BACKGROUND: Pancreatic cancer is a lethal malignancy prone to gemcitabine resistance. The single-strand selective monofunctional uracil DNA glycosylase (SMUG1), which is responsible for initiating base excision repair, has been reported to predict the outcomes of different cancer types. However, the function of SMUG1 in pancreatic cancer is still unclear. METHODS: Gene and protein expression of SMUG1 as well as survival outcomes were assessed by bioinformatic analysis and verified in a cohort from Fudan University Shanghai Cancer Center. Subsequently, the effect of SMUG1 on proliferation, cell cycle, and migration abilities of SMUG1 cells were detected in vitro . DNA damage repair, apoptosis, and gemcitabine resistance were also tested. RNA sequencing was performed to determine the differentially expressed genes and signaling pathways, followed by quantitative real-time polymerase chain reaction and Western blotting verification. The cancer-promoting effect of forkhead box Q1 (FOXQ1) and SMUG1 on the ubiquitylation of myelocytomatosis oncogene (c-Myc) was also evaluated. Finally, a xenograft model was established to verify the results. RESULTS: SMUG1 was highly expressed in pancreatic tumor tissues and cells, which also predicted a poor prognosis. Downregulation of SMUG1 inhibited the proliferation, G1 to S transition, migration, and DNA damage repair ability against gemcitabine in pancreatic cancer cells. SMUG1 exerted its function by binding with FOXQ1 to activate the Protein Kinase B (AKT)/p21 and p27 pathway. Moreover, SMUG1 also stabilized the c-Myc protein via AKT signaling in pancreatic cancer cells. CONCLUSIONS SMUG1 promotes proliferation, migration, gemcitabine resistance, and c-Myc protein stability in pancreatic cancer via protein kinase B signaling through binding with FOXQ1. Furthermore, SMUG1 may be a new potential prognostic and gemcitabine resistance predictor in pancreatic ductal adenocarcinoma.
Humans ; Pancreatic Neoplasms/pathology* ; Forkhead Transcription Factors/genetics* ; Signal Transduction/genetics* ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Proliferation/physiology* ; Mice ; Uracil-DNA Glycosidase/genetics* ; Female ; Male ; Gemcitabine ; Mice, Nude ; Apoptosis/physiology* ; Deoxycytidine/analogs & derivatives* ; Cell Movement/genetics*

OBJECTIVE: To investigate the relationship between Ig class switch recombination (CSR) and mismatch repair (MMR) protein, microsatellite phenotype in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). METHODS: Forty cases of MALT lymphoma archived in the Department of Pathology, Jiading District Central Hospital, Shanghai University of Medicine & Health Sciences were selected as the observation group, and twenty cases of benign lymphoid tissue hyperplasia were as the control group. The expressions of IgG, IgM, IgD, and IgA in both groups were detected by immunohistochemical double staining, and MMR proteins including MLH1, MSH2, MSH6, and PMS2 in both groups were detected by immunohistochemistry. Multiplex fluorescence PCR capillary electrophoresis was used to detect microsatellite phenotype in tumor and adjacent tissues of the experimental group. RESULTS: In the observation group, the proportions of single Ig heavy chain expression (modeⅠ), negative expression (modeⅡ), and multiple expression (mode Ⅲ) were 65% (26/40), 27.5% (11/40), and 7.5% (3/40), respectively, while in the control group were 0 (0/20), 5% (1/20), and 95% (19/20). The proportion of Ig heavy chain expression mode Ⅰ+Ⅱ in the observation group was 92.5%, which was significantly higher than 5% in the control group (P < 0.01). In the observation group, partial deletion of MMR protein was observed in 3 cases (7.5%), including 2 cases of MSH6 deletion and 1 case of both MSH6 and PMS2 deletion. In the control group, there was 1 case (5%) with PMS2 deletion. There was no significant difference in the deletion rate of MMR protein between the two groups ( P >0.05). A total of 5 cases of microsatellite instability (MSI) were detected in the observation group, including 1 case of low-frequency MSI (MSI-L), 4 cases of high-frequency MSI (MSI-H), and 2 cases of MSI-H with MSH6 deletion. When the loss expression of MSI-H or MMR protein was counted as a positive result, the MSI-H rate detected by PCR capillary electrophoresis was 10% (4/40), which was slightly higher than the MMR protein deletion rate detected by immunohistochemistry (7.5%, 3/40), but there was no statistically significant difference between the two groups (P >0.05). The MMR protein deletion rates among the Ig heavy chain protein expression mode Ⅰ, mode Ⅱ, and mode Ⅲ groups were 0 (0/26), 18.2% (2/11), and 33.3% (1/3), respectively. There was a statistically significant difference in the constituent ratios among the three groups (P < 0.05). The MMR protein deletion rates among the MSS, MSI-L, and MSI-H groups were 2.9% (1/35), 0 (0/1), and 50% (2/4), respectively. There was a statistically significant difference in the constituent ratios among the three groups (P < 0.05). MMR protein deficiency was positively correlated with Ig heavy chain expression pattern and MSI ( r =0.41, P < 0.05; r =0.48, P < 0.05), but Ig heavy chain expression pattern was not correlated with MSI ( r =0.02, P >0.05). CONCLUSION Ig heavy chain CSR detection is helpful for the differential diagnosis of MALT lymphoma. Low frequency MMR protein deletion and MSI-H phenotype exist in MALT lymphoma, which may be of certain value for the study of its occurrence, development and clinical treatment.
Humans ; Lymphoma, B-Cell, Marginal Zone/genetics* ; DNA Mismatch Repair ; Immunoglobulin Class Switching ; DNA-Binding Proteins/metabolism* ; MutS Homolog 2 Protein ; Microsatellite Repeats ; Phenotype ; MutL Protein Homolog 1 ; Mismatch Repair Endonuclease PMS2 ; Male

Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1-104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*

OBJECTIVES: To investigate the effect of nuclear protein (Nupr) 1 on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes. METHODS: Chondrocytes from mouse knees were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of acyl-CoA synthetase long-chain family (ACSL) 4, P53, glutathione peroxidase (GPX) 4 and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into four groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA control targeting Nupr1 (shNupr1) group, DMM+AAV5-shRNA control group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of matrix metallopeptidase (MMP) 13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5, cyclooxy-genase (COX) 2, and GPX4 were detected by qRT-PCR. RESULTS: In vitro experiments showed that knocking down Nupr1 alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered lipid peroxidation, downregulated ACSL4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all P<0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, in vivo animal experiments demonstrated that knocking down Nupr1 delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all P<0.01). CONCLUSIONS Knockdown of Nupr1 can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.
Animals ; Ferroptosis ; Mice ; Chondrocytes/metabolism* ; Osteoarthritis/pathology* ; RNA, Small Interfering/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Interleukin-1beta/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Coenzyme A Ligases/genetics* ; Tumor Suppressor Protein p53/metabolism* ; Mice, Inbred C57BL ; DNA-Binding Proteins ; Neoplasm Proteins ; Amino Acid Transport System y+ ; Nuclear Receptor Subfamily 1, Group D, Member 1

Objective To explore the role of the base mismatch repair gene Mutyh in retinopathy of prematurity(ROP). Methods Mutyh(-/-)and wild-type(WT)mice were used for the modeling of oxygen-induced retinopathy.The retinal oxidative stress was examined,and the ultrastructures of photoreceptors and mitochondria were observed.The biomarkers of photoreceptors and mitochondria were tested.Furthermore,the photoreceptor cell line 661W was treated with hydrogen peroxide for the modeling of oxidative stress.In the cell model,and the oxidative stress and photoreceptor functions in the cells were measured. Results In both the mouse and cell models,the expression of Mutyh was up-regulated.Mutyh knockout in mice and knockdown in cells exerted negative effects on photoreceptors and mitochondria.Mutyh overexpression showed protective functions in the cell model,indicating that Mutyh played a role in repairing photoreceptors and mitochondria. Conclusions Mutyh showed the potential to become a biomarker of ROP.Increasing Mutyh expression might have therapeutic effects on ROP,which needs further validation.
Oxidative Stress ; Animals ; Mice ; DNA Glycosylases/metabolism* ; Retinopathy of Prematurity/etiology* ; Mice, Knockout ; Disease Models, Animal ; Humans ; Mitochondria/metabolism*

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.
Vibrio parahaemolyticus/genetics* ; Uracil-DNA Glycosidase/genetics* ; Hot Temperature ; CRISPR-Cas Systems ; Food Safety

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26^+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.
Animals ; Mice ; DNA-Binding Proteins/genetics* ; Cell Differentiation ; Neural Stem Cells ; Translocation, Genetic ; Ubiquitins/genetics* ; Ligases/genetics*

OBJECTIVE: To explore the role of a new blood-based, multiomics and multidimensional method for evaluating the efficacy of patients with lymphoma. METHODS: 10 ml peripheral blood was extracted from each patient, and the genomic copy number aberrations (CNA) and fragment size (FS) were evaluated by low-depth whole genome sequencing of cfDNA, and the level of a group of plasma tumor marker (PTM) were detected at the same time. The cancer efficacy score (CES) was obtained by standardized transformation of the value of above three numerical indexes, and the changes of CES before and after treatment were compared to evaluate the patient's response to the treatment regimen. RESULTS: A total of 35 patients' baseline data were collected, of which 23 cases (65.7%) had elevated CES values. 18 patients underwent the first time test. The results showed that the CES value of 9 patients with positive baseline CES decreased significantly at the first test, and the efficacy evaluation was PR, which was highly consistent with the imaging evaluation results of the same period. At the same time, the CNA variation spectrum of all patients were evaluated and it was found that 23 patients had partial amplification or deletion of chromosome fragments. The most common amplification site was 8q24.21, which contains important oncogenes such as MYC. The most common deletion sites were 1p36.32, 4q21.23, 6q21, 6q27, 14q32.33, and tumor suppressor-related genes such as PRDM1, ATG5, AIM1, FOXO3 and HACE1 were expressed in the above regions, so these deletions may be related to the occurrence and development of lymphoma. CONCLUSION With the advantages of more convenience, sensitivity and non-invasive, this multiomics and multidimensional efficacy detection method can evaluate the tumor load of patients with lymphoma at the molecular level, and make more accurate efficacy evaluation, which is expected to serve the clinic better.
Humans ; Multiomics ; Lymphoma/genetics* ; Cell-Free Nucleic Acids ; Genomics/methods* ; DNA Copy Number Variations ; Ubiquitin-Protein Ligases

OBJECTIVE: To analyze the distribution characteristics of hereditary peripheral neuropathy (HPN) pathogenic genes in Chinese Han population, and to explore the potential pathogenesis and treatment prospects of HPN and related diseases. METHODS: Six hundred and fifty-six index patients with HPN were enrolled in Peking University Third Hospital and China-Japan Friendship Hospital from January 2007 to May 2022. The PMP22 duplication and deletion mutations were screened and validated by multiplex ligation probe amplification technique. The next-generation sequencing gene panel or whole exome sequencing was used, and the suspected genes were validated by Sanger sequencing. RESULTS: Charcot-Marie-Tooth (CMT) accounted for 74.3% (495/666) of the patients with HPN, of whom 69.1% (342/495) were genetically confirmed. The most common genes of CMT were PMP22 duplication, MFN2 and GJB1 mutations, which accounted for 71.3% (244/342) of the patients with genetically confirmed CMT. Hereditary motor neuropathy (HMN) accounted for 16.1% (107/666) of HPN, and 43% (46/107) of HPN was genetically confirmed. The most common genes of HMN were HSPB1, aminoacyl tRNA synthetases and SORD mutations, which accounted for 56.5% (26/46) of the patients with genetically confirmed HMN. Most genes associated with HMN could cause different phenotypes. HMN and CMT shared many genes (e.g. HSPB1, GARS, IGHMBP2). Some genes associated with dHMN-plus shared genes associated with amyotrophic lateral sclerosis (KIF5A, FIG4, DCTN1, SETX, VRK1), hereditary spastic paraplegia (KIF5A, ZFYVE26, BSCL2) and spinal muscular atrophy (MORC2, IGHMBP, DNAJB2), suggesting that HMN was a continuum rather than a distinct entity. Hereditary sensor and autosomal neuropathy (HSAN) accounted for a small proportion of 2.6% (17/666) in HPN. The most common pathogenic gene was SPTLC1 mutation. TTR was the main gene causing hereditary amyloid peripheral neuropathy. The most common types of gene mutations were p.A117S and p.V50M. The symptoms were characterized by late-onset and prominent autonomic nerve involvement. CONCLUSION CMT and HMN are the most common diseases of HPN. There is a large overlap between HMN and motor-CMT2 pathogenic genes, and some HMN pathogenic genes overlap with amyotrophic lateral sclerosis, hereditary spastic hemiplegia and spinal muscular atrophy, suggesting that there may be a potential common pathogenic pathway between different diseases.
Amyotrophic Lateral Sclerosis ; Charcot-Marie-Tooth Disease/genetics* ; DNA Helicases/genetics* ; DNA-Binding Proteins/genetics* ; Flavoproteins ; HSP40 Heat-Shock Proteins ; Humans ; Intracellular Signaling Peptides and Proteins/genetics* ; Kinesins ; Ligases/genetics* ; Molecular Chaperones ; Multifunctional Enzymes ; Muscular Atrophy, Spinal/genetics* ; Mutation ; Phosphoric Monoester Hydrolases ; Protein Serine-Threonine Kinases ; RNA Helicases/genetics* ; RNA, Transfer ; Transcription Factors/genetics*

Objective: To investigate the relationship between the expression of four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) and NTRK genetic fusions in colorectal cancer. Methods: The paraffin-embedded tissue blocks of 830 cases of colorectal cancer were collected at the Affiliated Drum Tower Hospital, Nanjing University Medical School, China, from 2015 to 2019. Immunohistochemical and fluorescence in situ hybridization(FISH) method were used respectively to detect the expression of mismatch repair proteins and the break-apart of NTRKs; and the relationship between the expression of mismatch repair proteins and the NTRK genetic fusions was analyzed. Results: The overall mismatch repair protein deficiency (dMMR) rate was 9.88% (82/830), the mismatch repair proteins proficiency (pMMR) rate was 90.12%(748/830). The total deficiency rate of MLH1 protein was 9.04% (75/830), hPMS2 protein deficiency rate was 9.04% (75/830), MSH2 protein deficiency rate was 2.53% (21/830), MSH6 protein deficiency rate was 4.10% (34/830), the deficiency rate of synchronous MLH1 and PMS2 were 8.67% (72/830) and the deficiency rate of synchronous MSH2 and MSH6 were 2.17% (18/830). The dMMR group was associated with tumor location, different histological subgroups, tumor differentiation, AJCC stage and N stage (P<0.05). There were six cases (7.32%) carrying NTRK fusion by FISH among the 82 cases of dMMR, but only seven cases (0.94%) carrying NTRK fusion among the 748 cases of PMMR. The NTRKs translocation by FISH in all 13 cases were further confirmed by next generation sequencing. Among the clinicopathological characteristics, only differentiation showed significant difference between NTRK fusion positive and negative groups (P<0.05). More importantly, NTRK fusion was enriched in dMMR group (7.32% vs. 0.94%). Conclusion: In dMMR colorectal cancer group, the prevalence of NTRK fusion is higher than that in pMMR group.
Colonic Neoplasms ; Colorectal Neoplasms/genetics* ; DNA Mismatch Repair/genetics* ; Humans ; In Situ Hybridization, Fluorescence ; Mismatch Repair Endonuclease PMS2/metabolism* ; MutL Protein Homolog 1/metabolism* ; MutS Homolog 2 Protein/metabolism*