and rates of rectification (change from incorrect to correct answer) or confounding (correct to incorrect) after watching videos were analyzed. One hundred eighty-seven urologists responded to the survey. The accuracy rate of stone identification with pictures was 43.8% vs 46.1% with videos (P =.27). Accuracy for individual stones was low and highly variable. Video only improved accuracy for 1 cystine stone. After viewing videos, participants were more likely to rectify vs confound their answers. Urologists were more likely to be confident with videos than pictures alone (65.4% vs 53.7%, respectively; P <.001); however, confident answers were not more likely to yield accurate predictions with videos vs still pictures. Stone identification by urologists is marginally improved with videos vs pictures alone. Overall, accuracy in stone identification is low irrespective of confidence level, picture, and lithotripsy video visualization. Urologists should be cautious in using endoscopic stone appearance to direct metabolic management. [ABSTRACT FROM AUTHOR]

Elevated circulating homocysteine (Hcy) is a well-known risk factor for cardiovascular diseases (CVDs), including coronary artery disease (CAD) and heart failure (HF). It remains unclear how Hcy and its derivatives relate to left ventricular (LV) diastolic function. The aim of the present study was to investigate the relationship between plasma Hcy-related metabolites and diastolic dysfunction (DD) in patients with heart disease (HD). A total of 62 HD patients with preserved LV ejection fraction (LVEF ≥ 50%) were enrolled. Plasma Hcy and its derivatives were measured by liquid chromatography‒mass spectrometry (LC–MS/MS). Spearman's correlation test and multiple linear regression models were used to analyze the associations between metabolite levels and LV diastolic function. The cystine/methionine (CySS/Met) ratio was positively correlated with LV diastolic function, which was defined from the ratio of mitral inflow E and mitral e′ annular velocities (E/e′) (Spearman's r = 0.43, p < 0.001). When the subjects were categorized into two groups by E/e′, the high-E/e′ group had a significantly higher CySS/Met ratio than the low-E/e′ group (p = 0.002). Multiple linear regression models revealed that the CySS/Met ratio was independently associated with E/e′ after adjustment for age, sex, body mass index (BMI), diabetes mellitus, hypertension, chronic kidney disease (CKD), hemoglobin, and lipid peroxide (LPO) {standardized β (95% CI); 0.14 (0.04–0.23); p = 0.005}. Hcy, CySS, and Met did not show a significant association with E/e′ in the same models. A high plasma CySS/Met ratio reflected DD in patients with HD. [ABSTRACT FROM AUTHOR]

Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). To examine the hypothesis that the malfunctioning of the cystinotic RPT arises from defective differentiation, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts from an individual with infantile cystinosis, as well as a normal individual. The results indicate that both the cystinotic and normal hiPSCs are pluripotent and can form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to a differentiation regime that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+ Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). The formation of tubules with lumens was less pronounced in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an impairment in differentiation and/or by other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death. [ABSTRACT FROM AUTHOR]

Cystinosis is an autosomally inherited rare genetic disorder in which cystine accumulates in the lysosome. The defect arises from a mutation in the lysosomal efflux pump, cystinosin (or CTNS). Despite the disease being known for more than a century, research, diagnosis, and treatment in India have been very minimal. In recent years, however, some research on cystinosis has been carried out on understanding the pathophysiology and in the development of a humanized yeast model for interrogating the CTNS protein. There has also been a greater awareness of the disease that has been facilitated by the formation of the Cystinosis Foundation of India just over a decade ago. Awareness among primary physicians is critical for early diagnosis, which in turn is critical for proper treatment. Eight different mutations have been observed in cystinosis patients in India, and the mutation spectrum seems different to what has been seen in the US and Europe. Despite these positive developments, there are immense hurdles still to be surmounted. This includes ensuring that the diagnosis is done sooner, making cysteamine more easily available, and, also for the future, to make accessible the promise of gene therapy to cystinosis patients. [ABSTRACT FROM AUTHOR]

Background: Urinary tract problems are a common complaint in small animal medicine and urolithiasis is considered to be an important cause of urinary tract disease in dogs. In this study the main aim was to investigate whether the occurrence of cystine urolithiasis increased during a five-year period. A second aim was to evaluate possible risk-factors as breed, age and gender. This study also evaluated how urine specific gravity, pH and level of cystine in urine responded to preventive strategies. Medical records of dogs with urolithiasis presented at nine Norwegian animal clinics and one animal hospital between 2015 and 2020 were retrospectively reviewed. Results: The incidence of cystine uroliths increased significantly during the five study years (R2 = 0.72, P = 0.0199). Dogs with cystine uroliths were significantly younger (5.0 years (n = 84, 95% CI [4.4–5.6])) when they were diagnosed with cystine uroliths compared to dogs with other types of uroliths (8.1 years (n = 255, 95% CI[7.8-8.5]) P < < 0.0001). Cystine levels in urine were increased in 93% of the dogs with cystine urolithiasis. Cystinuria decreased significantly after neutering (P < 0.0001). Breeds most commonly affected with cystine urolithiasis in this study were Staffordshire bull terrier, Danish Swedish farmdog and Chihuahua. Conclusions: The results from this study supports a suggested genetic basis for cystine urolithiasis as described in previous studies. Neutering is considered an important part of preventing reoccurrence since cystine values decreased significantly after neutering. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Glutamate-induced cytotoxicity (excitotoxicity) has been detected in pancreatic beta cells. The cystine/glutamate antiporter System xc- exports glutamate to the extracellular space and is therefore implicated as driving excitotoxicity. As of yet, it has not been investigated whether System xc- contributes to pancreatic islet function. Methods: This study describes the implications of deficiency of System xc- on glucose metabolism in both constitutive and myeloid cell-specific knockout mice using metabolic tests and diet-induced obesity. Pancreatic islets were isolated and analysed for beta cell function, glutathione levels and ER stress. Results: Constitutive System xc- deficiency led to an approximately threefold decrease in glutathione levels in the pancreatic islets as well as cystine shortage characterised by upregulation of Chac1. This shortage further manifested as downregulation of beta cell identity genes and a tonic increase in endoplasmic reticulum stress markers, which resulted in diminished insulin secretion both in vitro and in vivo. Myeloid-specific deletion did not have a significant impact on metabolism or islet function. Conclusions/interpretation: These findings suggest that System xc- is required for glutathione maintenance and insulin production in beta cells and that the system is dispensable for islet macrophage function. [ABSTRACT FROM AUTHOR]

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by l- and d-stereoisomers. In cystinotic mice, 2-month diets with 5x-l-lysine and 5x-l-arginine were overall well tolerated, while 5x-d-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-d-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by d-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo. [ABSTRACT FROM AUTHOR]

System xc-, encoded by Slc7a11, is an antiporter responsible for exporting glutamate while importing cystine, which is essential for protein synthesis and the formation of thiol peptides, such as glutathione. Glutathione acts as a co-factor for enzymes responsible for scavenging reactive oxygen species. Upon exposure to bacterial products, macrophages exhibit a rapid upregulation of system xc-. This study investigates the impact of Slc7a11 deficiency on the functionality of peritoneal and bone marrow-derived macrophages. Our findings reveal that the absence of Slc7a11 results in significantly reduced glutathione levels, compromised mitochondrial flexibility, and hindered cytokine production in bone marrow-derived macrophages. Conversely, system xc- has a lesser impact on peritoneal macrophages in vivo. These results indicate that system xc- is essential for maintaining glutathione levels, mitochondrial functionality, and cytokine production, with a heightened importance under atmospheric oxygen tension. [ABSTRACT FROM AUTHOR]

The prevalence of cystinuria as an inborn error of metabolism in dogs is unknown. The purpose of this study was to evaluate the prevalence of cystinuria in dogs of various breeds in the Czech Republic. In total, 326 voided urine samples from client owned dogs were obtained. Samples were divided into four groups according to the breed - Irish Terriers (n = 58), Dachshunds (n = 67), French Bulldogs (n = 64) and a group of various breeds (n = 137). Urinary cystine concentration was measured using liquid chromatography/mass spectrometry, urinary creatinine concentration was determined by Jaffe method. Samples with urinary cystine concentrations above 178 µmol/g creatinine were considered as cystinuric. Urinary cystine concentration above the upper limit was found in 71 dogs (21.8%) with significantly higher incidence in intact males of Irish Terriers. In general, cystinuria was more common in intact males. In Irish Terriers, Dachshunds, French Bulldogs, German Shorthaired Pointer, Pomeranian, and Fox Terrier, cystinuria was identified in females. Cystinuria appears to be a relatively common finding in dogs in the Czech Republic, with some breeds being more affected. The finding of cystinuria in two female Irish Terriers calls into question the classification of cystinuria in this breed as androgen dependent only. The detection of cystinuria in related dogs suggests that other individuals in the same family should be examined as part of the management of a cystinuric patient. [ABSTRACT FROM AUTHOR]

Cystinosis is a lysosomal storage disease that is characterized by the accumulation of dipeptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7Δ). We demonstrated that this mutant is retained in the ER and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the roles of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7Δ) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones previously used for treating CFTR F508Δ and demonstrated that they could facilitate the folding and trafficking of cystinosin(7Δ). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by approximately 70%. We believe that our study conclusively establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7Δ). [ABSTRACT FROM AUTHOR]

The prevalence of cystine urolithiasis in domestic ferrets (Mustela putorius furo) has increased dramatically in the past decade. Genetic mutations associated with a defect in the active resorption of cysteine (which is oxidized to cystine) and dibasic amino acids in the proximal renal tubules leading to cystinuria were described in several species; however, the etiology in ferrets is unknown. This study aimed to characterize cystinuria in pet ferrets. Urine samples were aseptically acquired from 27 domestic client-owned ferrets with known dietary and medical histories. The samples underwent a routine urinalysis and microscopic evaluation. Urine aliquots were deproteinized with 6% sulfosalicylic acid and analyzed for amino acid concentrations by ion exchange chromatography. Urine cystine concentrations ranged from 8 to 2486 nmol/mL (median 243; SD 540.02). Principal component analyses revealed correlations among urinary amino acid concentrations, which were explained mostly by three components. Analyses revealed significant correlations between cystine and citrulline, ornithine, and lysine concentrations. Linear regression revealed a negative correlation between age and urinary cystine concentrations and no significant correlation between cystinuria and diet or treats. This is the 1st description of urine amino acid analysis in a cohort of pet ferrets. The detected correlations between cystine, ornithine, and citrulline could indicate that the pathology responsible for cystine urolithiasis in ferrets is like in other species. Result may indicate young ferrets are at increased risk of cystine urolithiasis Further research is needed to evaluate the possible association between diet and cystinuria. [ABSTRACT FROM AUTHOR]

Cystinuria is a known genetic disorder. To date, two genes, SLC3A1 and SLC7A9, have been identified as causes of cystinuria. In this study of 10 patients with cystinuria, which is the largest Korean cohort ever studied, we examined the patients' phenotypes, clinical courses, and genetic analyses. A total of 10 patients with cystinuria diagnosed with cystine stones in a single tertiary medical center (Severance Hospital, Seoul, Republic of Korea) from April 2000 to July 2023 were included in the study. All of the patients participated in mutational studies, and the clinical presentation and consecutive laboratory findings of the patients were analyzed retrospectively. After the initial stone-related surgery or procedure at our hospital, 6 of the 10 patients underwent additional surgery at least once for recurrent stones. Genetic analyses identified six new mutations, of which only two patients had type B mutations. The most common genotype was compound heterozygous type A. We investigated the genotypes and clinical courses of 10 Korean patients with cystinuria who had not been previously reported. More data are needed to statistically analyze the genotype and phenotype of cystinuria. [ABSTRACT FROM AUTHOR]

Cystinuria is one of various disorders that cause biomineralization in the urinary system, including bladder stone formation in humans. It is most prevalent in children and adolescents and more aggressive in males. There is no cure, and only limited disease management techniques help to solubilize the stones. Recurrence, even after treatment, occurs frequently. Other than a buildup of cystine, little is known about factors involved in the formation, expansion, and recurrence of these stones. This study sought to define the growth of bladder stones, guided by micro-computed tomography imaging, and to profile dynamic stone proteome changes in a cystinuria mouse model. After bladder stones developed in vivo, they were harvested and separated into four developmental stages (sand, small, medium and large stone), based on their size. Data-dependent and data-independent acquisitions allowed deep profiling of stone proteomics. The proteomic signatures and pathways illustrated major changes as the stones grew. Stones initiate from a small nidus, grow outward, and show major enrichment in ribosomal proteins and factors related to coagulation and platelet degranulation, suggesting a major dysregulation in specific pathways that can be targeted for new therapeutic options.

Differentiation and fate decisions are critical for the epithelial cells lining the proximal tubule (PT) of the kidney, but the signals involved remain unknown. Defective cystine mobilization from lysosomes through CTNS (cystinosin, lysosomal cystine transporter), which is mutated in cystinosis, triggers the dedifferentiation and dysfunction of the PT cells, causing kidney disease and severe metabolic complications. Using preclinical models and physiologically relevant cellular systems, along with functional assays and a generative artificial intelligence (AI)-powered engine, we found that cystine storage imparted by CTNS deficiency stimulates Ragulator-RRAG GTPase-dependent recruitment of MTORC1 and its constitutive activation. In turn, this diverts the catabolic trajectories and differentiating states of PT cells toward growth and proliferation, disrupting homeostasis and their specialized functions. Therapeutic MTORC1 inhibition by using low doses of rapamycin corrects lysosome function and differentiation downstream of cystine storage and ameliorates PT dysfunction in preclinical models of cystinosis. These discoveries suggest that cystine may act as a lysosomal fasting signal that tailors MTORC1 signaling to direct fate decisions in the kidney PT epithelium, highlighting novel therapeutic paradigms for cystinosis and other lysosome-related disorders. [ABSTRACT FROM AUTHOR]

Background: Crystallization experiments of renal-calculi-forming compounds (calcium oxalate, calcium phosphates, uric acid) are normally performed by monitoring these processes during periods of time similar to the residence of urine inside the kidney. Nevertheless, cystine requires high supersaturation for its crystallization, and most experiments last for longer periods. It must be considered that at high supersaturation, the inhibitors of crystalline development have poor effects. Methods: The induction time of crystallization (ti) of cystine in experimental conditions similar to those of the formation of cystine renal calculi and the effect of different cystine-binding thiol agents was determined through turbidimetric measurements. We also studied the macro- and microstructure of 30 cystine kidney stones through stereoscopic microscopy and scanning electron microscopy. Results: Under the studied conditions, the ti in absence of crystallization inhibitors was 15 min, and the presence of 9 mM of penicillamine, tiopronin, or N-acetylcysteine totally inhibited crystallization, as their effects relate to the formation of complexes with cystine, although N-acetylcysteine also delayed cystine crystalline development and modified cystine crystal morphology. Cystine stones have traditionally been classified as smooth and rough. The study of their structure shows that all of them begin their formation from a few crystals that generate a compact radial structure. Their subsequent growth, depending on the renal cavity where they are located, gives rise to the rough structure in the form of large blocks of cystine crystals or the smooth structure with small crystals. Conclusions: To prevent the development of cystine renal stones, the formation of small crystals must be avoided by reducing urinary cystine supersaturation, with N-acetylcysteine being the most effective among the studied cystine-binding thiol agents. Also, the removal of cystine crystals through increased water intake and physical activity can be a very important preventive measure.

Thomas Hughes,1 Lazaros Tzelves,2 Bhaskar K Somani3 1Department of Urology, Warwick Hospital, Warwick, UK; 2Department of Urology, Sismanogleio Hospital, National and Kapodistrian University of Athens, Athens, Greece; 3Department of Urology, University Hospital Southampton, Southampton, UKCorrespondence: Bhaskar K Somani, University Hospital Southampton NHS Trust, Southampton, UK, Tel +44-2381206873, Email bhaskarsomani@yahoo.comAbstract: Cystinuria is a rare genetic condition that is responsible for cystine stones. Besides stone recurrence, patients with cystine stones have reduced health-related quality of life, increased rates of chronic kidney disease and hypertension. Although lifestyle measures, medical therapy and close follow up are essential to reduce and monitor cystine stone recurrences, surgical intervention is frequently needed for most cystinuria patients. Shock wave lithotripsy, ureteroscopy, percutaneous nephrolithotomy and active surveillance all have a role, and technological advances in endourology are vital in achieving a stone-free status and to prevent recurrences. The complexity of managing cystine stones necessitates a multidisciplinary team discussion, patient involvement and an individualised approach in a specialist centre for optimum management. Thulium fibre laser and virtual reality may have an increasing role in the future of cystine stone management.Keywords: kidney calculi, ureteroscopy, percutaneous nephrolithotomy, laser, cystine, recurrence

Background: The upregulation of antioxidant mechanisms is a common occurrence in cancer cells, as they strive to maintain balanced redox state and prevent oxidative damage. This includes the upregulation of the cystine/glutamate antiporter xCT, which plays a crucial role in protecting cancer cells from oxidative stress. Consequently, targeting xCT has become an attractive strategy for cancer treatment. However, xCT is also expressed by several types of immune cells where it has a role in proliferation and effector functions. In light of these observations, a comprehensive understanding of the specific role of xCT in the initiation and progression of cancer, as well as its potential impact on the immune system within the tumor microenvironment and the anti-tumor response, require further investigation. Methods: We generated xCTnull BALB/c mice to investigate the role of xCT in the immune system and xCTnull/Erbb2-transgenic BALB-neuT mice to study the role of xCT in a mammary cancer-prone model. We also used mammary cancer cells derived from BALB-neuT/xCTnull mice and xCTKO 4T1 cells to test the contribution of xCT to malignant properties in vitro and in vivo. Results: xCT depletion in BALB-neuT/xCTnull mice does not alter autochthonous tumor initiation, but tumor cells isolated from these mice display proliferation and redox balance defects in vitro. Although xCT disruption sensitizes 4T1 cells to oxidative stress, it does not prevent transplantable tumor growth, but reduces cell migration in vitro and lung metastasis in vivo. This is accompanied by an altered immune cell recruitment in the pre-metastatic niche. Finally, systemic depletion of xCT in host mice does not affect transplantable tumor growth and metastasis nor impair the proper mounting of both humoral and cellular immune responses in vivo. Conclusions: xCT is dispensable for proper immune system function, thus supporting the safety of xCT targeting in oncology. Nevertheless, xCT is involved in several processes required for the metastatic seeding of mammary cancer cells, thus broadening the scope of xCT-targeting approaches. [ABSTRACT FROM AUTHOR]

Abdominal radiography is an important diagnostic to detect uroliths. Cystine and urate uroliths were historically characterized as nonmineral opaque on survey radiographs. However, recent research and clinical observations indicate that pure urate and cystine uroliths may be detected with digital radiography. The primary purpose of this prospective, in vitro, diagnostic accuracy study was to determine the sensitivity of digital radiography in detecting cystoliths of varying size and composition. Forty canine uroliths of pure composition (10 each of calcium oxalate, struvite, cystine, and urate), acquired from Minnesota Urolith Center and ranging from 1 to 10 mm, were placed in phantoms of three various sizes and radiographed. The radiographs, including three sets of each urolith separately, were evaluated by three blinded radiologists on two separate occasions. Evaluation included presence or absence of urolith, number of uroliths, and maximum diameter of the urolith(s). For all four types of uroliths and all readers, the specificity and PPV were 100% with an associated very high sensitivity (94.4%–98.9%) and NPV (94.8%–98.9%). Calcium oxalate uroliths were the most accurately measured and struvite were the least accurately measured when compared with the gross measurement. Smaller uroliths were more accurately measured than larger uroliths. Uroliths placed in smaller bladder phantoms were more accurately measured than in larger bladder phantoms. Though accurate measurement of uroliths is complicated by and dependent on numerous variables, our results reveal that urate and cystine uroliths are visualized on digital radiography making them a relevant differential diagnosis when seen clinically. [ABSTRACT FROM AUTHOR]

Background: Cystine stone is a Mendelian genetic disease caused by SLC3A1 or SLC7A9. In this study, we aimed to estimate the genetic prevalence of cystine stones and compare it with the clinical prevalence to better understand the disease etiology. Methods: We analyzed genetic variants in the general population using the 1000 Genomes project and the Human Gene Mutation Database to extract all SLC3A1 and SLC7A9 pathogenic variants. All variants procured from both databases were intersected. Pathogenic allele frequency, carrier rate, and affected rate were calculated and estimated based on Hardy–Weinberg equilibrium. Results: We found that 9 unique SLC3A1 pathogenic variants were carried by 26 people and 5 unique SLC7A9 pathogenic variants were carried by 12 people, all of whom were heterozygote carriers. No homozygote, compoun d heterozygote, or double heterozygote was identified in the 1000 Genome database. Based on the Hardy–Weinberg equilibrium, the calculated genetic prevalence of cystine stone disease is 1 in 30,585. Conclusion: The clinical prevalence of cystine stone has been previously reported as 1 in 7,000, a notably higher figure than the genetic prevalence of 1 in 30,585 calculated in this study. This suggests that the etiology of cystine stone is more complex than what our current genetic knowledge can explain. Possible factors that may contribute to this difference include novel causal genes, undiscovered pathogenic variants, alternative inheritance models, founder effects, epigenetic modifications, environmental factors, or other modifying factors. Further investigation is needed to fully understand the etiology of cystine stone. [ABSTRACT FROM AUTHOR]