Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Analysis of circRNA-mRNA expression profiles and functional enrichment in diabetes mellitus based on high throughput sequencing.

To study the pathogenesis of diabetes mellitus (DM) and identify new biomarkers, high-throughput RNA sequencing provides a technical means to explore the regulatory network of MD gene expression. To better elucidate the genetic basis of DM, we analysed the circRNA and mRNA expression profiles in serum samples from diabetic patients. The circRNAs and mRNAs with abnormal expression in the DM group and non-diabetic group (NDM) were classified by RNA sequencing and differential expression analysis. The circRNA-miRNA-mRNA regulatory network reveals the mechanism by which competitive endogenous RNAs (ceRNAs) regulate gene expression. The biological functions and interactions of circRNA and mRNA were analysed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differential expression analysis showed that 441 circRNAs (366 up-regulated, 75 down-regulated) and 683 mRNAs (354 up-regulated, 329 down-regulated) were significantly differentially expressed in the DM group compared with the NDM group. Screening of the differential genes at the nodes of the interaction network showed that a single circRNA could interact with multiple miRNAs and then jointly regulate more mRNAs. In addition, the expressions of circRNA CNOT6 and AXIN1 as well as mRNA STAT3, MYD88, and B2M were associated with the progression of diabetes. Enrichment pathway analysis indicated that differentially expressed circRNA and mRNA may participate in Nod-like receptor signalling pathway, insulin signalling pathway, sphinolipid metabolism pathway, and ribosome pathway, and play a role in the pathogenesis of diabetes. This study provides a theoretical basis for elucidating the molecular mechanism of DM occurrence and development at circRNA and mRNA levels.

Combined analysis of circRNA and mRNA profiles and interactions in patients with Diabetic Foot and Diabetes Mellitus.

In order to elucidate the pathogenesis and explore new biomarkers for diabetes and diabetic foot (DF), an analysis using RNA sequencing affords broader insights into gene expression regulatory networks in DF. To better explore the molecular basis of DF, we carried out an analysis of circular RNA (circRNA) and messenger RNA (mRNA) expression profiles of serum samples from DF patients and diabetes mellitus (DM) patients. The potential roles and interactions of differentially expressed circRNAs and mRNAs were classified by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Compared with diabetes patients, 279 mRNAs were upregulated and 353 mRNAs were downregulated in the serum of DF patients, and 33 circRNAs were differently expressed. The differential genes at the nodes of the interaction network were screened, and TLR6 RUNX1 and ST2 were found to be related to the progression of diabetes and DF. The enrichment pathway analysis revealed that the lysosomal pathway played a critical role in the occurrence and development of DF. TLR6, RUNX1, and ST2 mRNA expressions and the lysosomal pathway may be involved in the pathogenesis of diabetes and DF. In addition, methane metabolism and Chagas disease pathways were observed in the occurrence and development of DF, which is a new discovery in this study. This study provides clues on the molecular mechanisms of DF at the circRNA and mRNA levels.

The REST gene signature predicts drug sensitivity in neuroblastoma cell lines and is significantly associated with neuroblastoma tumor stage.

Neuroblastoma is the most common and deadly solid tumor in children, and there is currently no effective treatment available for neuroblastoma patients. The repressor element-1 silencing transcription (REST) factor has been found to play important roles in the regulation of neural differentiation and tumorigenesis. Recently, a REST signature consisting of downstream targets of REST has been reported to have clinical relevance in both breast cancer and glioblastoma. However it remains unclear how the REST signature works in neuroblastoma. Publicly available datasets were mined and bioinformatic approaches were used to investigate the utility of the REST signature in neuroblastoma with both preclinical and real patient data. The REST signature was found to be associated with drug sensitivity in neuroblastoma cell lines. Further, neuroblastoma patients with enhanced REST activity are significantly associated with higher clinical stages. Loss of heterozygosity on chromosome 11q23, which occurs in a large subset of high-risk neuroblastomas, tends to be correlated with high REST activity, with marginal significance. In conclusion, the REST signature has important implications for targeted therapy, and it is a prognostic factor in neuroblastoma patients.

Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

BACKGROUND: Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. METHODS: Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. RESULTS: As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P < 0.05) and muscle (P < 0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. CONCLUSIONS: We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.

In silico development and in vitro validation of a novel five-gene signature for prognostic prediction in colon cancer.

Colon cancer is one of the most common cancers in digestive system, and its prognosis remains unsatisfactory. Therefore, this study aimed to identify gene signatures that could effectively predict the prognosis of colon cancer patients by examining the data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. LASSO-Cox regression analysis generated a five-gene signature (DCBLD2, RAB11FIP1, CTLA4, HOXC6 and KRT6A) that was associated with patient survival in the TCGA cohort. The prognostic value of this gene signature was further validated in two independent GEO datasets. GO enrichment revealed that the function of this gene signature was mainly associated with extracellular matrix organization, collagen-containing extracellular matrix, and extracellular matrix structural constituent. Moreover, a nomogram was established to facilitate the clinical application of this signature. The relationships among the gene signature, mutational landscape and immune infiltration cells were also investigated. Importantly, this gene signature also reliably predicted the overall survival in IMvigor210 anti-PD-L1 cohort. In addition to the bioinformatics study, we also conducted a series of in vitro experiments to demonstrate the effect of the signature genes on the proliferation, migration, and invasion of colon cancer cells. Collectively, our data demonstrated that this five-gene signature might serve as a promising prognostic biomarker and shed light on the development of personalized treatment in colon cancer patients.

Association of differential body water composition by sex, measured using BIA, with survival in a real­world lung cancer cohort.

OBJECTIVE: The aim of this study was to evaluate the association of differential body water composition with survival in patients with lung cancer. METHODS: This retrospective cohort study included 1314 patients diagnosed with lung cancer in 80 Chinese institutions from May 2013 to August 2020. We calculated hazard ratios (HRs) to evaluate the associations of all-cause mortality with extracellular water (ECW) and intracellular water (ICW). Cox proportional risk regression models were adjusted for sociodemographic characteristics, tumor characteristics, treatment, body mass index (BMI), and body composition measures. We also evaluated cross-classification of the dichotomy of ECW and ICW with outcomes. The association among ECW, ICW, and survival was evaluated via Cox regression and the restricted cubic-spline model using a two-sided P value. RESULTS: The study included 819 (62%) men and 495 (28%) women. The HR of lung cancer mortality significantly decreased as ECW increased (HR, 0.96; 95% confidence interval [CI], 0.93-1.00) and ICW (HR, 0.97; 95% CI, 0.95-1.00) with cutoff values of 10.5 and 16.3 L, respectively. When patients were cross-classified into categories of sex, age, BMI, visceral fat index, pathology, tumor stage, tumor burden, total bilirubin, and neutrophil count, ICW and ECW were protective factors. Only sex interacted significantly with ICW or ECW. High ICW and ECW had significant protective effects, and women had a greater risk for death than men in the case of either poor ICW or poor ECW. Sensitivity analysis showed the protective effect of the higher dichotomy of ICW (HR, 0.52; 95% CI, 0.35-0.78) and ECW (HR, 0.45; 95% CI, 0.31-0.66) on female lung cancer patients by removing patients who died within 12 mo of diagnosis. CONCLUSION: Greater ICW and ECW, especially ICW, were independent predictors for better survival in patients with lung cancer. Female patients were more vulnerable to dehydration than male patients.

Comprehensive molecular characterization of inhibitors of apoptosis proteins (IAPs) for therapeutic targeting in cancer.

BACKGROUND: Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic proteins modulating cell cycle, signal transduction and apoptosis. Dysregulated IAPs have been reported to contribute to tumor progression and chemoresistance in various cancers. However, existing studies were sporadic and only focus on one specific cancer with one particular gene in the IAPs family. A systematic investigation on the co-expression pattern, regulation frameworks on various pathways, prognostic utility on patient outcomes, and predictive value on drug sensitivity among all the IAPs across multiple tumor types was lacking. METHODS: Leveraging The Cancer Genome Atlas data with comprehensive genomic characterizations on 9714 patients across 32 tumor types and the Genomics of Drug Sensitivity in Cancer data with both genomic characterizations and drug sensitivity data on > 1000 cell lines, we investigated the co-expression pattern of IAPs, their regulations of apoptosis as well as other pathways and clinical relevance of IAPs for therapeutics development. RESULTS: We discovered diverse expression pattern among IAPs, varied spectrum of apoptosis regulations through IAPs and extensive regulations beyond apoptosis involving immune response, cell cycle, gene expression and DNA damage repair. Importantly, IAPs were strong prognostic factors for patient survival and tumor stage in several tumor types including brain, liver, kidney, breast and lung cancer. Further, several IAPs were found to be predictive of sensitivity to BCL-2 inhibitors (BIRC3, BIRC5, BIRC6, and BIRC7) as well as RIPK1 inhibitors (BIRC3 and BIRC6). CONCLUSION: Together, our work revealed the landscape of regulations, prognostic utilities and therapeutic relevance of IAPs across multiple tumor types.

Next-Generation Sequencing Analysis of ctDNA for the Detection of Glioma and Metastatic Brain Tumors in Adults.

Background and aims: The next-generation sequencing technologies and their related assessments of circulating tumor DNA in both glioma and metastatic brain tumors remain largely limited. Methods: Based largely on a protocol approved by the institutional review board at Peking University International Hospital, the current retrospective, single-center study was conducted. Genomic DNA was extracted from blood samples or tumor tissues. With the application of NextSeq 500 instrument (Illumina), Sequencing was performed with an average coverage of 550-fold. Paired-end sequencing was employed utilized with an attempt to achieve improved sensitivity of duplicate detection and therefore to increase the detection reliability of possible fusions. Results: A total of 28 patients (21 men and 7 women) with brain tumors in the present study were involved in the study. The patients enrolled were assigned into two groups, including glioma group (n = 21) and metastatic brain tumor group (n = 7). The mean age of metastatic brain tumor group (59.86 ± 8.85 y), (43.65 ± 13.05 y) reported significantly higher results in comparison to that of glioma group (45.3 ± 12.3 years) (P < 0.05). The mutant genes in metastatic brain tumor group included ALK, MDM2, ATM, BRCA1, FGFR1, MDM4 and KRAS; however, there were no glioma-related mutant genes including MGMT, IDH1, IDH2, 1p/19q, and BRAF et al. Interesteringly, only two patient (28.3%) was detected blood ctDNA in metastatic brain tumor group; In contrast, blood ctDNA was found in ten glioma patients (47.6%) including 1p/19q, MDM2, ERBB2, IDH1, CDKN2A, CDK4, PDGFRA, CCNE1, MET. The characterizations of IDH mutations in the glioma included IDH1 mutation (p.R132H) and IDH2 mutation (p.R172K). The mutation rate of IDH in tumor tissues was 37.06 ± 8.32%, which was significantly higher than blood samples (P < 0.05). Conclusion: The present study demonstrated that the mutant genes among glioma and metastatic brain tumors are shown to be different. Moreover, the ctDNAs in the metastatic brain tumors included ALK and MDM2, and glioma-related ctDNAs included 1p/19q and MDM2 followed by frequencies of ERBB2, IDH1, CDKN2A, CDK4, PDGFRA, CCNE1, MET. These ctDNAs might be biomarkers and therapeutic responders in brain tumor.

Topical Application of a Mast Cell Stabilizer Improves Impaired Diabetic Wound Healing.

Impaired wound healing in the diabetic foot is a major problem often leading to amputation. Mast cells have been shown to regulate wound healing in diabetes. We developed an indole-carboxamide type mast cell stabilizer, MCS-01, which proved to be an effective mast cell degranulation inhibitor in vitro and can be delivered topically for prolonged periods through controlled release by specifically designed alginate bandages. In diabetic mice, both pre- and post-wounding, topical MCS-01 application accelerated wound healing comparable to that achieved with systemic mast cell stabilization. Moreover, MCS-01 altered the macrophage phenotype, promoting classically activated polarization. Bulk transcriptome analysis from wounds treated with MCS-01 or placebo showed that MCS-01 significantly modulated the mRNA and microRNA profile of diabetic wounds, stimulated upregulation of pathways linked to acute inflammation and immune cell migration, and activated the NF-κB complex along with other master regulators of inflammation. Single-cell RNA sequencing analysis of 6,154 cells from wounded and unwounded mouse skin revealed that MCS-01 primarily altered the gene expression of mast cells, monocytes, and keratinocytes. Taken together, these findings offer insights into the process of diabetic wound healing and suggest topical mast cell stabilization as a potentially successful treatment for diabetic foot ulceration.

Integrated Skin Transcriptomics and Serum Multiplex Assays Reveal Novel Mechanisms of Wound Healing in Diabetic Foot Ulcers.

Nonhealing diabetic foot ulcers (DFUs) are characterized by low-grade chronic inflammation, both locally and systemically. We prospectively followed a group of patients who either healed or developed nonhealing chronic DFUs. Serum and forearm skin analysis, both at the protein expression and the transcriptomic level, indicated that increased expression of factors such as interferon-γ (IFN-γ), vascular endothelial growth factor, and soluble vascular cell adhesion molecule-1 were associated with DFU healing. Furthermore, foot skin single-cell RNA sequencing analysis showed multiple fibroblast cell clusters and increased inflammation in the dorsal skin of patients with diabetes mellitus (DM) and DFU specimens compared with control subjects. In addition, in myeloid cell DM and DFU upstream regulator analysis, we observed inhibition of interleukin-13 and IFN-γ and dysregulation of biological processes that included cell movement of monocytes, migration of dendritic cells, and chemotaxis of antigen-presenting cells pointing to an impaired migratory profile of immune cells in DM skin. The SLCO2A1 and CYP1A1 genes, which were upregulated at the forearm of nonhealers, were mainly expressed by the vascular endothelial cell cluster almost exclusively in DFU, indicating a potential important role in wound healing. These results from integrated protein and transcriptome analyses identified individual genes and pathways that can potentially be targeted for enhancing DFU healing.

Protein kinase C and protein kinase A are involved in the protection of recombinant human glucagon-like peptide-1 on glomeruli and tubules in diabetic rats.

AIMS/INTRODUCTION: Blockade or reversal the progression of diabetic nephropathy is a clinical challenge. The aim of the present study was to examine whether recombinant human glucagon-like peptide-1 (rhGLP-1) has an effect on alleviating urinary protein and urinary albumin levels in diabetic rats. MATERIALS AND METHODS: Streptozotocin-induced diabetes rats were treated with rhGLP-1 insulin and saline. Using immunostaining, hematoxylin-eosin, electron microscopy and periodic acid-Schiff staining to study the pathology of diabetic nephropathy, and we carried out quantitative reverse transcription polymerase chain reaction, western blot and immunohistochemistry to identify the differentially expressed proteins. The mechanism was studied through advanced glycation end-products-induced tubular epithelial cells. RESULTS: rhGLP-1 inhibits protein kinase C (PKC)-ß, but increases protein kinase A (PKA), which reduces oxidative stress in glomeruli and in cultured glomerular microvascular endothelial cells. In tubules, rhGLP-1 increased the expression of two key proteins related to re-absorption - megalin and cubilin - which was accompanied by downregulation of PKC-ß and upregulation of PKA. On human proximal tubular epithelial cells, rhGLP-1 enhanced the absorption of albumin, and this was blocked by a PKC activator or PKA inhibitor. CONCLUSIONS: These findings suggest that rhGLP-1 can reverse diabetic nephropathy by protecting both glomeruli and tubules by inhibiting PKC and activating PKA.

An expression based REST signature predicts patient survival and therapeutic response for glioblastoma multiforme.

Proper regulation of neuronal gene expression is crucial for the development and differentiation of the central nervous system. The transcriptional repressor REST (repressor element-1 silencing transcription factor) is a key regulator in differentiation of pluripotent stem cells to neuronal progenitors and mature neurons. Dysregulated REST activity has been implicated in various diseases, among which the most deadly is glioblastoma multiforme (GBM). Here we have developed an expression-based REST signature (EXPREST), a device providing quantitative measurements of REST activity for GBM tumors. EXPREST robustly quantifies REST activity (REST score) using gene expression profiles in absence of clinic-pathologic assessments of REST. Molecular characterization of REST activity identified global alterations at the DNA, RNA, protein and microRNA levels, suggesting a widespread role of REST in GBM tumorigenesis. Although originally aimed to capture REST activity, REST score was found to be a prognostic factor for overall survival. Further, cell lines with enhanced REST activity was found to be more sensitive to IGF1R, VEGFR and ABL inhibitors. In contrast, cell lines with low REST score were more sensitive to cytotoxic drugs including Mitomycin, Camptothecin and Cisplatin. Together, our work suggests that therapeutic targeting of REST provides a promising opportunity for GBM treatment.

Endothelial progenitor cells from human fetal aorta cure diabetic foot in a rat model.

OBJECTIVE: Recent evidence has suggested that circulating endothelial progenitor cells (EPCs) can repair the arterial endothelium during vascular injury. However, a reliable source of human EPCs is needed for therapeutic applications. In this study, we isolated human fetal aorta (HFA)-derived EPCs and analyzed the capacity of EPCs to differentiate into endothelial cells. In addition, because microvascular dysfunction is considered to be the major cause of diabetic foot (DF), we investigated whether transplantation of HFA-derived EPCs could treat DF in a rat model. METHODS: EPCs were isolated from clinically aborted fetal aorta. RT-PCR, fluorescence-activated cell sorting, immunofluorescence, and an enzyme-linked immunosorbent assay were used to examine the expressions of CD133, CD34, CD31, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), von Willebrand Factor (vWF), and Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1). Morphology and Dil-uptake were used to assess function of the EPCs. We then established a DF model by injecting microcarriers into the hind-limb arteries of Goto-Kakizaki rats and then transplanting the cultured EPCs into the ischemic hind limbs. Thermal infrared imaging, oxygen saturation apparatus, and laser Doppler perfusion imaging were used to monitor the progression of the disease. Immunohistochemistry was performed to examine the microvascular tissue formed by HFA-derived EPCs. RESULTS: We found that CD133, CD34, and VEGFR2 were expressed by HFA-derived EPCs. After VEGF induction, CD133 expression was significantly decreased, but expression levels of vWF and ELAM-1 were markedly increased. Furthermore, tube formation and Dil-uptake were improved after VEGF induction. These observations suggest that EPCs could differentiate into endothelial cells. In the DF model, temperature, blood flow, and oxygen saturation were reduced but recovered to a nearly normal level following injection of the EPCs in the hind limb. Ischemic symptoms also improved. Injected EPCs were preferentially and durably engrafted into the blood vessels. In addition, anti-human CD31+-AMA+-vWF+ microvasculars were detected after transplantation of EPCs. CONCLUSION: Early fetal aorta-derived EPCs possess strong self-renewal ability and can differentiate into endothelial cells. We demonstrated for the first time that transplanting HFA-derived EPCs could ameliorate DF prognosis in a rat model. These findings suggest that the transplantation of HFA-derived EPCs could serve as an innovative therapeutic strategy for managing DF.

Endoscopic versus surgical treatment of downstream pancreatic duct stones in chronic pancreatitis.

Pancreatic duct stone is thought not only to be the cause of abdominal pain but also to be a risk factor for pancreatic cancer. Several treatment options have been implemented in the treatment of pancreatic duct stones. Stone location is a critical factor in selecting treatment. We present the results of 27 endoscopic treatments and 35 surgical treatments performed in three hospitals at a single university between January 2000 and January 2005. The results were compared retrospectively in terms of success rate of stone removal, length of hospital stay, complications, pain relief, and changes of pancreatic functions. In our study, endoscopy resulted in a similar success rate of stone removal and short-term pain relief rate as the surgical approach and with a shorter length of hospital stay. However, the surgical group had a more favorable long-term clinical result, as well as a lower number of hospital readmissions at the 5-year follow-up point. Based on long-term results, surgical treatment is more effective than endoscopy.