👤 Xinyi He

Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation. Show less

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene. A second copy, SMN2, is similar to SMN1 but produces ∼10% SMN protein because of a single-point mutation that causes splicing defects. Chronic low levels of SMN cause accumulation of co-transcriptional R-loops and DNA damage leading to genomic instability and neurodegeneration in SMA. Severity of SMA disease correlates inversely with SMN levels. SMN2 is a promising target to produce higher levels of SMN by enhancing its expression. Mechanisms that regulate expression of SMN genes are largely unknown. We report that zinc finger protein ZPR1 binds to RNA polymerase II, interacts in vivo with SMN locus and upregulates SMN2 expression in SMA mice and patient cells. Modulation of ZPR1 levels directly correlates and influences SMN2 expression levels in SMA patient cells. ZPR1 overexpression in vivo results in a systemic increase of SMN levels and rescues severe to moderate disease in SMA mice. ZPR1-dependent rescue improves growth and motor function and increases the lifespan of male and female SMA mice. ZPR1 reduces neurodegeneration in SMA mice and prevents degeneration of cultured primary spinal cord neurons derived from SMA mice. Further, we show that the low levels of ZPR1 associated with SMA pathogenesis cause accumulation of co-transcriptional RNA-DNA hybrids (R-loops) and DNA damage leading to genomic instability in SMA mice and patient cells. Complementation with ZPR1 elevates senataxin levels, reduces R-loop accumulation and rescues DNA damage in SMA mice, motor neurons and patient cells. In conclusion, ZPR1 is critical for preventing accumulation of co-transcriptional R-loops and DNA damage to avert genomic instability and neurodegeneration in SMA. ZPR1 enhances SMN2 expression and leads to SMN-dependent rescue of SMA. ZPR1 represents a protective modifier and a therapeutic target for developing a new method for the treatment of SMA. Show less

Agmatine, an endogenous biogenic amine, is considered to be a central neurotransmitter. And it plays an important role in mammal feeding behavior. However, there were few studies on the effect of agmatine on feeding behavior in fishes. Here, we investigated the impact of intracerebroventricular (ICV) injections of agmatine (1.25-20 nmol/fish) on food intake in mandarin fish (Siniperca chuatsi). At 1-h post-injection, food intake showed a significant decrease in agmatine-treated fishes compared with the saline treated. Furthermore, the food intake in agmatine treatment mostly did not differ from that in saline treatment at 4--24-h post-injection as well as the results of genes expression of neuropeptide Y (NPY), agouti-regulated peptide (AgRP), and anorexigenic melanocortin 4 receptor (MC4R). In accordance with the insulin level increasing in liver, the gene expression of insulin receptor substrate (IRS2) was significantly higher in agmatine treatment compared to saline treatment at 1-h post-injection. Thus, the anorexigenic effect of agmatine is likely to decrease NPY and AgRP expression levels and increase MC4R and IRS2 levels which was coupled with stimulation of insulin secretion. Although these initial findings are limited in dose, the data firstly provides evidence for the anorectic effects of agmatine in fish. Show less

Obesity is one of the major health problems strongly influenced by lifestyle, genetic and environmental factors. Previous studies have reported many single-nucleotide polymorphisms (SNPs) are associated with obesity in different races. This study aimed to explore the genetic associations between LEPR, MC4R polymorphisms and overweight/obesity in Chinese Han adolescents. 400 adolescents including 222 health controls and 178 overweight/obese adolescents were genotyped and their body compositions were also analyzed in this study. We found that allelic and genotypic frequencies of LEPR SNP rs8179183 were significantly different between controls and cases (allelic frequency p < 0.001; genotypic frequency p = 0.004). These difference was still significant (allelic frequency p < 0.011; genotypic frequency p = 0.024) after Bonferroni correction. Moreover, we found that rs8179183 was associated with serum triglyceride level after adjusting for age and body mass index (BMI) (p = 0.037). In summary, our results found a significant association between LEPR SNP rs8179183 and overweight/obesity in Chinese Han adolescent. This study may provide a reference for future studies of obesity. Show less

Many achievements have been made to develop quantitative trait loci (QTLs) and gene-associated single nucleotide polymorphisms (SNPs) to facilitate practical marker-assisted selection (MAS) in aquatic animals. However, the systematic studies of SNPs associated with extreme threshold traits were poor in populations lacking of parental genomic information. Coupling next generation sequencing with bulked segregant analysis (BSA) should allow identification of numerous associated SNPs with extreme phenotypes. In the present study, using combination of SNP frequency difference and Euclidean distance, we conducted linkage analysis of SNPs located in genes involved in immune responses, and identified markers associated with Vibrio anguillarum resistance in turbot (Scophthalmus maximus). A total of 221 SNPs was found as candidate SNPs between resistant and susceptible individuals. Among these SNPs, 35 loci located in immune related genes were genotyped in verification population and 7 of them showed significant association with V. anguillarum resistance in both alleles and genotypes (P < 0.05). Among these 7 genes, PIK3CA-like, CYLD, VCAM1, RhoB and RhoGEF are involved in PI3K/Akt/mTOR pathway and NF-κB pathway, which influence the efficiency of bacteria entering the host and inflammation. SNP-SNP interaction analysis was performed by generalized multifactor dimensionality reduction (GMDR). The combination of SNP loci in RhoB, PIK3CA-like and ADCY3 showed a significant effect on V. anguillarum resistance with the verification rate in the sequencing population up to 70.8%. Taken all, our findings demonstrated the feasibility of BSA-seq approach in identifying genes responsible for the extreme phenotypes and will aid in performing MAS in turbot. Show less

Inflammation is an important element of the pathophysiological process of heart failure (HF) and is correlated with subtypes of HF. The association between multiple biomarkers of inflammation and HF subtypes in Chinese subjects remains unclear. This study aimed to compare the differences in inflammation biomarkers among Chinese patients with different subtypes of HF who have been identified to date. We included 413 consecutive patients with HF, including 262 with preserved ejection fraction (HFpEF), 55 with middle-ranged ejection fraction (HFmrEF) and 96 with reduced ejection fraction (HFrEF). Ten inflammation biomarkers were analyzed and compared according to the HF subtypes. One hundred contemporary non-HF subjects were also recruited as the control group. Moreover, the correlations between the inflammatory biomarkers and left ventricular ejection fraction of the HF subtypes were assessed. The mean age of the HF patients was 65.0 ± 12.0 years, 65.8% were male. Distinct subtypes of HF demonstrated different inflammation biomarker panels. IL-6, PTX-3, ANGPTL-4 and TNF-α were correlated with HFrEF; IL-1β and PTX-3 were correlated with HFmrEF; and IL-1β and IL-6 were correlated with HFpEF. The multivariable logistic regression showed that IL-1β [relative ratio (RR) = 1.08, 95% CI: 1.02-1.15, Diverse inflammation biomarkers have multifaceted presentations according to the subtype of HF, which may illustrate the diverse mechanisms of inflammation in Chinese HF patients. IL-6, PTX-3, and ANGPTL-4 were independent inflammation factors associated with HFrEF and HF. Show less

Focal and segmental glomerular sclerosis (FSGS) is a common cause of nephrotic syndrome and end-stage renal disease. It has been reported that overproduction of reactive oxygen species (ROS) and cell apoptosis are associated with the development of FSGS. Epigallocatechin-3-gallate (EGCG) is a bioactive constituent accounting for more than 50% of the total catechins in green tea, which have anti-oxidative and anti-apoptotic effects. Based on this, this study was designed to evaluate the renoprotective effect of EGCG treatment on Adriamycin-induced FSGS. -Methods: In C57BL/6 mice, Adriamycin nephropathy (AN) was induced by Adriamycin (10 mg/kg body weight, diluted in normal saline) via a tail vein on day 0. Then the mice were given with EGCG (20 mg/kg body weight) or YC-1 (Lificiguat, a specific inhibitor of hypoxia-inducible factor-1α [HIF-1α], 50 mg/kg body weight) or both intraperitoneally. Both the EGCG and YC-1 were given on the day of Adriamycin injection and continued for 6 weeks. The animals were organized into the following 5 groups for the animal experiments: the control group, the AN group, the AN + EGCG group, the AN + YC-1 group and the AN + EGCG + YC-1 group. At 6 weeks, the mice were sacrificed; kidneys and blood samples were collected for further analysis. The HIF-1α and the angiopoietin-like 4 (ANGPTL4) expression were detected by Western blot, real-time PCR, immunohistochemistry or immunofluorescence. Dihydroethidium staining and NADPH oxidase 1 (Nox1) measurement were used to detect ROS production. Terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining and caspase-3 measurement was used to detect cell apoptosis. When the animals were treated with Adriamycin, both the ROS production and TUNEL positive cells increased. Besides, the expression of HIF-1α, ANGPTL4, and caspase-3 were also up-regulated, while EGCG treatment could attenuate these changes. Interestingly, compared with treatment with YC-1 or EGCG alone, more pronounced inhibition of ANGPTL4, caspase-3 and Nox1 were obtained when YC-1 and EGCG were administered simultaneously. EGCG attenuates FSGS through the suppression of Oxidant Stress and apoptosis by targeting the HIF-1α/ANGPTL4 pathway. Show less

Gastric cancer is still among the leading causes of cancer deaths worldwide. Despite the improvements in diagnostic methods, the status of early detection has not been achieved so far. Early diagnosis of gastric cancer may significantly improve the cure rate of patients. Therefore, a new diagnostic method is needed. In this study, subtractive SELEX was performed to screen gastric cancer serum-specific DNA aptamers by using gastric cancer serum and normal serum as the target and negative serum, respectively. Four highly specific aptamers generated for gastric cancer serum, Seq-3, Seq-6, Seq-19 and Seq-54, were developed using whole-serum subtractive SELEX technology with Show less

The present study aims to discover novel serum biomarkers of early-onset myocardial infarction (MI) using proteomic analysis. In the first stage, the iTRAQ-coupled LC-MS/MS technique is utilized to investigate protein profiles of patients with early-onset MI. In the second stage, these candidate proteins are validated using ELISA. A total of 538 proteins are quantified, with pregnancy zone protein (PZP), leucine-rich α-2-glycoprotein (LRG) and Apolipoprotein C-I (Apo C-I) being upregulated and Apolipoprotein A-I (Apo A-I) and Apolipoprotein A-IV (Apo A-IV) downregulated in early-onset MI patients. Results from the validation stage demonstrate that the serum concentrations of PZP and LRG are significantly increased in the early-onset MI group. The correlation between the concentrations of C-reactive protein (CRP) and the two candidate biomarkers is positive. Area under the curve values used to diagnose early-onset MI for LRG and PZP are 0.939 and 0.874, respectively. Five differential serum proteins are identified in early-onset MI using proteomic analysis. Lipoprotein-related biomarkers further demonstrate the close relationship between lipid metabolism and the disease. Inflammation-associated LRG and PZP may be novel biomarkers of the disease. In addition, changes in these proteins may partly reveal the possible mechanisms in the pathogenesis and pathophysiology of early-onset MI. Show less

Triglyceride (TG) concentrations >2000 mg/dL are extremely elevated and increase the risk of pancreatitis. We characterized five cases and two kindreds and ascertained prevalence in a reference laboratory population. Plasma lipids and DNA sequences of LPL, GPIHBP1, APOA5, APOC2, and LMF1 were determined in cases and two kindreds. Hypertriglyceridemia prevalence was assessed in 440,240 subjects. Case 1 (female, age 28 years) had TG concentrations >2000 mg/dL and pancreatitis since infancy. She responded to diet and medium-chain triglycerides, but not medications. During two pregnancies, she required plasma exchange for TG control. She was a compound heterozygote for a p.G236Gfs*15 deletion and a p.G215E missense mutation at LPL, as was one sister with hypertriglyceridemia and pancreatitis during pregnancy. Her father was heterozygous for the deletion and had hypertriglyceridemia and recurrent pancreatitis. Other family members had either the missense mutation or the deletion, and had hypertriglyceridemia but no pancreatitis. In kindred 2, three preschool children had severe hypertriglyceridemia and were homozygous for a GPIHBP1 p.T108R missense mutation. Case 5 (male, age 43 years) presented with pancreatitis and TG levels >5000 mg/dL and had heterozygous GPIHBP1 p.G175R and APOC2 intron 2-4G>C mutations. On diet, fenofibrate, fish oil, and atorvastatin, his TG concentration was 2526 mg/dL, but normalized to <100 mg/dL with added pioglitazone. In our population study, 60 subjects (0.014%) of 440,240 had TG concentrations >2000 mg/dL, and 66.7% were diabetic and had elevated insulin levels. Extreme hypertriglyceridemia is rare (0.014%); and during pregnancy, it may require plasma exchange. Show less

Hypertriglyceridemia severity is linked to acute pancreatitis prognosis, but it remains unknown why a portion of severe hypertriglyceridemia patients do not develop severe acute pancreatitis. To investigate whether hypertriglyceridemia subtypes affect acute pancreatitis progression, we analyzed two genetically modified hypertriglyceridemia mouse models-namely, glycosylphosphatidylinositol high-density lipoprotein binding protein 1 knockout (Gpihbp1-/-) and apolipoprotein C3 transgenic (ApoC3-tg) mice. Acute pancreatitis was induced by 10 intraperitoneal caerulein injections. Biochemical assays and pathological analysis were performed for the severity evaluation of acute pancreatitis. Plasma triglyceride-rich lipoproteins (TRLs), including chylomicrons and very low-density lipoprotein (VLDL), were collected via ultracentrifugation to evaluate their cytotoxic effects on primary pancreatic acinar cells (PACs). We found that the particle sizes of Gpihbp1-/- TRLs were larger than ApoC3-tg TRLs. Severe pancreatic injury with large areas of pancreatic necrosis in the entire lobule was induced in Gpihbp1-/- mice when plasma triglyceride levels were greater than 2000 mg/dL. However, ApoC3-tg mice with the same triglyceride levels did not develop large areas of pancreatic necrosis, even upon the administration of poloxamer 407 to further increase triglyceride levels. Meanwhile, in the acute pancreatitis model, free fatty acids (FFAs) in the pancreas of Gpihbp1-/- mice were greater than in ApoC3-tg mice. TRLs from Gpihbp1-/- mice released more FFAs and were more toxic to PACs than those from ApoC3-tg mice. Chylomicrons from patients showed the same effects on PACs as TRLs from Gpihbp1-/- mice. Gpihbp1-/- mice with triglyceride levels below 2000 mg/dL had milder pancreatic injury and less incidence of pancreatic necrosis than those with triglyceride levels above 2000 mg/dL, similar to Gpihbp1-/-mice with triglyceride levels above 2000 mg/dL but with fenofibrate administration. These findings demonstrated that hypertriglyceridemia subtypes with large TRL particles could affect acute pancreatitis progression and that chylomicrons showed more cytotoxicity than VLDL by releasing more FFAs. Show less

Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes. Show less

Nitidine chloride (NC) has been demonstrated to have an anticancer effect in hepatocellular carcinoma (HCC). However, the mechanism of action of NC against HCC remains largely unclear. In this study, three pairs of NC-treated and NC-untreated HCC xenograft tumour tissues were collected for circRNA sequencing analysis. In total, 297 circRNAs were differently expressed between the two groups, with 188 upregulated and 109 downregulated, among which hsa_circ₀₀₈₈₃₆₄ and hsa_circ₀₀₉₀₀₄₉ were validated by real-time quantitative polymerase chain reaction. The in vitro experiments showed that the two circRNAs inhibited the malignant biological behaviour of HCC, suggesting that they may play important roles in the development of HCC. To elucidate whether the two circRNAs function as "miRNA sponges" in HCC, we identified circRNA-miRNA and miRNA-mRNA interactions by using the CircInteractome and miRwalk, respectively. Subsequently, 857 miRNA-associated differently expressed genes in HCC were selected for weighted gene co-expression network analysis. Module Eigengene turquoise with 423 genes was found to be significantly related to the survival time, pathology grade and TNM stage of HCC patients. Gene functional enrichment analysis showed that the 423 genes mainly functioned in DNA replication- and cell cycle-related biological processes and signalling cascades. Eighteen hubgenes (SMARCD1, CBX1, HCFC1, RBM12B, RCC2, NUP205, ECT2, PRIM2, RBM28, COPS7B, PRRC2A, GPR107, ANKRD52, TUBA1B, ATXN7L3, FUS, MCM8 and RACGAP1) associated with clinical outcomes of HCC patients were then identified. These findings showed that the crosstalk between hsa_circ₀₀₈₈₃₆₄ and hsa_circ₀₀₉₀₀₄₉ and their competing mRNAs may play important roles in HCC, providing interesting clues into the potential of circRNAs as therapeutic targets of NC in HCC. Show less

Primary liver cancer ranks among the leading causes of cancer death worldwide. Risk factors are closely linked to inflammation, such as viral hepatitis and alcoholic as well as non-alcoholic steatohepatitis. Among the pathways involved in the pathogenesis of malignant liver tumors, dysregulation of NF-κB signaling plays a prominent role. It provides a link between inflammation and cancer. To examine the role of NF-κB in a MYC-induced model of hepatocellular carcinoma we deleted NEMO (IKKγ) specifically from hepatocytes. NEMO deletion accelerated tumor development and shortened survival, suggesting a tumor-suppressive function of NF-κB signaling. We observed increased proliferation, inflammation and fibrosis, as well as activation of MAPK and STAT signaling. Importantly, deletion of NEMO modified the tumor phenotype from hepatocellular carcinoma to combined hepatocellular cholangiocarcinoma. The intrahepatic cholangiocarcinoma tumor component showed increased expression of progenitor markers such as Sox9 and reduced expression of mature hepatic markers such as CPS1. In both cases tumorigenesis was reversible by turning off MYC expression. To our knowledge this is the first mouse model of combined hepatocellular cholangiocarcinoma and may provide insights into the development of this rare malignant tumor. Show less

Background Endometrial adenocarcinoma (EAC) is one of the most commonly diagnosed gynaecological malignancies among female population of the developed countries. DUSP6 is a negative regulator of ERK signaling, which is a molecular switch involved in MAPK signaling during the progress of malignancies. DUSP6 was previously found to inhibit tumorigenesis and EMT-associated properties in several cancers, however, its exact role in EAC remains unclear Methods The level of DUSP6, (E-cad) and (N-cad) in EAC cancerous tissues and respective adjacent non-cancerous tissues were examined by western-blot or immunohistochemistry. The cell growth, invasion and migration abilities were measured in Ishikawa 3H12 endometrial cancer cell lines with overexpressed or knock down DUSP6. Protein levels of EMT-associated markers E-cadherin, N-cadherin and Vimentin were also determined. The impacts of DUSP6 on ERK signaling was assessed by detection of ERK and p-ERK. Results Down-regulation of DUSP6 was observed in EAC compared with the normal controls. The overexpression of DUSP6 significantly attenuated tumor cell growth, invasion, migration abilities and inhibited EMT-associated markers, while knock down of DUSP6 showed opposite trends. Overexpression of DUSP6 also down-regulated p-ERK and the knock down of DUSP6 inversely up-regulated p-ERK level. Conclusions DUSP6 inhibited cell growth, invasion and migration abilities in Ishikawa 3H12 cells as well as attenuating EMT-associated properties. This tumor suppressive effect of DUSP6 in EAC is achieved by inhibiting ERK signaling pathway. Show less

Non-planar sensor arrays are used to determine solar orientation based on the orientation matrix formed by orientation vectors of the sensor planes. Solar panels or existing photodiodes can be directly used without increasing the size or mass of the spacecraft. However, a limiting factor for the improvement of the accuracy of orientation lies with the lack of an assessment-based approach. A formulation was developed for the supremum (i.e., the least upper bound) of orientation error of an arbitrary orientation matrix in terms of its influencing factors. The new formulation offers a way to evaluate the supremum of orientation error considering interference with finite energy and interference with infinite energy but finite average energy. For a given non-planar sensor array, a sub-matrix of the full orientation matrix would reach the optimal accuracy of orientation if its supremum of orientation error is the least. Principles for designing an optimal sensor array relate to the configuration of the orientation matrix, which can be pre-determined for a given number of sensors. Simulations and field experiment tested and validated the methods, showing that our sensor array optimization method outperforms the existing methods, while providing a way of assessment and optimization. Show less

Autologous bone grafts used for surgical reconstruction are limited by infection or insufficient supply of host material. Experimental agents that promote differentiation of stem cells into mature bone are currently being studied for future use in the repair of bone defects. The authors hypothesized that imiquimod, a synthetic immune response modifier, increases Notch pathway gene expression and acts synergistically with bone morphogenetic protein (BMP) 9 to induce differentiation of mesenchymal stem cells toward an osteogenic phenotype. Alkaline phosphatase activity was used to assess the osteogenic potential of cultured mouse immortalized multipotent adipose-derived cells (iMADs) treated with 0, 4, 6, and 8 μg/ml of imiquimod with and without BMP9. Adenoviral vectors expressing human BMP9 and a dominant-negative mutant of mouse Notch1 were used to assess BMP9 and Notch blockade on osteogenic activity, respectively. Expression of Notch signaling mediators and osteogenic markers were assayed by quantitative polymerase chain reaction. Alizarin red staining was used to assess the synergism between BMP9 and imiquimod. Imiquimod exposure enhanced osteogenic differentiation of iMADs by 2.8-fold (p < 0.001) and potentiated BMP9-induced osteogenic differentiation of iMADs by 1.6-fold (p < 0.001), shown by increased alkaline phosphatase activity and augmented matrix mineralization. Quantitative-real time polymerase chain reaction analysis demonstrated that imiquimod induced the expression of downstream genes (p < 0.01) of the Notch signaling pathway Hey1, Hey2, and Hes1, by increases of 9.7-, 22-, and 2.7-fold, respectively. These findings identify a novel role for imiquimod to shift mesenchymal stem cells toward an osteogenic phenotype. Imiquimod may be useful clinically when scaffolds are applied to treat bone defects. Show less

Although the genotype-phenotype for familial medullary thyroid carcinoma (FMTC) is well studied, only few low susceptibility risk loci were identified for familial non-medullary thyroid carcinoma (FNMTC). The aim of this study is to screen and identify high-penetrate genes for FNMTC. A total of 34 families with more than two first-degree relatives diagnosed as papillary thyroid cancer without other familial syndrome were recruited. Whole exome and target gene sequencing were performed for candidate variants. These variants were screened and analyzed with ESP6500, ExAC, 1000 genomes project, and the Cancer Genome Atlas (TCGA) with SIFT score and Polyphen2 prediction. Finally, we identified recurrent genetic mutation of MAP2K5 variants c.G961A and c.T1100C (p. A321T and p.M367 T) as susceptibility loci for FNMTC. The frequencies of MAP2K5 c.G961A and c.T1100C were found, 0.0385 and 0.0259 in FNMTC and 0 and 0.00022523 in healthy Chinese controls (n = 2200, P < 0.001), respectively. Both variants were located in the protein kinase domain. The functional study showed that MAP2K5 A321T or M367 T could consistently phosphorylate downstream protein ERK5 on site Ser731 + Thr733 or Ser496, promoting nuclear translocation and subsequently altering target gene expressions. Our data revealed that MAP2K5 variants A321T or M367 T can activate MAP2K5-ERK5 pathway, alter downstream gene expression, and subsequently induce thyroid epithelial cell malignant transformation. While classic MAP2K1/2(MEK1/2)-ERK1/2 signaling is well known for driving sporadic NMTC, our research indicated that MAP2K5 (MEK5) is a susceptibility gene for FNMTC. These findings highlight the potential application of MAP2K5 for molecular diagnosis as well as early prevention. Show less

Mono-2-ethylhexyl phthalate (MEHP) is a major bioactive metabolite in the widely used industrial plasticizer diethylhexyl phthalate (DEHP) that has been found to be toxic to the liver. The aim of this study is to determine whether MEHP exposure can change the expression of fatty acid metabolism-related genes in HepG2 cells, which might be related to non-alcoholic fatty liver disease (NAFLD). The results revealed that exposure to MEHP promoted lipid accumulation in HepG2 cells. The levels of intracellular triglycerides in the hepatocytes increased after exposure to 0.8-100 μM MEHP for 24 h and 48 h. The genetic expressions of SREBP-1c, ChREBP, ACC1, FASN, and SCD significantly increased at 6 h after exposure to MEHP. At 24 h, the expression of the SREBP-1c and ChREBP genes remained increased, while the expression of the FASN and SCD genes decreased. At 48 h, the expression of SREBP-1c, ChREBP, ACC1, FASN, and SCD decreased. Furthermore, the levels of proteins including ACC1, FASN, SCD, and ChREBP (except SREBP-1c) increased at 24 h. These findings suggest that MEHP exposure can promote fatty acid synthesis in hepatocytes by regulating the expression of relevant genes and proteins, contributing to NAFLD. Show less

Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs. Show less

Targeting of extrinsic apoptosis pathway by TNF-related apoptosis-inducing ligand (TRAIL) is an attractive approach for cancer therapy. However, two TRAIL drug candidates failed in clinical trials due to lack of efficacy. We identified 17-hydroxy wortmannin (17-HW) in a drug repurposing screen that resensitized TRAIL's response in the resistant colon cancer cells. The deficiency of caspase-8 in drug-resistant cells along with defects in apoptotic cell death was corrected by 17-HW, an inhibitor of PIK3C3-beclin 1 (BECN1) complex and autophagy activity. Further study found that BECN1 significantly increased in the TRAIL-resistant cells, resulting in increased autophagosome formation and enhanced autophagy flux. The extracellular domain (ECD) of BECN1 directly bound to the caspase-8 catalytic subunit (p10), leading to sequestration of caspase-8 in the autophagosome and its subsequent degradation. Inhibition of BECN1 restored the caspase-8 level and TRAIL's apoptotic response in the resistant colon cancer cells. An analysis of 120 colon cancer patient tissues revealed a correlation of a subgroup of patients (30.8%, 37/120) who have high BECN1 level and low caspase-8 level with a poor survival rate. Our study demonstrates that the increased BECN1 accompanied by enhanced autophagy activity is responsible for the TRAIL resistance, and a combination of TRAIL with a PIK3C3-BECN1 inhibitor is a promising therapeutic approach for the treatment of colon cancer. Show less

Pulmonary arterial hypertension (PAH) is a life-threatening disease without effective therapies. PAH is associated with a progressive increase in pulmonary vascular resistance and irreversible pulmonary vascular remodeling. SUMO1 (small ubiquitin-related modifier 1) can bind to target proteins and lead to protein SUMOylation, an important post-translational modification with a key role in many diseases. However, the contribution of SUMO1 to PAH remains to be fully characterized. In this study, we explored the role of SUMO1 in the dedifferentiation of vascular smooth muscle cells (VSMCs) involved in hypoxia-induced pulmonary vascular remodeling and PAH in vivo and in vitro. In a mouse model of hypoxic PAH, SUMO1 expression was significantly increased, which was associated with activation of autophagy (increased LC3b and decreased p62), dedifferentiation of pulmonary arterial VSMCs (reduced α-SMA, SM22 and SM-MHC), and pulmonary vascular remodeling. Similar results were obtained in a MCT-induced PAH model. Overexpression of SUMO1 significantly increased VSMCs proliferation, migration, hypoxia-induced VSMCs dedifferentiation, and autophagy, but these effects were abolished by inhibition of autophagy by 3-MA in aortic VSMCs. Furthermore, SUMO1 knockdown reversed hypoxia-induced proliferation and migration of PASMCs. Mechanistically, SUMO1 promotes Vps34 SUMOylation and the assembly of the Beclin-1-Vps34-Atg14 complex, thereby inducing autophagy, whereas Vps34 mutation K840R reduces Vps34 SUMOylation and inhibits VSMCs dedifferentiation. Our data uncovers an important role of SUMO1 in VSMCs proliferation, migration, autophagy, and phenotypic switching (dedifferentiation) involved in pulmonary vascular remodeling and PAH. Targeting of the SUMO1-Vps34-autophagy signaling axis may be exploited to develop therapeutic strategies to treat PAH. Show less

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

Deep brain stimulation of the subthalamic nucleus (STN-DBS) stimulation produces significant improvement of overall pain related to Parkinson disease; however, the mechanisms underlying analgesic effects of STN-DBS are still unknown. This report describes direct neuroanatomical evidence for the central melanocortinergic-opioidergic circuits in the STN. We investigated melanocortin-4 receptor (MC4R) and mu-opioid receptor (MOR)-positive expression of the STN in MC4R-GFP transgenic mice using fluorescence immunohistochemical detection. Immunohistochemistry showed a large number of MC4R-GFP- and MOR-positive neurons within the STN region, and approximately 50% of MC4R-GFP-positive neurons coexpressed MOR. The results of this study showed direct neuroanatomical evidence for the central melanocortinergic-opioidergic signaling in the STN region. These findings contribute to the view of melanocortinergic-opioidergic circuits in the subthalamic nucleus as a reliable source of modulating of nociception with therapeutic potential for alleviating pain. Show less

Obesity is increasingly becoming a major public health problem worldwide. Peripheral LKB1 inhibits white fat generation, but the effect of central LKB1 on diet-induced obesity (DIO) is unknown. Therefore, we examined whether LKB1 over-expression in the hypothalamus can inhibit the development of obesity. Adult male Sprague-Dawley rats were anesthetized and placed in a stereotaxic apparatus. LKB1-AAV-EGFP (2.0 × 108 or 2.0 × 1010 vector genomes) or Control-AAV-EGFP (2.0 × 108 vector genomes) was injected into the third ventricle. After administration, the rats were fed a high-fat diet (HFD) for 9 weeks to induce obesity. Rats fed a chow fat diet were used as normal controls. LKB1 delivery decreased body weight, energy intake, fat mass, and serum lipid levels. LKB1 also improved HFD-induced hepatic fatty degeneration. Interestingly, LKB1 over-expression in the hypothalamus activated the AMPK-POMC neurons-sympathetic nervous system (SNS) axis, which can release epinephrine to promote white fat browning. Conversely, the elevated expression of MC3R/MC4R inhibited food intake. These two factors worked together to inhibit the development of obesity. LKB1 in the hypothalamus may have therapeutic potential for DIO through the activation of the AMPK-POMC neurons-SNS axis. Show less

Military personnel and athletes exposed to traumatic brain injury may develop chronic traumatic encephalopathy (CTE). Brain pathology in CTE includes intracellular accumulation of abnormally phosphorylated tau proteins (p-tau), the main constituent of neurofibrillary tangles (NFTs). Recently, we found that cholinergic basal forebrain (CBF) neurons within the nucleus basalis of Meynert (nbM), which provide the major cholinergic innervation to the cortex, display an increased number of NFTs across the pathological stages of CTE. However, molecular mechanisms underlying nbM neurodegeneration in the context of CTE pathology remain unknown. Here, we assessed the genetic signature of nbM neurons containing the p-tau pretangle maker pS422 from CTE subjects who came to autopsy and received a neuropathological CTE staging assessment (Stages II, III, and IV) using laser capture microdissection and custom-designed microarray analysis. Quantitative analysis revealed dysregulation of key genes in several gene ontology groups between CTE stages. Specifically, downregulation of the nicotinic cholinergic receptor subunit β-2 gene (CHRNB2), monoaminergic enzymes catechol-O-methyltransferase (COMT) and dopa decarboxylase (DDC), chloride channels CLCN4 and CLCN5, scaffolding protein caveolin 1 (CAV1), cortical development/cytoskeleton element lissencephaly 1 (LIS1), and intracellular signaling cascade member adenylate cyclase 3 (ADCY3) was observed in pS422-immunreactive nbM neurons in CTE patients. By contrast, upregulation of calpain 2 (CAPN2) and microtubule-associated protein 2 (MAP2) transcript levels was found in Stage IV CTE patients. These single-population data in vulnerable neurons indicate alterations in gene expression associated with neurotransmission, signal transduction, the cytoskeleton, cell survival/death signaling, and microtubule dynamics, suggesting novel molecular pathways to target for drug discovery in CTE. Show less