Search

Background This study aimed to identify new protein markers that can evaluate the metastatic ability of Pancreatic neuroendocrine tumors (PanNETs) and predict prognosis together with Ki-67. Methods Multicentric series analysis was performed to evaluate the effect of metastasis on overall survival (N = 1,109). Single-cell RNA sequencing, proteomics (32 PanNETs and 10 normal pancreas tissues), and immunohistochemical staining (152 PanNETs) were conducted to screen proteins associated with PanNET metastatic capability. Associations between expression of screened proteins and survival were examined using Cox regression. In vitro experiments were performed to verify the function of candidate protein. A survival signature was constructed using recursive partitioning and survival forest analyses. Results The combination of G grade and metastasis predicted long-term survival more accurately though retrospective analysis. Single-cell RNA-seq highlighted the PanNET characteristics of metastasis that do not correspond with G grade and identified high metastatic capability subgroups in PanNET. Five proteins (PIN1, POSTN, SEMA4F, ASPN, and KCDT12), which may be related to the metastatic capability of PanNETs, were identified by proteomics. Random survival forest analysis showed that PIN1 could be a marker of PanNET metastatic ability. PIN1 overexpression promoted metastasis through LAMIN in PanNET cells. Finally, a survival signature with a consistency index of 0.921 and strong calibration was established, with good ability to predict patient survival. Conclusion In summary, we found that PIN1 can precisely evaluate the metastatic potential of PanNETs and, together with the proliferation marker Ki-67, can accurately predict the survival of PanNET patients.

AJCC TNM stage and WHO grade (G) are two widely used staging systems to guide clinical management for pancreatic neuroendocrine neoplasms (panNENs), based on clinical staging and pathological grading information, respectively. We proposed to integrate TNM stage and G grade into one staging system (TNMG) and to evaluate its clinical application as a prognostic indicator for panNENs. Accordingly, 5254 patients diagnosed with panNENs were used to evaluate and to validate the applicability of TNMG to panNENs. The predictive accuracy of TNMG system was compared with that of each separate staging/grading system. We found that TNM stage and G grade were independent risk factors for survival in both the Surveillance, Epidemiology, and End Result (SEER) and multicenter series. The interaction effect between TNM stage and G grade was significant. Twelve subgroups combining the TNM stage and G grade were proposed in the TNMG stage, which were classified into five stages TNMG. According to the TNMG staging classification in the SEER series, the estimated median survival for stages I, II, III, IV, and V were 203, 174, 112, 61, and 8 months, respectively. The predictive accuracy of TNMG stage was higher than that of TNM stage and G grade used independently. The TNMG stage classification was more accurate in predicting panNEN patient's prognosis than either the TNM stage or G grade.

Inhibition of immunocyte infiltration and activation has been proven to effectively ameliorate hepatic inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Paired immunoglobulin-like receptor B (PirB) and its human orthologue receptor, leukocyte immunoglobulin-like receptor B (LILRB2), are immune-inhibitory receptors with unknown roles in NASH. Here, we demonstrate that PirB/LILRB2 regulates the migration of macrophages in NASH pathogenesis and fibrogenesis by binding to its NASH-associated ligand angiopoietin-like protein 8 (ANGPTL8). Mechanistically, PirB facilitates the ANGPTL8-induced infiltration of monocyte-derived macrophages (MDMs) into the liver by regulating the phosphorylation of P38, AKT, and P65. Hepatocyte-specific knockout of its ligand ANGPTL8 reduces MDM infiltration and resolves lipid accumulation and fibrosis progression in the livers of NASH mice. In addition, PirB−/− bone marrow (BM) chimaeras abrogated ANGPTL8-induced MDM migration to the liver. PirB ectodomain protein can ameliorate the lipid accumulation inflammatory response and fibrosis of NASH by sequestering ANGPTL8. Furthermore, LILRB2-ANGPTL8-axis-associated MDM migration and inflammatory activation are also observed in human peripheral blood monocytes. Taken together, our findings reveal a novel role of PirB/LILRB2 in NASH pathogenesis and identify PirB/LILRB2-ANGPTL8 signalling as a potential target for the management or treatment of NASH.

The aim of this study was to compare the short- and long-term outcomes of laparoscopic surgery (LS) and open surgery (OP) for perihilar cholangiocarcinoma (PHC) using a large real-world dataset in China.Data of patients with PHC who underwent LS and OP from January 2013 to October 2018, across 10 centers in China, were extracted from medical records. A comparative analysis was performed before and after propensity score matching (PSM) in the LS and OP groups and within the study subgroups. The Cox proportional hazards mixed-effects model was applied to estimate the risk factors for mortality, with center and year of operation as random effects.A total of 467 patients with PHC were included, of whom 161 underwent LS and 306 underwent OP. Postoperative morbidity, such as hemorrhage, biliary fistula, abdominal abscess, and hepatic insufficiency, was similar between the LS and OP groups. The median overall survival (OS) was longer in the LS group than in the OP group (NA vs. 22 months; hazard ratio [HR] 1.19, 95% confidence interval [CI] 1.02-1.39, p = 0.024). Among the matched datasets, OS was comparable between the LS and OP groups (NA vs. 35 months; HR 0.99, 95% CI 0.77-1.26, p = 0.915). The mixed-effect model identified that the surgical method was not associated with long-term outcomes and that LS and OP provided similar oncological outcomes.Considering the comparable long-term prognosis and short-term outcomes of LS and OP, LS could be a technically feasible surgical method for PHC patients with all Bismuth-Corlett types of PHC.

Non-alcoholic steatohepatitis (NASH) is an advanced form of non-alcoholic fatty liver disease characterized by hepatosteatosis, liver cell injury, and inflammation. The pathogenesis of NASH involves dysregulated transcription of genes involved in critical processes in the liver, including metabolic homeostasis and inflammation. Chromatin immunoprecipitation (ChIP) utilizes antibody-mediated immunoprecipitation followed by the detection of associated DNA fragments via real-time PCR or high-throughput sequencing to quantitatively profile the interactions of proteins of interest with functional chromatin elements. Here, we present a detailed protocol to study the interactions of DNA and chromatin-associated proteins (e.g., transcription factors, co-activators, co-repressors, and chromatin modifiers) and modified histones (e.g., acetylated and methylated) in isolated primary mouse hepatocytes and mouse liver. The application of these methods can enable the identification of molecular mechanisms that underpin dysregulated hepatic processes in NASH.

Although non-alcoholic steatohepatitis (NASH) can progress to liver cancer and liver failure, no FDA-approved drugs exist to treat NASH. Deciphering the molecular mechanisms underlying the pathogenesis of NASH will facilitate the development of effective treatments for NASH, and requires loss- or gain-of-function experimental approaches. While genetically modified animals provide important information about the function of a gene, adenovirus is a fast, effective, and versatile tool that allows transient knockdown, knockout, or overexpression of one or more genes of interest (GOIs) in primary hepatocytes in vitro and in mouse liver in vivo. In addition, adenovirus is a promising treatment method in preclinical animal models, including rodents and non-human primates, and is used in many clinical trials. Here, we describe a step-by-step protocol to generate adenovirus for basic medical research. We discuss critical steps during virus propagation and purification and provide notes about how to avoid common pitfalls.

Copper deficiency has emerged to be associated with various lipid metabolism diseases, including non-alcoholic fatty liver disease (NAFLD). However, the mechanisms that dictate the association between copper deficiency and metabolic diseases remain obscure. Here, we reveal that copper restoration caused by hepatic ceruloplasmin (Cp) ablation enhances lipid catabolism by promoting the assembly of copper-load SCO1-LKB1-AMPK complex. Overnutrition-mediated Cp elevation results in hepatic copper loss, whereas Cp ablation restores copper content to the normal level without eliciting detectable hepatotoxicity and ameliorates NAFLD in mice. Mechanistically, SCO1 constitutively interacts with LKB1 even in the absence of copper, and copper-loaded SCO1 directly tethers LKB1 to AMPK, thereby activating AMPK and consequently promoting mitochondrial biogenesis and fatty acid oxidation. Therefore, this study reveals a mechanism by which copper, as a signaling molecule, improves hepatic lipid catabolism, and it indicates that targeting copper-SCO1-AMPK signaling pathway ameliorates NAFLD development by modulating AMPK activity.

Laparoscopic duodenum-preserving total pancreatic head resection (LDPPHRt) is used for treating benign or low-grade malignant tumors of the pancreatic head. However, preservation of the duodenum and biliary tract integrity remains challenging. We present a new approach for LDPPHRt and evaluate its feasibility and safety.From April 2020 to December 2020, 30 patients successfully underwent LDPPHRt using the intracapsular approach in our center. Their medical records were reviewed for relevant clinical characteristics, pathologic findings, postoperative complications, and survival.The median diameter of the lesions was 3.6 cm (range, 2.0-5.5 cm). The median operative time was 234.7 min (range, 195-310 min). The median blood loss was 66.7 ml (range, 20-250 ml). The morbidity rate was 26.7%, including POPF, hemorrhage, lymphatic leakage, wound infection, pulmonary infection, and delayed gastric emptying. Five patients developed pancreatic fistula type A, and two patients had type B, classified according to the International Study Group on Pancreatic Fistula. No biliary tract injury or duodenal leakage was observed. The median postoperative hospital stay was 11.5 days (range, 6-25), and the operative mortality rate was 0%.The intracapsular approach is a feasible and safe surgical procedure in LDPPHRt for patients with benign or low-grade malignant tumors, especially those without severe pancreatic head fibrosis or peripancreatic adhesions.

Copper is an essential nutrient and a co-factor of numerous enzymes governing a wide range of intracellular processes. Copper deficiency has emerged to be associated with various lipid metabolism diseases, including non-alcoholic fatty liver disease (NAFLD). However, the molecular mechanisms of how copper regulates lipid metabolism and is sensed remain elusive. Here, we reveal that copper elevation caused by hepatic ceruloplasmin (CP) ablation enhances lipid catabolism by promoting the assembly of copper-load SCO1/AMPK complex. We report that overnutrition-mediated CP elevation results in hepatic copper loss, and that liver-specific CP ablation counteracts this reduction in copper levels and ameliorates NAFLD in mice. Mechanistically, SCO1 constitutively interacts with LKB1 even in the absence of copper, and copper-loaded SCO1 directly tethers LKB1 to AMPK, thereby activating AMPK and consequently promoting mitochondrial biogenesis and fatty acid oxidation in hepatocytes. Therefore, this study reveals an unexpected role for AMPK to sense copper alteration via SCO1 and uncovers a previously unidentified mechanism by which copper, as a signaling molecule, improves hepatic lipid catabolism, and indicates that targeting copper-AMPK signaling pathway ameliorates NAFLD development by modulating AMPK activity.

The elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiological and pathological states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ is abundantly expressed in pericentral hepatocytes and its expression is markedly reduced by fasting. TAZ interacts via its WW domain with the ligand-binding domain of GR to limit the binding of GR to the GR response element in gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate that hepatic TAZ inhibits GR transactivation of gluconeogenic genes and coordinates gluconeogenesis in response to physiological fasting and feeding.

Due to the high-efficiency energy absorption and high-tension strength material properties of spider silk, many researchers have studied the mechanical properties and microstructure of the spider web. The concept of spider web structure has been recognized to be adopted for structural engineering aspect. The structure of spider web and its material properties have been studied for decades. However, the fundamental free vibration mode shapes and their corresponding frequencies have never been fully investigated. This study investigates the nonlinear characteristics in the large-amplitude free vibration of imperfect spider web structures using finite element analysis. The spider web applies the concept of elastic cables taking only axial deformation into account. The finite element models of a spider web considering geometric nonlinearities are employed. It should be noted that spider web could experience large deformation when the spider uses its silk to catch prey. This research aims at analyzing the linear and geometric nonlinear behaviour of imperfect spider web structure. Four different types of imperfect spider web: spiral imperfect spider web, radial imperfect spider web, central imperfect spider web, and circular rings imperfect spider web, are considered. It is found that pretension in spider silk plays a significant role in nonlinear vibration characteristics of the spider web. Moreover, the radial thread damaged tends to have a greater effect on structural free vibration of spider web in comparison with other imperfections. The outcome will help a structural engineer to adapt the concept of spider web, its properties, and damage patterns for any larger structures.

Background:There is no widely-accepted staging system for pancreatic neuroendocrine tumors (pNETs). The aim of this study was to validate the American Joint Committee on Cancer (AJCC) 8th edition staging system for well-differentiated (G1/G2) pNETs.Methods:A multicenter dataset (n=1086) was used to evaluate the application of the AJCC 7th and 8th, the European Neuroendocrine Tumor Society (ENETS), and the modified ENETS (mENETS) staging systems for well-differentiated pNETs.Results:The proportion of patients with stage III tumors was extremely low (1.1%) according to the AJCC 7th staging system. For the ENETS staging system, patients with stage IIIA disease had worse estimated mean survival than patients with stage IIIB disease (78.9 vs. 107.3 months). When comparing with patients in stage I, the AJCC 7th, ENETS, and mENETS staging systems showed good performance in discriminating between stages; however, there was no significant difference in some stages when the reference was defined as the earlier stage. When the reference was defined as stage I or the earlier stage, there was a significant inter-stage difference in the AJCC 8th staging system.Conclusions:The AJCC 8th staging system is more suitable for pNETs than other TNM staging systems and may be adopted in clinical practice.

Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2Kb, I-Ab) recipients pre-treated with BALB/c MDSCs were transplanted with either donor-type (BALB/c, H2Kd, I-Ad) or third-party (C3H, H2Kk, I-Ak) cardiac grafts. Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b+Gr1+ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b+Gr1+ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient’s endogenous MDSCs.

BackgroundPancreatic cancer is one of the most lethal human cancers.N6-methyladenosine (m6A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive.MethodsLC/MS was used to profile m6A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m6A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14.ResultsWe found that the m6A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m6A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo,via direct targeting of the downstreamPERPmRNA (p53 effector related to PMP-22) in an m6A-dependent manner. Methylation of the target adenosine lead to increasedPERPmRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells.ConclusionsOur data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m6A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.

Purpose: Cancer stem cells (CSCs) initiate and maintain tumorigenesis due to their unique pluripotency. However, pancreatic stem cell gene signatures are not completely revealed yet. Here, we isolated pancreatic cancer stem cells (P-CSCs) and exploited their distinct genome-wide mRNA and miRNA expression profiles using microarrays. Methods: CD24+ CD44+ ESA+ cells were isolated from two pancreatic xenograft cells by the flow cytometry and identified the stem cell-like properties by the tumor formation, self-renew and chemoresistance. Microarrays and qRT-PCR were used to exploit their distinct Genome-wide mRNA and miRNA expression profiles. The function and candidate target genes of key microRNA were detected after Ectopic restoration in the pancreatic cancer cell lines MIA Paca-2 (CSChigh) and BxPC-3 (CSClow). Results: In this study, we isolated P-CSCs from two xenografts cells. Genome-wide profiling experiments showed 479 genes and 15 microRNAs specifically expressed in the P-CSCs, including genes involved in TGF-β and p53 signaling pathways and particularly miR-146b-3p as the most significantly downregulated miRNA. We confirmed miR-146b-3p as a downregulated signature in pancreatic cancer tissues and cell line MIA Paca-2 (CSChigh) cells. Ectopic restoration of miR-146b-3p expression with pre-miR reduced cell proliferation, induced apoptosis, increased G1 phase and reduced S phase in cell cycle in MIA Paca-2 (CSChigh), but not in BxPC-3 (CSClow). Re-expression of miR-146b-3p with lentivirus significantly inhibited tumorigenicity in vivo in MIA Paca-2, but slightly in BxPC-3. Furthermore, we demonstrated that miR-146b-3p directly targeted MAP3K10 and might activate Hedgehog pathway as well through DYRK2 and GLI2. Conclusions: These results suggest that P-CSCs have distinct gene expression profiles. MiR-146b-3p inhibits proliferation and induced apoptosis in P-CSCs high cells lines by targeting MAP3K10. Targeting P-CSCs specific genes may provide novel strategies for therapeutic purposes.

The elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiologic and pathologic states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ interactsviaits WW domain with the ligand-binding domain of GR to limit the binding of GR to gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate distinct roles of the hippo pathway effector proteins yes-associated protein 1 (YAP) and TAZ in liver physiology: while deletion of hepatic YAP has little effect on glucose homeostasis, hepatic TAZ protein expression decreases upon fasting and coordinates gluconeogenesis in response to physiologic fasting and feeding.

The spinal cord is a cylinder structure in the vertebra and thought a simplified with the gray matter and white matter. Rexed lamination for the gray matter and regional sub-division for whiter matter are completely termed to date. Anterior commissure locates between the central canal and the anterior median fissure. However, some experimental data may still confront with new confined anatomical interpretation. By using NADPH diaphorase [N-d] enzyme histology, we found a vertical oriented neuronal pathway between the central canal and the anterior median fissure in the sacral spinal cord of young adult and aged dog. We used a term “supra fissure area” [SFA] to illustrate the region which consisted of the gray commissure and anterior white commissure. The N-d pathway was notably observable in aged animals. The vertical neurites revealed the cerebrospinal fluid [CSF] contacting neurites between the anterior median fissure and the central canal. We further examined the monkey, rat and pigeon in the region for better understanding of the structure and potential function. The neurodegeneration of N-d dystrophy was detected in the [SFA] in the thoracic spinal cord of the aged monkey. N-d positive fibers were detected in anterior fissure of the rat spinal cord. N-d fibrous structures were also detected in the pigeon spinal cord. These results suggested a new pathway of CSF contacting neurons and the neuronal communications about the central canal.

Additional file 6: Table S1. Association between clinicopathological features and m6A mRNA levels.

The Hippo signaling pathway plays a crucial role in regulating tissue homeostasis, organ size, tumorigenesis and cancer chemoresistance when deregulated. Physiologically, the Hippo core kinase cassette that consists of mamma-lian STE20-like protein kinase 1/2 (MST1/2), and large tumour suppressor 1/2 (LATS1/2), together with the adaptor proteins Salvador homologue 1 (SAV1) and MOB kinase activator 1 (MOB1), tightly restricts the activities of homologous oncoproteins Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) to low levels. However, how the Hippo kinase cassette core components are simultaneously inhibited, to exhibit constitutively inactivated Hippo signaling and activated YAP/TAZ in cancer remains puzzling. Herein, we reported that miR-181c directly repressed MST1, LATS2, MOB1 and SAV1 expression in human pancreatic cancer cells. Overexpression of miR-181c induced hyperactivation of the YAP/TAZ and enhanced expression of the Hippo signaling downstream genes CTGF, BIRC5 and BLC2L1, leading to pancreatic cancer cell survival and chemoresistance in vitro and in vivo. Importantly, high miR-181c levels were significantly correlated with Hippo signaling inactivation in pancreatic cancer samples, and predicted a poor patient overall survival. These findings provide a novel mechanism for Hippo signaling inactivation in cancer, indicating not only a potentially pivotal role for miR-181c in the progression of pancreatic cancer, but also may represent a new therapeutic target and prognostic marker.

Gate leakage of deep-submicron MOSFET with stack high-k dielectrics as gate insulator is studied by building a model of tunneling current. Validity of the model is checked for MOSFET's with SiO2 and high-k dielectric material as gate dielectric respectively, and simulated results exhibit good agreement with experimental data. The model is successfully used for a tri-layer gate-dielectric structure of HfON/HfO 2 /HfSiON with a U-shape nitrogen profile and a Si/SiO 2 -like interface, which is proposed to solve the problems of boron diffusion into channel region and high interface-state density between Si and high-k dielectric. By using the model, the optimum structural parameters of the tri-layer dielectric can be determined. For example, for an equivalent oxide thickness of 2.0 nm, the tri-layer gate-dielectric MOS capacitor with 0.3-nm HfON, 0.5-nm H f O2 and 1.2-nm HfSiON exhibits the lowest gate leakage.

Congenital adrenal hyperplasia (CAH) is a rare autosomal recessive disorder caused by mutations in the cytochrome P450 family 17 subfamily A member 1 (CYP17A1) gene located on chromosome 10q24.3, which leads to a deficiency in 17α-hydroxylase/17,20-lyase. The disorder is characterized by low blood levels of estrogens, androgens and cortisol, which leads to a compensatory increase in adrenocorticotropic hormone levels that stimulate the production of mineralocorticoid precursors. This subsequently leads to hypertension, hypokalemia, primary amenorrhea and sexual infantilism. Over 90 distinct genetic lesions have been identified in patients with this disorder. The prevalence of common mutation of CYP17A1 gene differs among ethnic groups. Treatment of this disorder involves replacement of glucocorticoids and sex steroids. Estrogen alone is prescribed for patients who are biologically male with 17α-hydroxylase deficiencies that identify as female. However, genetically female patients may receive estrogen and progesterone supplementation. In the present study, a 17-year-old female with 17α-hydroxylase/17,20-lyase deficiency that presented with primary amenorrhea and sexual infantilism and no hypertension, was examined. The karyotype of the patient was 46, XX, and genetic analysis revealed the presence of a compound heterozygous mutation in exons 6 and 8, leading to the complete absence of 17α-hydroxylase/17,20-lyase activity. The patient was treated with prednisolone and ethinyl estradiol. In addition, a summary of the recent literature regarding CAH is presented.