Acute pancreatitis (AP) is a sudden-onset disease of the digestive system caused by abnormal activation of pancreatic enzymes. Dual oxidase 2 (DUOX2) has been found to be elevated in the progression of a variety of inflammatory diseases. Therefore, we analyzed the specific roles of DUOX2 in AP development. Blood samples were collected from of AP patients and healthy people, and the caerulein- stimulated human pancreatic duct cells (H6C7) were utilized to establish an AP cell model. Cell growth and apoptosis were measured using an MTT assay and TUNEL staining. Additionally, RT-qPCR and western blot assays were conducted to assess the RNA and protein expressions of the cells. ELISA kits were used to determine TNF-α, IL-6, IL-8, and IL-1β levels. The interaction between DUOX2 and miR-605-3p was predicted using the Targetscan database and confirmed by dual-luciferase report assay. We found that DUOX2 increased while miR-605-3p decreased in the blood of AP patients and caerulein-stimulated H6C7 cells. DUOX2 was targeted by miR-605-3p. Furthermore, DUOX2 knockdown or miR-605-3p overexpression promoted cell viability, decreased the TNF-α, IL-6, IL-8, and IL-1β levels, and inhibited apoptosis rate in caerulein-stimulated H6C7 cells. DUOX2 knockdown or miR-605-3p overexpression also increased the Bcl-2 protein levels and down-regulated Bax, cleaved-caspase-1, NLRP3 and p-p65. Interestingly, DUOX2 overexpression reversed the miR-605-3p mimic function in the caerulein-treated H6C7 cells. In conclusion, our research demonstrated that DUOX2 knockdown relieved the injury and inflammation in caerulein-stimulated H6C7 cells.

Crohn\'s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as \'LND\') with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn\'s ileitis and colitis.

Thyroid dyshormonogenesis (THD) is a heterogeneous group of genetic diseases caused by the total or partial defect in the synthesis or secretion of thyroid hormones. Genetic variants in DUOX2 can cause partial to total iodination organification defects and clinical heterogeneity, from transient to permanent congenital hypothyroidism. The aim of this study was to undertake a molecular characterization and genotype-phenotype correlation in patients with THD and candidate variants in DUOX2. A total of 31 (19.38%) patients from the Catalan Neonatal Screening Program presented with variants in DUOX2 that could explain their phenotype. Fifteen (48.39%) patients were compound heterozygous, 10 (32.26%) heterozygous, and 4 (12.90%) homozygous. In addition, 8 (26.67%) of these patients presented variants in other genes. A total of 35 variants were described, 10 (28.57%) of these variants have not been previously reported in literature. The most frequent variant in our cohort was c.2895_2898del/p.(Phe966SerfsTer29), classified as pathogenic according to reported functional studies. The final diagnosis of this cohort was permanent THD in 21 patients and transient THD in 10, according to reevaluation and/or need for treatment with levothyroxine. A clear genotype-phenotype correlation could not be identified; therefore, functional studies are necessary to confirm the pathogenicity of the variants.

Congenital hypothyroidism (CHT) is a diverse condition with various genetic etiologies. This study aimed to investigate the utility of next-generation sequencing (NGS) analysis in guiding treatment decisions and predicting prognosis for CHT patients with gland in situ (GIS). A retrospective analysis was conducted on 33 CHT patients with GIS who underwent NGS analysis at a single institution between 2018 and 2023. Patients were classified as having permanent (PCH), transient congenital hypothyroidism, or ambiguous congenital hypothyroidism (ACH) CHT based on their response to levothyroxine discontinuation at 3 years of age. Among the 33 patients, genetic variants were identified in 26, with the most prevalent variants found in DUOX2 (26.92%), TSHR (30.77%), TG (19.35%), and DUOXA2 (19.23%). Patients with high initial thyroid-stimulating hormone levels (>50 mIU/L) and low free thyroxine levels (<0.89 ng/dL) at diagnosis tended to have compound heterozygous or homozygous variants in DUOX2, DUOXA2, and TG, and were more likely to develop PCH. In contrast, patients with heterozygous variants in these genes often exhibited ACH. TSHR variants were associated with diverse clinical manifestations, ranging from PCH to ACH, and were more common in patients with initial thyroid-stimulating hormone levels <50 mIU/L. The study highlights the potential utility of NGS analysis in predicting the clinical course and guiding treatment decisions for CHT patients with GIS. Genetic analysis may aid in determining the appropriate duration of levothyroxine therapy and monitoring strategies, particularly in cases where traditional clinical indicators are inconclusive.

BACKGROUND: Enterobacter cloacae insecticidal proteins have been reported to kill Galleria mellonella larvae through affecting their midgut microbiome. However, the mechanisms involved remain unclear. Here we aim to investigate how the insecticidal proteins act on the midgut Duox-ROS system and microbial community of G. mellonella larvae.
METHODS: Reverse transcription qPCR and fluorescence probes were utilized to assess the Duox expression levels and to evaluate quantitative changes of the ROS levels. Sequencing of the 16S rRNA gene sequences of the midgut bacteria of G. mellonella larvae was conducted for further analyses of bacterial diversity, composition, and abundance.
RESULTS: After the injection of the insecticidal proteins, the Duox expression levels first increased within 28 h, then dramatically peaked at 36 h, and slowly decreased thereafter. Simultaneously, the ROS levels increased significantly at 36 h, peaked at 48 h, and rapidly declined to the normal level at 60 h. Responsive to the change of the ROS levels, the structure of the midgut microbial community was altered substantially, compared to that of the untreated larvae. The relative abundance of Enterobacteriaceae and other specific pathogenic bacteria increased significantly, whereas that of Lactobacillus decreased sharply. Importantly, notable shifts were observed in the crucial midgut predicted metabolic functions, including membrane transportation, carbohydrate metabolism, and amino acid metabolism.
CONCLUSIONS: Insecticidal proteins of E. cloacae kill G. mellonella larvae mainly through generation of high oxidative stress, alterations of the midgut microbial community and function, and damage to the physiological functions. These findings provide insights into the inhibition mechanism of E. cloacae insecticidal proteins to G. mellonella larvae.

Hydrogen peroxide is a key element in redox signaling and in setting cellular redox tone. DUOX1 and DUOX2, that directly synthesize hydrogen peroxide, are the most abundant NADPH oxidase transcripts in most epithelia. DUOX1 and DUOX2 hydrogen peroxide synthesis is regulated by intracellular calcium transients and thus cells can respond to signals and initiate responses by increasing cellular hydrogen peroxide synthesis. Nevertheless, many details of their enzymatic regulation are still unexplored. DUOX1 and DUOXA1 were expressed in HEK293T cells and activity was studied in homogenates and membrane fractions. When DUOX1 homogenates or membranes were pre-incubated in NADPH and started with addition of Ca2+, to mimic intracellular activation, progress curves were distinctly different from those pre-incubated in Ca2+ and started with NADPH. The Ca2+ EC50 for DUOX1\'s initial rate when pre-incubated in Ca2+, was three orders of magnitude lower (EC50 ∼ 10-6 M) than with preincubation in NADPH (EC50 ∼ 10-3 M). In addition, activity was several fold lower with Ca2+ start. Identical results were obtained using homogenates and membrane fractions. The data suggested that DUOX1 Ca2+ binding in expected physiological signaling conditions only slowly leads to maximal hydrogen peroxide synthesis and that full hydrogen peroxide synthesis activity in vivo only can occur when encountering extremely high concentration Ca2+ signals. Thus, a complex interplay of intracellular NADPH and Ca2+ concentrations regulate DUOX1 over a wide extent and may limit DUOX1 activity to a restricted range and spatial distribution.

Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH.

Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M-1) while EF1 shows a weaker binding (Ka = 105 M-1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox\'s domain-domain interaction for electron transfer.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.

BACKGROUND: Oxidative stress is implicated in the pathogenesis of heart failure. Dual oxidase 1 (DUOX1) might be important in heart failure development through its mediating role in oxidative stress. This study was designed to evaluate the potential role of DUOX1 in heart failure.
METHODS: AC16 cells were treated with 2 µmol/L of doxorubicin (DOX) for 12, 24, and 48 h to construct a heart failure model. DUOX1 overexpression and silencing in AC16 cell were established. DUOX1 expression was detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Pyroptosis and reactive oxygen species (ROS) production were measured by flow cytometry.
RESULTS: Increased DUOX1 expression levels were observed after DOX treatment for 24 h in AC16 cells. DUOX1 silencing inhibited DOX-induced pyroptosis and ROS production. The release of IL-1β, IL-18, and lactate dehydrogenase (LDH), and expression levels of pyroptosis-related proteins were also decreased. DUOX1 overexpression increased pyroptosis, ROS production, IL-1β, IL-18, and LDH release, and pyroptosis-related protein expression. N-acetyl-cysteine (NAC) significantly reversed DUOX1-induced pyroptosis, ROS, and related factors.
CONCLUSIONS: These results suggest that DUOX1-derived genotoxicity could promote heart failure development. In the process, oxidative stress and pyroptosis may be involved in the regulation of DUOX1 in heart failure.