Copper-transporting ATPase Research Articles

Aberrant copper metabolism and hepatic inflammation cause neurological manifestations in a mouse model of Wilson’s disease

Pathogenic germline mutations in the P-type copper-transporting ATPase (ATP7B) gene cause Wilson’s disease (WD), a hereditary disorder characterized by disrupted copper metabolism. The Arg778Leu (R778L) mutation in exon 8 is prevalent among individuals with WD in East Asia and is associated with more severe phenotypes. In this study, we generated a WD mouse model harboring R778L mutation (R778L mice) using CRISPR/Cas9. R778L mice exhibit a range of pathological characteristics resembling those of patients with WD and the same point mutations, including aberrant copper metabolism, pathological cellular injury, inflammation, and severe hepatic fibrosis. At 3–5 months of age, these mice started to display neurological deficits in motor coordination and cognitive dysfunction, accompanied by increased expression of inflammatory cytokines in the central nervous system. Microglia in the striatum and cortex exhibit significant activation, shorter processes, and decreased branch points. However, the Cu2+ levels in the brain tissue of R778L mice did not differ significantly from those of wild-type mice. Notably, inhibition of hepatic inflammation with PJ34 or siNfkb markedly alleviated the deficiencies in cognitive performance and improved locomotor activity in R778L mice. Thus, this study establishes a novel murine model to investigate the pathophysiology of WD, highlights the liver-brain crosstalk responsible for neurological manifestations in individuals with WD caused by the R778L point mutation, and demonstrates the potential of modulating liver inflammation as a therapeutic strategy for alleviating the neurological manifestations of WD.

Clinical characteristics and prognosis of early diagnosed Wilson's disease: A large cohort study.

Few studies have focused on the outcomes of Wilson's disease (WD) diagnosed before age of 5 years. This study aimed to summarize the clinical features of early diagnosed WD and analyse treatment outcomes and the risk factors associated with treatment failure. A total of 139 children confirmed with WD before 5 years were enrolled in this study. Only patients with follow-up over 1 year were analysed with Kaplan-Meier survival analysis. The composite outcomes included death, progression to liver failure or acute hepatitis, development of renal or neurological symptoms and persistent elevation of alanine aminotransferase (ALT). The treatment failure was defined as occurrence of at least one of above outcomes. Among 139 WD patients at diagnosis, two (1.4%) WD patients presented with symptomatic liver disease, whereas 137 (98.6%) were phenotypically asymptomatic, including 135 with elevated ALT and 2 with normal liver function. Median serum ceruloplasmin (Cp) was 3.1 mg/dL, and urinary copper excretion was 87.4 μg/24-h. There were 71 variants identified in the the copper-transporting ATPase beta gene, and 29 were loss of function (LOF). 51 patients with LOF variant were younger at diagnosis and had lower Cp than 88 patients without LOF. Among 93 patients with over 1 year of follow-up, 19 (20.4%) received zinc monotherapy, and 74 (79.6%) received a zinc/D-penicillamine combination therapy. 14 (15.1%) patients underwent treatment failure, and its occurrence was associated with poor compliance (p < .01). Cp is a reliable biomarker for early diagnosis, and zinc monotherapy is an effective treatment for WD during early childhood. Good treatment compliance is critical to achieve a favourable outcome.

Impaired cellular copper regulation in the presence of ApoE4.

The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allelic variation of the APOE gene, with the following risk structure: ε4 > ε3 > ε2. The biochemical basis for this risk profile is unclear. Here, we reveal a new role for the APOE gene product, apolipoprotein E (ApoE) in regulating cellular copper homeostasis, which is perturbed in the AD brain. Exposure of ApoE target replacement (TR) astrocytes (immortalised astrocytes from APOE knock-in mice) to elevated copper concentrations resulted in exacerbated copper accumulation in ApoE4- compared to ApoE2- and ApoE3-TR astrocytes. This effect was also observed in SH-SY5Y neuroblastoma cells treated with conditioned medium from ApoE4-TR astrocytes. Increased intracellular copper levels in the presence of ApoE4 may be explained by reduced levels and delayed trafficking of the copper transport protein, copper-transporting ATPase 1 (ATP7A/Atp7a), potentially leading to impaired cellular copper export. This new role for ApoE in copper regulation lends further biochemical insight into how APOE genotype confers risk for AD and reveals a potential contribution of ApoE4 to the copper dysregulation that is a characteristic pathological feature of the AD brain.

A novel mutation in the ATP7B gene causing hepatolenticular degeneration in a Chinese family: A case report.

Hepatolenticular degeneration (Wilson disease) is an autosomal recessive monogenic disorder caused by mutations in the ATPase copper transporting beta (ATP7B) gene located on human chromosome 13. This gene encodes a copper-transporting P-type ATPase (ATP7B). Recent studies have revealed that the ATP7B gene is predominantly affected by a few hotspot mutations, with the His1069Gln mutation in exon 14 accounting for 50 to 80% of cases. In China, the Arg778Leu mutation in exon 8 is the most prevalent. However, the discovery of novel mutant genes persists. A 56-year-old Chinese female was referred to our hospital with a liver injury and cirrhosis. Her parents, 2 younger brothers, and children exhibited no signs of liver function impairment. Whole-exome sequencing was conducted on the proband's genomic DNA, and Sanger sequencing was performed on 6 family members for first-generation verification. We identified a novel c.3715G > T (p.Val1239Phe) variant mutation in the ATP7B gene in the patient. The ATP7B c.3715G > T (p.Val1239Phe) variant is predicted to impact the copper transport P-type ATPase. When combined with another mutant gene to form a compound heterozygous mutation, it can lead to hepatolenticular degeneration. This discovery broadens the range of pathogenic genes in the ATP7B gene.

Copper resistance in the cold: Genome analysis and characterisation of a PIB-1 ATPase in Bizionia argentinensis.

Copper homeostasis is a fundamental process in organisms, characterised by unique pathways that have evolved to meet specific needs while preserving core resistance mechanisms. While these systems are well-documented in model bacteria, information on copper resistance in species adapted to cold environments is scarce. This study investigates the potential genes related to copper homeostasis in the genome of Bizionia argentinensis (JUB59-T), a psychrotolerant bacterium isolated from Antarctic seawater. We identified several genes encoding proteins analogous to those crucial for copper homeostasis, including three sequences of copper-transport P1B-type ATPases. One of these, referred to as BaCopA1, was chosen for cloning and expression in Saccharomyces cerevisiae. BaCopA1 was successfully integrated into yeast membranes and subsequently extracted with detergent. The purified BaCopA1 demonstrated the ability to catalyse ATP hydrolysis at low temperatures. Structural models of various BaCopA1 conformations were generated and compared with mesophilic and thermophilic homologous structures. The significant conservation of critical residues and structural similarity among these proteins suggest a shared reaction mechanism for copper transport. This study is the first to report a psychrotolerant P1B-ATPase that has been expressed and purified in a functional form.

Unraveling Copper Exchange in the Atox1-Cu(I)-Mnk1 Heterodimer: A Simulation Approach.

Copper, an essential metal for various cellular processes, requires tight regulation to prevent cytotoxicity. Intracellular pathways crucial for maintaining optimal copper levels involve soluble and membrane transporters, namely, metallochaperones and P-type ATPases, respectively. In this study, we used a simulation workflow based on free-energy perturbation (FEP) theory and parallel bias metadynamics (PBMetaD) to predict the Cu(I) exchange mechanism between the human Cu(I) chaperone, Atox1, and one of its two physiological partners, ATP7A. ATP7A, also known as the Menkes disease protein, is a transmembrane protein and one of the main copper-transporting ATPases. It pumps copper into the trans-Golgi network for the maturation of cuproenzymes and is also essential for the efflux of excess copper across the plasma membrane. In this analysis, we utilized the nuclear magnetic resonance (NMR) structure of the Cu(I)-mediated complex between Atox1 and the first soluble domain of the Menkes protein (Mnk1) as a starting point. Independent free-energy simulations were conducted to investigate the dissociation of both Atox1 and Mnk1. The calculations revealed that the two dissociations require free energy values of 6.3 and 6.2 kcal/mol, respectively, following a stepwise dissociation mechanism.

A Case of Wilson's Disease Suspected to Involve a Novel Genetic Mutation of ATP7B Gene, Requiring a Differential Diagnosis with Non-alcoholic Steatohepatitis.

A 19-year-old Japanese man was referred for a further evaluation of liver dysfunction. Despite the absence of symptoms or obesity, the liver biopsy results were consistent with non-alcoholic steatohepatitis. Subsequent investigations revealed low serum ceruloplasmin, increased urinary copper excretion, and a known mutation c.3809A>G (p.Asn1270Ser) in the copper-transporting enzyme P-type ATPase (ATP7B) gene, leading to a diagnosis of Wilson's disease. A previously unreported variant, i.e., c.3866A>T (p.Asp1289Val) was detected on the patient's other allele and was considered a novel mutation, classified as 'likely pathogenic' according to the American College of Medical Genetics guidelines.

Identification of cuproptosis-related biomarkers and analysis of immune infiltration in allograft lung ischemia-reperfusion injury

Background: Allograft lung ischemia-reperfusion injury (ALIRI) is a major cause of early primary graft dysfunction and poor long-term survival after lung transplantation (LTx); however, its pathogenesis has not been fully elucidated. Cell death is a mechanism underlying ALIRI. Cuproptosis is a recently discovered form of programmed cell death. To date, no studies have been conducted on the mechanisms by which cuproptosis-related genes (CRGs) regulate ALIRI. Therefore, we explored the potential biomarkers related to cuproptosis to provide new insights into the treatment of ALIRI.Materials and methods: Datasets containing pre- and post-LTx lung biopsy samples and CRGs were obtained from the GEO database and previous studies. We identified differentially expressed CRGs (DE-CRGs) and performed functional analyses. Biomarker genes were selected using three machine learning algorithms. The ROC curve and logistic regression model (LRM) of these biomarkers were constructed. CIBERSORT was used to calculate the number of infiltrating immune cells pre- and post-LTx, and the correlation between these biomarkers and immune cells was analyzed. A competing endogenous RNA network was constructed using these biomarkers. Finally, the biomarkers were verified in a validation set and a rat LTx model using qRT-PCR and Western blotting.Results: Fifteen DE-CRGs were identified. GO analysis revealed that DE-CRGs were significantly enriched in the mitochondrial acetyl-CoA biosynthetic process from pyruvate, protein lipoylation, the tricarboxylic acid (TCA) cycle, and copper-transporting ATPase activity. KEGG enrichment analysis showed that the DE-CRGs were mainly enriched in metabolic pathways, carbon metabolism, and the TCA cycle. NFE2L2, NLRP3, LIPT1, and MTF1 were identified as potential biomarker genes. The AUC of the ROC curve for each biomarker was greater than 0.8, and the LRM provided an excellent classifier with an AUC of 0.96. These biomarkers were validated in another dataset and a rat LTx model, which exhibited good performance. In the CIBERSORT analysis, differentially expressed immune cells were identified, and the biomarkers were associated with the immune cells.Conclusion:NFE2L2, NLRP3, LIPT1, and MTF1 may serve as predictors of cuproptosis and play an important role in the pathogenesis of cuproptosis in ALIRI.

The copper P-type ATPase CtpA is involved in the response of Mycobacterium tuberculosis to redox stress

The functional difference among the three copper-transporting P-type ATPases (CtpA, CtpB, and CtpV) annotated in genome of Mycobacterium tuberculosis (Mtb) remains unclear. Thus, CtpA and CtpB are believed to deliver copper to extracytoplasmic proteins as a cofactor required to overcome redox and copper stress in the Mtb periplasm. This study investigates an alternative role of CtpA-mediated copper transportation and its possible interaction with the activity of the multicopper oxidase, (MmcO), in response to redox stress. Results from RT-qPCR experiments indicate that the ctpA gene is upregulated in low Cu2+ concentrations, and under oxidative (H2O2) and nitrosative (sodium nitroprusside) conditions in vitro, but not in high doses of Cu2+. Furthermore, the ctpA mutant strain (MtbΔctpA) showed impaired growth in the presence of oxidative and nitrosative stress in vitro. However, it did not display such growth impairments in response to high doses of copper in comparison to the wild-type strain. Disruption of the ctpA gene in the Mtb genome did not induce an accumulation of copper in cells under toxic doses of the metal, suggesting that CtpA is not directly involved in copper detoxification. On the other hand, whole-cell lysates of the MtbΔctpA mutant that were previously stimulated with Cu2+, H2O2 and SNP (sodium nitroprusside), displayed reduced ability to oxidize organic substrates (para-phenylenediamine (pPD) and 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) (ABTS). These finding strongly suggest that the efflux of copper transported by CtpA from the cytoplasm is relevant to the response to the redox stress and may be required for metalation and activity of MmcO in Mtb.

Severe early-onset Wilson disease caused by a common pathogenic variant in the Bukharan Jewish population in Israel

BackgroundWilson disease is caused by pathogenic variants in the ATP7B gene which encodes a copper-transporting ATPase. AimsDescribe a common founder pathogenic variant among Bukharan Jews and to assess its prevalence, clinical features, and outcome. MethodsThe cohort consisted of patients of Bukharan Jewish descent diagnosed with Wilson disease at a tertiary pediatric medical center in 2013–2018. Clinical and genetic data were collected and analyzed. ResultsSix patients from 4 unrelated families who were homozygous for the c.3784G > T p.(Val1262Phe) pathogenic variant in ATP7B were identified. Five presented with elevated aminotransferase levels, and one, with acute liver failure. Mean age at diagnosis was 8.7 years (5–12.5). Serum ceruloplasmin level was extremely low in all patients (1.9–7 mg/dL; mean 3.2(. The variant was identified in a heterozygous state in 5/153 Bukharan Jews; 2/33 from our local exome database and 3/120 healthy unrelated Bukharan Jews in another cohort, for an estimated carrier frequency of ∼1:30. ConclusionsWe report a common founder pathogenic variant in the ATP7B gene among Bukharan Jews associated with severe early-onset Wilson disease. Given the clinical severity, high frequency of the variant, and being a treatable disease, its inclusion in pre-symptomatic screening in the Bukharan Jewish community should be considered. Furthermore, WD should be part of future genetic newborn screening programs in Israel and worldwide, to enable early treatment and prevention of future life-threatening complications.

Effects of dietary copper (Cu) on growth performance, body composition, mineral content, hepatic histology and Cu transport of the GIFT strain of Nile tilapia (Oreochromis niloticus)

This study determined the influence of dietary copper (Cu) levels on growth and feed utilization, body composition, mineral content, liver histology and intestinal Cu transport, and identify dietary Cu requirement of the GIFT strain of Nile tilapia (Oreochromis niloticus). Six diets were prepared with Cu sulfate addition at levels of 0 (control), 5, 10, 20, 40, and 80 mg/kg, respectively. Analytical dietary Cu contents were 1.55, 2.97, 4.51, 7.25, 11.30, and 19.24 mg Cu/kg diet, respectively. Each experimental diet was fed to three replicates of GIFT tilapia (25 fish per tank, initial body weight 4.34 ± 0.01 g) in a recirculating freshwater system for eight weeks. GIFT tilapia fed 4.51 mg Cu/kg diet had higher growth performance and feed utilization. Cu contents in the whole-body, the liver and intestinal tract increased with dietary Cu levels. Moreover, dietary Cu addition also affected whole-body Mg content significantly, but did not affect whole body crude protein, lipid, zinc (Zn), iron (Fe), and calcium (Ca) content. GIFT tilapia fed with 4.51 mg Cu/kg diet had lower liver vacuoles relative area. Furthermore, dietary Cu addition also significantly influenced mRNA expression of genes involved in intestinal Cu transport, such as copper transporter 1 (ctr1), copper transporter 2 (ctr2), antioxidant 1 copper chaperone (atox1), cytochrome c oxidase copper chaperone (cox17) and copper-transporting ATPase alpha (atp7a). The regression analysis of weight gain and whole-body Cu retention against the dietary Cu levels suggested that 4.23 to 4.57 mg Cu/kg diet was optimal for GIFT tilapia. Statement of relevanceOur study determined the optimal dietary Cu requirement of GIFT strain of tilapia, which provided good references for dietary Cu addition for the fish species, and accordingly was helpful for its feed formulation and aquaculture production. Our study also demonstrated that dietary Cu addition influenced mRNA expression of genes involved in Cu transport, which provides a good basis for exploring Cu metabolism in fish.

P1B P-type ATPases: Cu&lt;sup&gt;+&lt;/sup&gt;-ATPases in GtoPdb v.2023.1

Copper-transporting ATPases convey copper ions across cell-surface and intracellular membranes. They consist of eight TM domains and associate with multiple copper chaperone proteins (e.g. ATOX1, O00244).

Clinical and genetic characterization of pediatric patients with Wilson's disease from Yunnan province where ethnic minorities gather.

Background: Wilson's disease (WD) is an autosomal recessive disease that is caused by mutations in the ATP7B (a copper-transporting P-type ATPase) gene. The disease has a low prevalence and is characterized by a copper metabolism disorder. However, various characteristics of the disease are determined by race and geographic region. We aimed to discover novel ATP7B mutations in pediatric patients with WD from Yunnan province, where there is a high proportion of ethnic minorities. We also performed a comprehensive analysis of ATP7B mutations in the different ethnic groups found in Southwest China. Methods: We recruited 45 patients who had been clinically diagnosed with WD, from 44 unrelated families. Routine clinical examinations and laboratory evaluations were performed and details of age, gender, ethnic group and symptoms at onset were collected. Direct sequencing of the ATP7B gene was performed in 39 of the 45 patients and their families. Results: In this study, participants came from seven different ethnic groups in China: Han, Bai, Dai, Zhuang, Yi, Hui and Jingpo. Three out of ten patients from ethnic minorities presented with elevated transaminases, when compared to the majority of the Han patients. Forty distinct mutations (28 missense, six splicing, three non-sense, two frameshift and one mutation of uncertain significance) were identified in the 39 patients with WD. Four of the mutations were novel and the most frequent mutation was c.2333G > T (p.R778L, allelic frequency: 15.38%). Using the phenotype-genotype correlation analysis, patients from ethnic minorities were shown to be more likely to have homozygous mutations (p = 0.035) than Han patients. The patients who carried the c.2310C > G mutation had lower serum ceruloplasmin levels (p = 0.012). In patients with heterozygous mutations, c.3809A > G was significantly associated with ethnic minorities (p = 0.042). The frequency of a protein-truncating variant (PTV) in Han patients was 34.38% (11/32), while we did not find PTV in patients from ethnic minorities. Conclusion: This study revealed genetic defects in 39 pediatric patients with WD from Yunnan province. Four novel mutations were identified and have enriched the WD database. We characterized the genotypes and phenotypes in different minorities, which will enhance the current knowledge on the population genetics of WD in China.

Molecular mapping of genomic regions and identification of possible candidate genes associated with gynoecious sex expression in bitter gourd

Bitter gourd is an important vegetable crop grown throughout the tropics mainly because of its high nutritional value. Sex expression and identification of gynoecious trait in cucurbitaceous vegetable crops has facilitated the hybrid breeding programme in a great way to improve productivity. In bitter gourd, gynoecious sex expression is poorly reported and detailed molecular pathways involve yet to be studied. The present experiment was conducted to study the inheritance, identify the genomic regions associated with gynoecious sex expression and to reveal possible candidate genes through QTL-seq. Segregation for the gynoecious and monoecious sex forms in the F2 progenies indicated single recessive gene controlling gynoecious sex expression in the genotype, PVGy-201. Gynoecious parent, PVGy-201, Monoecious parent, Pusa Do Mausami (PDM), and two contrasting bulks were constituted for deep-sequencing. A total of 10.56, 23.11, 15.07, and 19.38 Gb of clean reads from PVGy-201, PDM, gynoecious bulk and monoecious bulks were generated. Based on the ΔSNP index, 1.31 Mb regions on the chromosome 1 was identified to be associated with gynoecious sex expression in bitter gourd. In the QTL region 293,467 PVGy-201 unique variants, including SNPs and indels, were identified. In the identified QTL region, a total of 1019 homozygous variants were identified between PVGy1 and PDM genomes and 71 among them were non-synonymous variants (SNPS and INDELs), out of which 11 variants (7 INDELs, 4 SNPs) were classified as high impact variants with frame shift/stop gain effect. In total twelve genes associated with male and female gametophyte development were identified in the QTL-region. Ethylene-responsive transcription factor 12, Auxin response factor 6, Copper-transporting ATPase RAN1, CBL-interacting serine/threonine-protein kinase 23, ABC transporter C family member 2, DEAD-box ATP-dependent RNA helicase 1 isoform X2, Polygalacturonase QRT3-like isoform X2, Protein CHROMATIN REMODELING 4 were identified with possible role in gynoecious sex expression. Promoter region variation in 8 among the 12 genes indicated their role in determining gynoecious sex expression in bitter gourd genotype, DBGy-1. The findings in the study provides insight about sex expression in bitter gourd and will facilitate fine mapping and more precise identification of candidate genes through their functional validation.

The implications and prospect of cuproptosis-related genes and copper transporters in cancer progression.

Currently, cancer has become one of the major public health problems worldwide. Apoptosis is an important anti-cancer defense mechanism, which is used in the development of targeted drugs. Because cancer cells have endogenous resistance to apoptosis,the clinical efficacy of related drugs is not ideal. Therefore, non-apoptotic regulatory cell death may bring new therapeutic strategies for cancer treatment. Cuproptosis is a novel form of regulatory cell death which is copper-dependent, regulated and distinct from other known cell death regulatory mechanisms. FDX1,LIAS,and DLAT named cuproptosis-related genes play an essential role in regulating cuproptosis. Meanwhile, abnormal accumulation of copper can be observed in various malignant tumors. The correlation has been established between elevated copper levels in serum and tissues and the progression of several cancers. Copper transporters, CTR1 and Copper-transporting ATPases(ATP7A and ATP7B), are mainly involved in regulating the dynamic balance of copper concentration to maintain copper homeostasis. Thus,cuproptosis-related genes and copper transporters will be the focus of cancer research in future. This review elaborated the basic functions of cuproptosis-related genes and copper transporters by retrievalling PubMed. And then we analyzed their potential relationship with cancer aiming to provide theoretical support and reference in cancer progression, diagnosis and treatment for future study.

Diagnosis and treatment of Wilson disease: An update

Wilson’s illness is an autosomal dominant impairment of copper metabolism that results from the lack of or dysfunction of a copper-transporting P-type ATPase, which is expressed on chromosome 13. This ATPase is a component of the trans-Golgi network and is carried by hepatocytes. It transports copper into the secretory route where it is combined with ceruloplasmin and excreted as bile. Affected people gradually develop copper accumulation in the liver since the only pathway for copper excretion in physiological settings is through biliary clearance. Hepatocytes experience cell death when their storage capacity is exceeded, which causes hemolysis, copper leakage into the plasma, and tissue deposition. Children might experience rapid liver failure, silent cirrhosis, or chronic hepatitis. Among the neuropsychiatric symptoms induced by copper overload in the central nervous system that are frequently seen in young people are dystonia, tremors, temperamental disorders, and cognitive deficiencies. Test results showing lower ceruloplasmin levels in serum, elevated urine copper concentration, and elevated intrahepatic copper percentage indicate Wilson’s illness. Because there are more than 100 distinct mutations and most people are compound heterozygotes, molecular genetic analysis is difficult. Copper treatment with penicillamine is a successful therapy for the vast number of patients, and liver transplantation is beneficial in situations of chronic liver failure. The molecular genetic causes of Wilson’s disease have shed new light on the mechanisms governing cellular copper homeostasis.

Organic copper promoted copper accumulation and transport, enhanced low temperature tolerance and physiological health of white shrimp (Litopenaeus vannamei Boone, 1931)

This study was conducted to assess the effects of dietary copper source and level on hematological parameters, copper accumulation and transport, resistance to low temperature, antioxidant capacity and immune response of white shrimp (Litopenaeus vannamei Boone, 1931). Seven experimental diets with different copper sources and levels were formulated: C, no copper supplementation; S, 30 mg/kg copper in the form of CuSO4·5H2O; SO, 15 mg/kg copper in CuSO4·5H2O + 7.5 mg/kg copper in Cu-proteinate; O1, O2, O3 and O4, 10, 20, 30 and 40 mg/kg copper in the form of Cu-proteinate, respectively. A total of 840 shrimp (5.30 ± 0.04 g) were randomly distributed to 21 tanks (3 tanks/diet, 40 shrimp/tank). An 8-week feeding trial was conducted. The results showed that there was no significant difference in growth performance and whole shrimp chemical compositions among all groups. Compared with inorganic copper, dietary organic copper (O2 and O3) increased total protein, albumin, and glucose content of plasma, while decreased triglyceride and total cholesterol of plasma. Copper concentration in plasma and muscle and gene expression of metallothionein and copper-transporting ATPase 2 like in hepatopancreas were higher in shrimp fed organic copper (SO, O2, O3 and O4). The lowest mortality after low temperature (10 °C) challenge test was observed in the O2 and O3 groups. Organic copper (SO, O2, O3 and O4) significantly enhanced the antioxidant capacity (in terms of higher activities of total superoxide dismutase, copper zinc superoxide dismutase, catalase, glutathione peroxidase and total antioxidant capacity, lower malondialdehyde concentration of plasma, and up-regulated gene expression of superoxide dismutase, copper zinc superoxide dismutase, catalase and glutathione peroxidase of hepatopancreas). Organic copper (SO, O2, O3 and O4) enhanced the immune response (in terms of higher number of total hemocytes, higher activities of acid phosphatase, alkaline phosphatase, phenoloxidase, hemocyanin and lysozyme in plasma, and higher gene expressions of alkaline phosphatase, lysozyme and hemocyanin in hepatopancreas). Inorganic copper (Diet S) also had positive effects on white shrimp compared with the C diet, but the SO, O2, O3 and O4 diets resulted in better results, among which the O2 diet appeared to be the best one. In conclusion, organic copper was more beneficial to shrimp health than copper sulfate.

Oxysterol misbalance critically contributes to Wilson disease pathogenesis.

Wilson disease (WD) is a metabolic disorder caused by inactivation of the copper-transporting ATPase 2 (ATP7B) and copper (Cu) overload in tissues. Excess Cu causes oxidative stress and pathologic changes with poorly understood mechanistic connections. In Atp7b−/− mice with established liver disease, Cu overload activates the stress-sensitive transcription factor Nrf2 (nuclear factor erythroid-derived 2-like 2). Nrf2 targets, especially sulfotransferase 1e1 (Sult1e1), are strongly induced and cause elevation of sulfated sterols, whereas oxysterols are decreased. This sterol misbalance results in inhibition of the liver X receptor (LXR) and up-regulation of LXR targets associated with inflammatory responses. Pharmacological inhibition of Sult1e1 partially reverses oxysterol misbalance and LXR inhibition. Contribution of this pathway to advanced hepatic WD was demonstrated by treating mice with an LXR agonist. Treatment decreased inflammation by reducing expression of proinflammatory molecules, diminished fibrosis by down-regulating the noncanonical transforming growth factor–β signaling pathway, and improved liver morphology and function. Thus, the identified pathway is an important driver of WD.

The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a

BackgroundCadmium (Cd2+) is one of the highly toxic heavy metals and is considered as a carcinogenic agent. Our aim was to confirm the ability of Frankia alni ACN14a to resist Cd2+ and to determine the genes involved in the resistance mechanism. ResultsF. alni ACN14a and Frankia casuarinae CcI3 hyphae showed up to 10 and 22 times Cd2+ accumulation when exposed to 1 mM Cd2+, respectively. Scanning electron microscopy (SEM) exhibited a stable Cd2+ precipitate on the cell surface, and the increase in Cd2+ weight level reached 16.45% when evaluated with SEM-EDAX analysis. The following two potential Cd2+ operons were identified: 1. cadCA operon, which encodes a copper-transporting P-type ATPase A (cadA, FRAAL0989) and an ArsR family regulator (cadC, FRAAL0988), with 37- and 70-fold increase in their expression by qRT-PCR, respectively and 2. cadB/DX, which encodes a putative cobalt-zinc-cadmium resistance protein (cadD, FRAAL3628) and heavy metal-associated domain protein (cadX, FRAAL3626), with 22- and 16-fold upregulation when exposed to Cd2+ stress. ConclusionsCd2+ tolerance by F. alni ACN14a involved efflux of Cd2+ outside the cells and binding it to the membrane surface. Our results indicate the existence of two cadmium-resistance mechanisms in Frankia strains, which support the idea of using them as a bioremediation agent.How to cite: Rehan M, Alhusays A, Serag AM, et al. The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a. Electron J Biotechnol 2022;60. https://doi.org/10.1016/j.ejbt.2022.09.006.

OA30 Genetic analysis of whole exome sequencing in a cohort of children with refractory JIA reveals genetic risk factors for rare juvenile diseases

Introduction/BackgroundJuvenile idiopathic arthritis (JIA) encompasses a group of heterogeneous rheumatic diseases of childhood onset. JIA can result in long term disability and remission is the main goal of treatment. However refractory disease can occur, which is defined as the absence of response to a standard disease therapy. A genetic basis for refractory disease has yet to explored, where deleterious rare variants complicate diagnosis or treatment outcome. This study aimed to investigate, through genetic analysis, whether children with JIA that is refractory carry rare genetic risk factors in genes linked to monogenic diseases.Description/MethodWhole exome sequencing of 99 children with JIA was performed with the Agilent SureSelect Human All ExonV6 kit. All quality control, variant filtering and annotation was performed in Varseq (version 2.2.1). Variants with a read depth <30 and genotype quality <80 were removed. Rarity and pathogenicity filters were then applied to remove variants with an allele frequency >1% (based on ExAC, gnomAD, gnomAD exome, NHLBI and 1KGp phase 3), classified as benign or likely benign on ClinVar, with a CADD PHRED score <15 and a REVEL score >0.7. Variants were annotated if they appeared in a gene from the primary immunodeficiency PanelApp (Martin et al., 2019), in a gene associated with an arthritis phenotype or in a gene that appeared on a paediatric monogenic gene list. The variants were then classified using ACMG guidelines (Richards et al., 2015) and benign, or likely benign, classified variants were removed.Discussion/ResultsA total of 470 variants were identified and we found that 20 out of the 99 children screened were heterozygous for at least one recognised variant in a gene linked to a monogenic disease. Five of these children carried more than one recognised variant linked to monogenic genes. Here we provide a number of illustrative examples: three genes, ADAR, ATP7B and MVK, were prioritised based on prior evidence of associated disease. The variant p.Pro193Ala (gnomAD allele frequency (GAD) 2.2x10-3) of ADAR has previously been deemed pathogenic in a homozygous or compound heterozygous state for Aicardi-Goutières syndrome. Adenosine deaminases (ADARs) catalyse the hydrolytic deamination of adenosine to inosine in dsRNA and is suggested to act as a suppressor of type 1 interferon-stimulated genes. Within ATP7B, two distinct variants were detected; p.Gln1142His (GAD 1.6x10-5) and p.Ile1148Thr (GAD 4.0x10-5) have previously been reported as pathogenic, in combination with a third variant, for Wilson’s disease and were carried by one individual in this cohort. ATP7B encodes copper-transporting ATPase 2, which supplies copper to ceruloplasmin. Variant p.Val377Ile (GAD 1.6x10-3) of MVK was detected in eight individuals in this cohort, interestingly five of these individuals also carried at least one HLA-DRB1 stop-gained variant. This MVK mutation has been confirmed as pathogenic in a homozygous or compound heterozygous state for mevalonate kinase deficiency. MVK converts mevalonic acid into mevalonate-5-phosphate in the cholesterol synthesis pathway. Additionally, two stop-gained loss of function HLA-DRB1 variants, p.Tyr107Ter and p.Gln125Ter, were detected in five and 20 individuals, respectively, in this cohort. HLA-DRB1 is a recognised susceptibility locus for JIA.Key learning points/ConclusionScreening of a cohort of 99 children with JIA that have refractory disease has revealed that individuals carry deleterious variants in genes linked to monogenic forms of disease. These results highlight that the genetic basis for refractory disease needs to be further investigated. Carrying additional genetic risk factors to disease may complicate disease outcome and genetic screening of children with refractory JIA may improve treatment outcome in the future.