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Abstract Background Hereditary hemochromatosis (HH) is an iron overload disorder and can be caused by variants in non-HFE genes in Chinese patients. However, there is still a considerable proportion of patients suffering from unexplained iron overload. In our previous study, we had identified the p.R269Q variant in exon 4 of the Bone morphogenetic protein 4 (BMP4) gene in Chinese patients with unexplained primary iron overload by Whole Exome sequencing, and then the BMP4 p.H251Y variant was identified by Sanger sequencing in a Chinese patient with secondary iron overload. Our study aimed to explore the pathogenicity and underlying mechanism of BMP4 p.H251Y and BMP4 p.R269Q variants in patients with iron overload. Methods Sanger sequencing was conducted to identify the novel variants in the BMP4 gene of patients with unexplained iron overload. MRI and liver biopsy were used to display iron overload in the liver of the patient harboring the BMP4 p.H251Y variant. The BMP4 and hepcidin levels in BMP4 knockdown and BMP4 variant cells were examined by enzyme-linked immunosorbent assay. The effects of BMP4 p.H251Y and BMP4 p.R269Q variants on the hepcidin-regulation pathway were studied. Results One of 54 HH patients (1.85%) harbored the BMP4 p.R269Q variant. One of 148 patients (0.68%) with secondary hemochromatosis harbored the BMP4 p.H251Y variant, and these two variants were not found in 100 Chinese general population. For the patient harboring the BMP4 p.H251Y variant, abdominal MRI and Perl's staining of liver tissue displayed iron overload in the liver. Cells transfected with the BMP4 p.H251Y and p.R269Q variants showed down-regulation of hepcidin level and BMP/SMAD pathway compared with cells transfected with the wild-type BMP4 vector. Conclusion The BMP4 p.H251Y and p.R269Q variants can downregulate hepcidin levels by inhibiting the BMP/SMAD axis, suggesting they may play pathogenic roles in iron overload.

Background & Aims: The association between Wilson disease and various ATP7B mutations is well-established; however, the molecular mechanism underlying the functional consequence of these mutations, particularly the splicing mutations, remains unclear. This study focused on the ATP7B c.1543+1G>C variant, to reveal a universal pathogenic mechanism of the ATP7B mutants with altered N-terminus. Methods: The splicing assay and RNA pull-down were performed to explore the mechanism of the aberrant splicing. The ATP7B knockout HuH-7 cell line and Atp7b-/- mice were created, and the functional consequence of the mutant ATP7B were evaluated in vitro and in vivo. Results: The c.1543+1G>C mutation resulted in the skipping of ATP7B exon 3, and the mutant ATP7B showed a loss of trans-Golgi network localization and was degraded via the ubiquitin-proteasome pathway, facilitated by enhanced interactions with COMMD1. Elevated intercellular copper concentration and reduced survival rate were observed in HuH-7 cells expressing mutant ATP7B. Restoration of wild-type ATP7B in Atp7b-/- mice resulted in a substantial improvement in phenotype, whereas mice treated with mutant ATP7B did not demonstrate equivalent benefits. Conclusions: Our research investigated the pathogenicity and mechanism of ATP7B c.1543+1G>C variant, with particular focus on its enhanced interaction with COMMD1 as a potential universal mechanism contributing to the dysfunction of various ATP7B variants. These findings provide a foundation for the development of innovative therapeutic strategies that target abnormal splicing events in a range of hereditary diseases, including Wilson disease.

Abstract Background The epithelial‐mesenchymal transition (EMT), featured by abatement of cell–cell contact, is related to exacerbating non–small cell lung cancer (NSCLC) by inducing metastasis. MAL and relevant proteins for vesicle trafficking and membrane link domain 3 (MARVELD3) is a novel tight junction protein participated in the EMT. There is limited information available about the mechanism of MARVELD3 in NSCLC. In this trial, the inhibition effect of MARVELD3 on human NSCLC cells will be discussed. Methods MARVELD3 expression was measured in NSCLC tissues and para‐carcinoma tissues. The expression of MARVELD3 and EMT‐related genes were examined in transforming growth factor (TGF)‐β1‐induced NSCLC cells. NSCLC cells with MARVELD3‐knockdown and overexpressed were established to analyze the relationship between MARVELD3 and EMT and cell migration. The Wnt/β‐catenin pathway expression was also analyzed in NSCLC cell models and clinic species. Results Lower protein levels of MARVELD3 were observed in NSCLC samples than para‐carcinoma specimens, and the decreased expression of MARVELD3 in NSCLC specimens was associated with tumor metastasis. E‐Cadherin and MARVELD3 expression was reduced in TGF‐β1 treated NSCLC cells, whereas increased Vimentin expression was detected. MARVELD3 changed the EMT‐related genes and induced cell migration. In addition, Wnt/β‐catenin pathway and target genes, MYC and CCND1, expressions were inhibited in MARVELD3 overexpressed NSCLC cells. Conclusions TGF‐β1 induced EMT in human NSCLC cells can be suppressed by MARVELD3 through Wnt/β‐catenin signaling pathway. These results indicate that MARVELD3 might be a potential therapeutic modality useful in the treatment of NSCLC.

Abstract Backgrounds Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. Methods Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. Results Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. Conclusions Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

BackgroundEarly diagnosis of esophageal squamous cell carcinoma (ESCC) is critical for effective treatment and optimal prognosis; however, less study on serum biomarkers for the early ESCC detection has been reported. The aim of this study was to identify and evaluate several serum autoantibody biomarkers in early ESCC.MethodsWe initially screened candidate tumor-associated autoantibodies (TAAbs) associated with ESCC by serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (nano-LC-Q-TOF-MS/MS), and the TAAbs were further subjected to analysis by Enzyme-linked immunosorbent assay (ELISA) in a clinical cohort (386 participants, including 161 patients with ESCC, 49 patients with high-grade intraepithelial neoplasia [HGIN] and 176 healthy controls [HC]). Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance.ResultsThe serum levels of CETN2 and POFUT1 autoantibodies which were identified by SERPA were statistically different between ESCC or HGIN patients and HC in ELISA analysis with the area under the curve (AUC) values of 0.709 (95%CI: 0.654-0.764) and 0.741 (95%CI: 0.689-0.793), 0.717 (95%CI: 0.634-0.800) and 0.703 (95%CI: 0.627-0.779) for detection of ESCC and HGIN, respectively. Combining these two markers, the AUCs were 0.781 (95%CI: 0.733-0.829), 0.754 (95%CI: 0.694-0.814) and 0.756 (95%CI: 0.686-0.827) when distinguishing ESCC, early ESCC and HGIN from HC, respectively. Meanwhile, the expression of CETN2 and POFUT1 was found to be correlated with ESCC progression.ConclusionsOur data suggest that CETN2 and POFUT1 autoantibodies have potential diagnostic value for ESCC and HGIN, which may provide novel insights for early ESCC and precancerous lesions detection.

Abstract Background Hereditary hemochromatosis (HH) is widely recognized and clinical manifestations of hemochromatosis-related (HFE-related) HH is well studied in European populations. Less is known about the clinical and laboratory characteristics of non-HFE related HH in Asian population. We aimed to explore the relationship between genotype and clinical phenotype in Chinese patients with non-HFE related hereditary hemochromatosis. Methods Peripheral blood samples and clinical data of patients with primary iron overload were collected from the China Registry of Genetic/Metabolic Liver Diseases. Sanger sequencing was performed in cases with primary iron overload, for 5 known HH related genes (HFE, HJV, HAMP, TFR2 and SLC40A1) and 2 novel iron homeostasis-related genes (DENND3 and SUGP2). The correlation of genotype and clinical phenotype in these patients was analyzed. Results Of the 32 patients with primary iron overload (23 were males and 9 were females), non-HFE variants were detected in 31 (31/32, 97%), including 8 pathogenic variants in HJV, 7 pathogenic variants in SLC40A1, 8 likely pathogenic variants in SUGP2 and 5 likely pathogenic variants in DENND3 cases. Among these 31 cases, 4 cases harbored homozygous variants, 2 cases harbored homozygous + heterozygous variants, 19 cases harbored heterozygous or combined heterozygous variants, and 6 cases harbored no any damaging variants. None of investigated cases carried damaging HAMP and TFR2 variants were found. 8 cases were classified as type 2A HH and 6 cases as type 4 HH, 10 cases as non-classical genotype, and 6 cases had no pathogenic variants from 31 cases. During the statistical analysis, we excluded one case (SLC40A1 IVS3 + 10delGTT + SUGP2 p. R639Q(homo)) with difficulty in grouping due to combined damaging variants. Cases with type 2A HH have an earlier age at diagnosis (p = 0.007). The iron index of cases in type 2A HH and type 4 HH was higher than that in other groups (p = 0.01). Arthropathy was relatively rare in all groups. None of cases with type 2A HH developed cirrhosis. Cirrhosis and diabetes are more prevalent in type 4 HH. The incidence of cirrhosis (p = 0.011), cardiac involvement (p = 0.042), diabetes (p = 0.035) and hypogonadism (p = 0.020) was statistically significant in the four groups. However, due to the limited sample size, the pairwise comparison showed no significant difference. Conclusions This is the first comprehensive analysis about the gene variant spectrum and phenotypic aspects of non-HFE HH in China. The results will be useful to the identification, diagnosis and management of HH in China.

MarvelD3, a recently identified tight junction membrane protein, could be associated with hepatocellular carcinoma (HCC). We aimed to investigate the role of marvelD3 in Epithelial–Mesenchymal Transition (EMT) and migration of HCC and explore the underlying molecular mechanisms. First, we assessed marvlD3 expression in HCC and normal liver tissues and found loss of marvelD3 expression was significantly correlated with the occurrence and TNM stage of HCC. Second, we detected that marvelD3 was downregulated in HCC cells with transforming growth factor β1 and snail/slug-induced EMT. Finally, we analyzed expression of marvelD3 protein was significantly associated with EMT and the NF-κB signaling pathway. Our study demonstrated that MarvelD3 inhibited EMT and migration of HCC cells along with inhibiting NF-κB signaling pathway. Abbreviations: HCC, Hepatocellular carcinoma; TJ, Tight junction; MARVEL, MAL and related proteins for vesicle trafficking and membrane link; EMT, Epithelial–mesenchymal transition; NF-κB, Nuclear factor kappa B; TAMPs, Tight junction-associated marvel proteins; TGF-β1, Transforming growth factor-β1; MMP9, matrix metallopeptidase 9; RT-PCR, Real-time PCR; IHC, Immunohistochemistry; IF, Immunofluorescence.

Abstract Background Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder characterized by predominantly conjugated hyperbilirubinemia that is caused by pathogenic mutations in the adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2) gene, which encodes multidrug resistance-associated protein 2 (MRP2). However, little is known about the causative mutation of DJS in China. Recently, we have reported ABCC2 p.G693R mutation in two unrelated cases. In the present study, we investigated the pathogenicity of the ABCC2 p.G693R mutation in DJS in China. Methods Clinical and genetic analysis was conducted for the two patients with the ABCC2 p.G693R mutation. Whole exome sequencing for mutations in other known hyperbilirubinemia-related genes was conducted for the cases with ABCC2 p.G693R. Expression and cellular localization of the mutant MRP2 p.G693R were analyzed by Western blotting and immunofluorescence assay, respectively. Organic anion transport activity was evaluated by the analysis of glutathione-conjugated-monochlorobimane. Results The two DJS patients with ABCC2 p.G693R mutation, which was conserved among different species, showed typical hyperbilirubinemia phenotype. No pathogenic mutation was identified in the other known hyperbilirubinemia related genes. Functional studies in three cell lines showed that the expression, localization and the organic anion transport activity were significantly compromised by MRP2 p.G693R mutation compared with wild-type MRP2. Conclusions The recurrent ABCC2 p.G693R mutation is associated with loss of function of the MRP2 protein and may result in hyperbilirubinemia in DJS in China.

Abstract Background Early‐stage lung adenocarcinoma that radiologically manifests as part‐solid nodules, consisting of both ground‐glass and solid components, has distinctive growth patterns and prognosis. The characteristics of the tumour microenvironment and transcriptional features of the malignant cells of different radiological phenotypes remain poorly understood. Methods Twelve treatment‐naive patients with radiological part‐solid nodules were enrolled. After frozen pathology was confirmed as lung adenocarcinoma, two regions (ground‐glass and solid) from each of the 12 part‐solid nodules and 5 normal lung tissues from 5 of the12 patients were subjected to single‐cell sequencing by 10x Genomics. We used Seurat v3.1.5 for data integration and analysis. Results We comprehensively dissected the multicellular ecosystem of the ground‐glass and solid components of part‐solid nodules at the single‐cell resolution. In tumours, these components had comparable proportions of malignant cells. However, the angiogenesis, epithelial‐to‐mesenchymal transition, KRAS, p53, and cell‐cycle signalling pathways were significantly up‐regulated in malignant cells within solid components compared to those within ground‐glass components. For the tumour microenvironment, the relative abundance of myeloid and NK cells tended to be higher in solid components than in ground‐glass components. Slight subtype composition differences existed between the ground‐glass and solid components. The T/NK cell subsets’ cytotoxic function and the macrophages’ pro‐inflammation function were suppressed in solid components. Moreover, pericytes in solid components had a stronger communication related to angiogenesis promotion with endothelial cells and tumour cells. Conclusion The cellular landscape of ground‐glass components is significantly different from that of normal tissue and similar to that of solid components. However, transcriptional differences exist in the vital signalling pathways of malignant and immune cells within these components.

Krüppel-like factor 5 (KLF5) can both promote and suppress cell migration, but the underlying mechanisms have not been elucidated. In this study, we show that the function of KLF5 in epithelial-mesenchymal transition (EMT) and migration of liver cancer cells depends on the status of the cellular tumor antigen p53 (p53). Furthermore, zinc finger E-box-binding homeobox 2 (ZEB2) is the main regulator of KLF5 in EMT in liver cancer cells in the context of p53 loss. Most importantly, the regulation of ZEB2 by p53 and KLF5 is indirect and that miR-192 mediates this regulation. Finally, we find that in invasive liver cancer, KLF5 is absent in the context of p53 loss or mutation.

Serum autoantibodies against tumor-associated antigen have important value in the early diagnosis of hepatocellular carcinoma (HCC), but the mechanism of autoantibody production is poorly understood. We previously showed that autoantibodies against the centromere protein F (CENPF) may be useful as an early diagnostic marker for HCC. Here we explored the mechanism of cell apoptosis-based CENPF autoantibody production and verified the correlation of CENPF autoantibody level with HCC development. We demonstrated that CENPF was overexpressed and aberrantly localized throughout the nuclei and cytoplasm in human HCC cells compared with hepatic cells. CENPF overexpression promoted the production of CENPF autoantibodies in a manner that correlated with tumor growth of mouse HCC model. During apoptosis of HCC cells, CENPF protein translocated to apoptotic vesicles and relocalized at the cell surface. Through isolating apoptotic components, we found apoptotic body and blebs with lower CD31 and CD47 expression more effectively induced DC phagocytosis and maturation compared with apoptotic intact cells in vitro, and this DC response was independent of CENPF expression. Moreover, injection of mice with apoptotic bodies and blebs effectively induced an immune response and the production of CENPF-specific antibodies. Our findings provide a first elucidation of mechanisms underlying the CENPF autoantibody production via cell apoptosis-induced CENPF translocation, and demonstrate a direct correlation between CENPF autoantibody levels and HCC progression, suggesting the potential of CENPF autoantibody as an HCC diagnostic marker.

Background: Colorectal cancer (CRC) comprises a large proportion of malignant tumors, and early detection of CRC is critical for effective treatment and optimal prognosis. We aimed to discover and validate serum autoantibodies for early detection of CRC.Methods: Combined with CRC-associated autoantibodies discovered by serological proteome and multiplex analyses, 26 predefined autoantibodies were evaluated in 315 samples (130 CRCs, 75 advanced adenomas, and 110 healthy controls) by protein microarray analysis. Autoantibodies with potential detection value were verified by enzyme-linked immunosorbent assays (ELISAs). Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the accuracy of the biomarkers.Results: Four serum autoantibodies (ALDH1B1, UQCRC1, CTAG1, and CENPF) showed statistically different levels between patients with advanced neoplasm (CRC or advanced adenoma) and controls in microarray analysis, which were validated by ELISAs. Among the four biomarkers, the ALDH1B1 autoantibody showed the highest detection value with area under the curve (AUC) values of 0.70 and 0.74 to detect CRC and advanced adenoma with sensitivities of 75.68 and 62.31% and specificities of 63.06 and 73.87%, respectively. By combining the four biomarkers, the performance was improved with an AUC of 0.79 to detect CRC and advanced adenomas.Conclusion: The ALDH1B1 autoantibody has a good potential for early detection of CRC and advanced adenoma, and measuring serum autoantibodies against tumor-associated antigens may improve detection of early CRC.

The actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM. Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell

Rotor syndrome, a rare autosomal-recessive genetic disorder characterized by conjugated hyperbilirubinemia, is caused by biallelic pathogenic variants in both SLCO1B1 and SLCO1B3 genes. Long interspersed nuclear elements (LINEs) make up about 17% of the human genome and insertion of LINE-1 in genes can result in genetic diseases. In the current study, we examined SLCO1B1 and SLCO1B3 genes in two Chinese patients diagnosed with Rotor syndrome based on laboratory tests. In one patient, a novel exon 4 inversion variant was identified. This variant may have been induced by LINE-1 retrotransposon insertion into SLCO1B3 intron 3, and was identified using genome walking. Splicing assay results indicated that the exon inversion, resulting in SLCO1B3 exon 4 (122 bp) exclusion in the mature mRNA, might generate a premature termination codon. Here, we describe an exon inversion contributing to the molecular etiology of Rotor syndrome. Our results may inform future diagnoses and guide drug prescriptions and genetic counseling.

Objectives: The present study was aimed to investigate the antibiotic resistance, virulence potential and phylogenetic grouping of ESBL-producing uropathogenic Escherichia coli (EP-UPEC) isolated from long-term hospitalized patients. Materials and Methods: EP-UPEC isolates from September 2013 to June 2014 at a tertiary care hospital of China were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic resistance testing, PCR of different ESBL and virulence genes, and phylogenetic grouping. Results: One hundred and twenty EP-UPEC were isolated from long-term hospitalized patients. All EP-UPEC isolates were resistant to Ampicillin, Cefazolin, Cefuroxime, Cefotaxime, Cefoperazone and Ceftriaxone, and the majority of EP-UPEC isolates were resistant to Piperacillin (82.5%), Ciprofloxacin (81.2%), TrimethoprimSulfamethoxazole (72.5%). The isolates showed the highest sensitivity against Imipenem (98.4%), Piperacillin/tazobactam (96.7%), Cefoperazone/sulbactam (91.7%), Amikacin (90.8%) and Cefepime (75.8%). Nine different ESBL genotype patterns were observed and CTX-M type was the most prevalent ESBL genotype (42.5%, 51/120). Majority of EP-UPEC isolates possess more than one ESBL genes. EP-UPEC isolates belonged mainly to phylogenetic group B2(36.7%) and D(35.0%). The prevalence of traT, ompT, iss, PAI, afa, fimH and papC were 75.8%, 63.3%, 63.3%, 60.8%, 40.8%, 19.2% and 6.7%, respectively. The number of virulence genes (VGs) detected was significantly higher in group B2 than in group A (ANOVA, p

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that causes a variety of infections. MRSA has evolved resistance to multiple antibiotics. Genetic background and virulence differs in different geographic regions. The present study was aimed to investigate the prevalence of enterotoxin genes and spa genotypes of hospitalassociated methicillin-resistant Staphylococcus aureus (HAMRSA) isolated from a tertiary care hospital of Jiangsu province, China. Materials and Methods: HA-MRSA isolates from August 2013 to April 2014 at a tertiary care hospital of China were collected. We investigated antimicrobial pattern, spa types, SCCmec types and the presence of 14 virulence genes. Results: Eighty HA-MRSA isolates were collected. Results from SCCmec typing revealed that 73.8% were type II; 13.8% were type III; 12.5% were type V. There were 19 different spa types. Spa type t2460 was the most common (35.0%), followed by t002 (11.3%). CC5 was the predominant MLST CCs type (50%). The most frequent toxin genes were sea, seb, sed, sel, sen and seo (100.0%). None of the investigated isolates carried the sec or tst. Conclusion: Genotypic and virulence evaluation of the isolated HA-MRSA revealed that the isolates with CC5 and SCCmec II were the predominant type and highly homological. The virulence profiles mainly existed in the genes of sea, seb, sed, sel, sen, seo and ser. The prevalence of t2460 was an outbreak and the predominant spa type.

The purpose of this paper is to provide a new proof of real interpolations for martingale Hardy-Orlicz-Lorentz spaces. Different from the usual proof, our approach does not rely on any atomic decompositions. Instead, we establish the result via new Holmstedt's formulae in martingale Hardy-Orlicz type spaces. [ABSTRACT FROM AUTHOR]