Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the Transwell^TM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
Humans ; Cyclin D1/genetics* ; HEK293 Cells ; Interferon-gamma ; RNA, Small Interfering ; Apoptosis/genetics* ; Cadherins ; Cell Proliferation/genetics* ; Glioma/genetics* ; Proto-Oncogene Proteins c-bcl-2 ; Oxidoreductases Acting on Sulfur Group Donors ; STAT1 Transcription Factor/genetics*

OBJECTIVE: To explore the clinical features and genetic basis for a child with early-onset Isolated sulfite oxidase deficiency (ISOD). METHODS: A child with ISOD who was admitted to Weihai Hospital Affiliated to Qingdao University on May 10, 2020 was selected as the study subject. Clinical data of the child was analyzed. The child and her parents were subjected to trio-whole exome sequencing, and candidate variants were verified by Sanger sequencing. RESULTS: The female neonate was transferred to the intensive care unit due to "secondary pollution of amniotic fluid and laborious breathing for 11 minutes", and had developed frequent convulsions. Genetic testing revealed that she has harbored c.1200C>G and c.188G>A compound heterozygous variants of the SUOX gene, which were inherited from her mother and father, respectively. The c.1200C>G has been described previously and was rated as pathogenic based on guidelines from the American College of Medical Genetics and Genomics, whilst the c.188G>A variant was unreported previously and rated as variant of unknown significance. CONCLUSION The compound heterozygous variants of the SUOX gene probably underlay the ISOD in this child. Above finding has enriched the spectrum of SUOX gene variants and provided a basis for the clinical diagnosis and genetic counseling.
Female ; Humans ; Infant, Newborn ; Amino Acid Metabolism, Inborn Errors/diagnosis* ; Genetic Counseling ; Genetic Testing ; Mutation ; Oxidoreductases Acting on Sulfur Group Donors/genetics* ; Sulfite Oxidase/genetics*

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.
Catalysis ; Drug Development ; Oxidation-Reduction ; Thioredoxin Reductase 1/metabolism* ; Vitamin K 3/metabolism*

OBJECTIVE: To analyze the clinical and genetic characteristics of a patient with dihydrolipoamide dehydrogenase deficiency. METHODS: Potential variants of the DLD gene were detected by whole exome sequencing and verified by Sanger sequencing. RESULTS: Compound heterozygous variants, c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr), were detected in the DLD gene. The c.1058T>C (p.Ile353Thr) variant was derived from his mother and known to be pathogenic. The c.704_705delTT (p.Leu235Argfs*8) variant was derived from his father and was unreported previously. CONCLUSION The compound heterozygous variants of c.704_705delTT (p.Leu235Argfs*8) and c.1058T>C (p.Ile353Thr) of the DLD gene probably underlay the disease in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for the family.
Acidosis, Lactic/genetics* ; Dihydrolipoamide Dehydrogenase/genetics* ; Female ; Genetic Testing ; Genetic Variation ; Humans ; Male ; Maple Syrup Urine Disease/genetics* ; Pregnancy ; Whole Exome Sequencing

OBJECTIVE: To detect potential variation in an ethnic Han Chinese family affected with late-onset lipid storage myopathy. METHODS: Next generation sequencing (NGS) was used to screen disease-related genes in the proband. Suspected mutation was validated with PCR and Sanger sequencing in two patients, their father, and 100 healthy controls. RESULTS: Heterozygous c.770A>G (p.Tyr257Cys) and c.1395dupT (p.Gly466Tryfs) mutation were detected in the two patients. Their father was found to be heterozygous for the c.770A>G (p.Tyr257Cys) mutation, while the c.1395dupT (p.Gly466Tryfs) variation was not reported previously and not found among the healthy controls. CONCLUSION Mutations of the ETFDH gene probably underlie the pathogenesis in this family. The novel c.1395dupT (p.Gly466Tryfs) has enriched the mutation spectrum of EDFDH gene.
Asian Continental Ancestry Group ; Electron-Transferring Flavoproteins ; genetics ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Iron-Sulfur Proteins ; genetics ; Lipid Metabolism, Inborn Errors ; genetics ; Muscular Dystrophies ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics

BACKGROUND: Late-onset multiple acyl-coA dehydrogenase deficiency (MADD) is an autosomal recessive inherited metabolic disorder. It is still unclear about the muscle magnetic resonance image (MRI) pattern of the distal lower limb pre- and post-treatment in patients with late-onset MADD. This study described the clinical and genetic findings in a cohort of patients with late-onset MADD, and aimed to characterize the MRI pattern of the lower limbs. METHODS: Clinical data were retrospectively collected from clinic centers of Peking University People's Hospital between February 2014 and February 2018. Muscle biopsy, blood acylcarnitines, and urine organic acids profiles, and genetic analysis were conducted to establish the diagnosis of MADD in 25 patients. Muscle MRI of the thigh and leg were performed in all patients before treatment. Eight patients received MRI re-examinations after treatment. RESULTS: All patients presented with muscle weakness or exercise intolerance associated with variants in the electron transfer flavoprotein dehydrogenase gene. Muscle MRI showed a sign of both edema-like change and fat infiltration selectively involving in the soleus (SO) but sparing of the gastrocnemius (GA) in the leg. Similar sign of selective involvement of the biceps femoris longus (BFL) but sparing of the semitendinosus (ST) was observed in the thigh. The sensitivity and specificity of the combination of either "SO+/GA-" sign or "BFL+/ST-" sign for the diagnosis of late-onset MADD were 80.0% and 83.5%, respectively. Logistic regression model supported the findings. The edema-like change in the SO and BFL muscles were quickly recovered at 1 month after treatment, and the clinical symptom was also relieved. CONCLUSIONS This study expands the clinical and genetic spectrums of late-onset MADD. Muscle MRI shows a distinct pattern in the lower limb of patients with late-onset MADD. The dynamic change of edema-like change in the affected muscles might be a potential biomarker of treatment response.
Adolescent ; Adult ; Biopsy ; methods ; Carnitine ; analogs & derivatives ; blood ; Electron-Transferring Flavoproteins ; genetics ; Female ; Hamstring Muscles ; diagnostic imaging ; metabolism ; pathology ; Humans ; Iron-Sulfur Proteins ; genetics ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; diagnostic imaging ; genetics ; pathology ; Muscle, Skeletal ; diagnostic imaging ; metabolism ; pathology ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; Retrospective Studies ; Young Adult

This article reports the results of tandem mass spectrometry and the mutation features of the ETFDH gene for an infant with multiple acyl-CoA dehydrogenase deficiency. The results of tandem mass spectrometry showed that C14 : 1, C8, C6, C10, and C12 increased. Exon sequencing was performed on this infant and his parents and revealed double heterozygous mutations in the ETFDH gene of the infant: c.992A>T and c.1450T>C. The former was inherited from his mother, and the latter was inherited from his father. c.1450T>C was shown to be the pathogenic mutation in the HGMD database. PolyPhen2, SIFT, and PROVEAN all predicted that the novel mutation c.992A>T might be pathogenic, and the mutant amino acids were highly conserved across various species. The findings expand the mutation spectrum of the ETFDH gene, and provide molecular evidence for the etiological diagnosis of the patient with multiple acyl-CoA dehydrogenase deficiency as well as for the genetic counseling and prenatal diagnosis in the family.
Base Sequence ; Electron-Transferring Flavoproteins ; genetics ; Exons ; Humans ; Infant, Newborn ; Iron-Sulfur Proteins ; genetics ; Male ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; enzymology ; genetics ; Mutation ; Oxidoreductases Acting on CH-NH Group Donors ; genetics

The thioredoxin system plays a role in a variety of physiological functions, including cell growth, differentiation, apoptosis, tumorigenesis, and immunity. We previously confirmed that butaselen (BS), a novel thioredoxin reductase inhibitor, can inhibit the growth of various human cancer cell lines, yet the underlying mechanism remains elusive. In this study, we investigated the anti-tumor effect of BS in vivo through regulating the immune system of KM mice. We found that BS inhibits tumor proliferation by promoting the activation of splenic lymphocytes in mice. BS can elevate the percentage of CD₄^-CD₈⁺ T lymphocytes and the secretion of downstream cytokines in mice via down-regulating the expression of programmed death-ligand 1 (PD-L1) on the tumor cells' surface in vivo. Further study in HepG2 and BEL-7402 cells showed that decrease of PD-L1 level after BS treatment was achieved by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation. Taken together, our results suggest that BS has a role in promoting the immune response by reducing PD-L1 expression via the STAT3 pathway, and subsequently suppresses tumorigenesis.
Animals ; Antineoplastic Agents/pharmacology* ; B7-H1 Antigen/antagonists & inhibitors* ; Benzene Derivatives/therapeutic use* ; CD8-Positive T-Lymphocytes/drug effects* ; Hep G2 Cells ; Humans ; Liver Neoplasms/pathology* ; Male ; Mice ; Organoselenium Compounds/therapeutic use* ; STAT3 Transcription Factor/physiology* ; Thioredoxin-Disulfide Reductase/antagonists & inhibitors* ; Tumor Burden/drug effects*

The present study evaluated the effects of Androctonus amoreuxi scorpion venom, Cerastes cerastes snake venom and their mixture on prostate cancer cells (PC3). An MTT assay was used to determine the anti-proliferative effect of the venoms, while quantitative real time PCR was used to evaluate the expression of apoptosis-related genes (Bax and Bcl-2). Furthermore, colorimetric assays were used to measure the levels of malondialdehyde (MDA) and antioxidant enzymes. Our results show that the venoms significantly reduced PC3 cell viability in a dose-dependent manner. On the other hand, these venoms significantly decreased Bcl-2 gene expression. Additionally, C. cerastes venom significantly reduced Bax gene expression, while A. amoreuxi venom and a mixture of A. amoreuxi & C. cerastes venoms did not alter Bax expression. Consequently, these venoms significantly increased the Bax/Bcl-2 ratio and the oxidative stress biomarker MDA. Furthermore, these venoms also increased the activity levels of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. Overall, the venoms have cytotoxic and anti-proliferative effects on PC3 cells.
Apoptosis ; Catalase ; Cell Survival ; Gene Expression ; Genes, bcl-2 ; Glutathione Peroxidase ; Glutathione Reductase ; Hand ; Humans* ; Malondialdehyde ; Oxidative Stress ; Prostate* ; Prostatic Neoplasms* ; Real-Time Polymerase Chain Reaction ; Scorpion Venoms ; Scorpions* ; Snake Venoms* ; Snakes* ; Superoxide Dismutase ; Venoms ; Viper Venoms ; Viperidae*

BACKGROUN/OBJECTIVES: Although studies have revealed that black garlic is a potent antioxidant, its antioxidant mechanism remains unclear. The objective of this study was to determine black garlic's antioxidant activities and possible antioxidant mechanisms related to nuclear factor erythroid 2-like factor 2 (Nrf2)-Keap1 complex. METHODS/MATERIALS: After four weeks of feeding rats with a normal fat diet (NF), a high-fat diet (HF), a high-fat diet with 0.5% black garlic extract (HF+BGE 0.5), a high-fat diet with 1.0% black garlic extract (HF+BGE 1.0), or a high-fat diet with 1.5% black garlic extract (HF+BGE 1.5), plasma concentrations of glucose, insulin,homeostatic model assessment of insulin resistance (HOMA-IR) were determined. As oxidative stress indices, plasma concentrations of thiobarbituric acid reactive substances (TBARS) and 8-isoprostaglandin F2α (8-iso-PGF) were determined. To measure antioxidant capacities, plasma total antioxidant capacity (TAC) and activities of antioxidant enzymes in plasma and liver were determined. The mRNA expression levels of antioxidant related proteins such as Nrf2, NAD(P)H: quinone-oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutathione reductase (GR), and glutathione S-transferase alpha 2 (GSTA2) were examined. RESULTS: Plasma glucose level, plasma insulin level, and HOMA-IR in black garlic supplemented groups were significantly (P < 0.05) lower than those in the HF group without dose-dependent effect. Plasma TBARS concentration and TAC in the HF+BGE 1.5 group were significantly decreased compared to those of the HF group. The activities of catalase and glutathione peroxidase were significantly (P < 0.05) increased in the HF+BGE 1.0 and HF+BGE 1.5 groups compared to those of the HF group. The mRNA expression levels of hepatic Nrf2, NQO1, HO-1, and GSTA2 were significantly (P < 0.05) increased in the HF with BGE groups compared to those in the HF group. CONCLUSIONS: The improvements of blood glucose homeostasis and antioxidant systems in rats fed with black garlic extract were related to mRNA expression levels of Nrf2 related genes.
Animals ; Blood Glucose ; Catalase ; Diet ; Diet, High-Fat ; Garlic* ; Glucose ; Glutathione Peroxidase ; Glutathione Reductase ; Glutathione Transferase ; Heme Oxygenase-1 ; Homeostasis ; Insulin ; Insulin Resistance ; Liver ; Oxidative Stress ; Plasma ; Rats ; RNA, Messenger ; Thiobarbituric Acid Reactive Substances