Search

[Background and Aims] NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD., [Approach and Results] We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation., [Conclusions] These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD., Supported by C0120R3166, C0245R4032, and BH182173 from Newcastle University. M. G.-M. is a Sara Borrell Postdoctoral fellow (CD18/00203) from the Ministerio de Ciencia, Innovación y Universidades (Spain). J. P. B. is funded by the Agencia Estatal de Investigación, grants PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018-102576-T, Instituto de Salud Carlos III (CB16/10/00282), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA, and Fundación Ramón Areces. V. I. K. acknowledges support from Biotechnology and Biological Sciences Research Council (BB/M023389/1, BB/R008167/1, BB

Building on theory suggesting that loneliness is distinct from living arrangements, social isolation, and perceived social support, we examined change in loneliness for older people at the onset of the COVID-19 pandemic. Analyzing 14-years of data with multilevel mixed-effects models, we found higher levels of loneliness among people living alone, people more socially isolated, and people with less perceived support. Gender affected changes in loneliness, controlling for social isolation, perceived support, living arrangements, age, education, income, health, and marital status. Women, whether living alone or with others, experienced increases in loneliness; women living alone reported the greatest increase in loneliness. Men living alone reported high levels of loneliness prior to the pandemic, but only a slight increase over time. These analyses, which demonstrate that loneliness changed at the onset of the pandemic as a function of gender and living arrangement identify older people most likely to benefit from intervention.

Background Pancreatic cancer cells exist in a hypoxic microenvironment containing numerous factors that impact tumor survival, proliferation, and metastasis. MicroRNAs (miRs) are differentially expressed in cancer but also altered by hypoxia. We hypothesized that hypoxia could induce expression of miR-21, an oncomir in pancreatic cancer cells. Materials and methods We examined how hypoxia regulates miR-21 expression in pancreatic cancer cell lines (BxPC-3, AsPC-1) by stem-loop RT-PCR. Chromatin immunoprecipitation assays were used to study how hypoxia alters hypoxia-inducible factor (HIF)-1α binding to the hypoxia response element of miR-21. BxPC-3 and AsPC-1 cells were transfected with a constitutively stable HIF-1α subunit or vector control (pcDNA3.1) to determine the influence of miR-21 in normoxia. The effect of mature miR-21 sense and antisense oligonucleotides on proliferation and apoptosis in hypoxic and normoxic conditions was assessed via WST-1 assay and flow cytometry. Results MiR-21 levels increased in all cell lines grown in hypoxic conditions versus normoxia, whereas siRNA targeting HIF-1α reduced miR-21 expression. Hypoxic conditions resulted in direct binding of HIF-1α to the predicted binding site in miR-21. Transfection with a constitutively stable HIF-1α expression plasmid in normoxia resulted in upregulated miR-21, similar to that seen in hypoxia. Cells transfected with antisense constructs targeting miR-21 had reduced proliferation and increased apoptosis in normoxia, whereas miR-21 overexpression abrogated hypoxia-associated reductions in proliferation. Conclusions MiR-21 is induced by hypoxia in pancreatic cancer cells via HIF-1α upregulation. MiR-21 overexpression allows cells to avoid apoptosis in a hypoxic microenvironment. Inhibition of miR-21 expression may increase cellular susceptibility to hypoxia in pancreatic cancer.

Cancers of the bile duct and the pancreas are virtually indistinguishable using conventional histopathological and clinical characteristics. We sought to use microRNA (miR) profiling to differentiate these two cancers.RNA was harvested from the tumors of patients undergoing curative resection for cholangiocarcinoma or pancreatic adenocarcinoma and compared with adjacent normal bile duct or pancreas, respectively. There were 31 pairs of cholangiocarcinoma with matched tumor and adjacent bile duct and nine pairs of pancreatic cancer with matched tumor and adjacent uninvolved pancreas that had sufficient quantity of RNA that were included in the final analysis. Differential microRNA expression profiles were determined using the nCounter System from nanoString Technologies (Seattle, WA,USA).A total of 41 differentially expressed miRs were identified in cholangiocarcinoma (25 overexpressed, 16 underexpressed) and 52 differentially expressed miRs were found in pancreatic adenocarcinoma (30 overexpressed, 22 underexpressed) relative to adjacent normal tissue. Of these two profiles, 15 miRs were commonly dysregulated between tumor types. Also, eight miRs were similarly overexpressed or underexpressed in cholangiocarcinoma and pancreatic adenocarcinoma, whereas the other seven miRs had inverse expression levels.Cholangiocarcinoma has a distinct miR profile from pancreatic adenocarcinoma. Discrimination between these two tumor types may be possible with as few as seven miRs.

Osteopontin (OPN) is a secreted protein of the extracellular matrix. It has been used as a marker for tumor aggressiveness and correlated with clinical outcomes in several solid tumors, such as liver, lung, and breast. We determined the OPN expression and its influence on survival in patients with resected pancreatic adenocarcinoma.Tissue microarrays were constructed from 245 resected pancreatic adenocarcinomas. Immunohistochemical staining for OPN was undertaken and compared to normal pancreas (n = 12). OPN expression was then correlated with patient demographics, tumor size, grade, node, and margin status. Survival curves were created by the Kaplan-Meier method and compared by log rank analysis.In total, 181 (74 %) of pancreatic adenocarcinoma tissues expressed OPN compared to 7 (58 %) of normal controls (p = 0.004). Expression was observed predominantly in the cytoplasm of the tumor cells. The median and 2 year overall survival was longer when OPN was expressed (17.1 vs. 11.6 months, and 38 vs. 24 %, respectively, p = 0.04). Multivariate analysis showed OPN expression and T stage to be independent predictors of overall survival, while other histopathologic factors such as tumor grade, tumor size, and nodal status were not.These results suggest that the presence of OPN expression in pancreatic adenocarcinoma may have a protective effect independent of tumor stage. This emphasizes the importance of the interaction between pancreatic cancer cells and their stromal elements.

Objectives: The authors examined the resilience of self-esteem after loss in the lives of older adults. Specifically, the authors investigated the relationship between loss and change in self-esteem during a 3-year period.Method: A subsample of older adults ( n = 1,278) from the Americans’ Changing Lives Study was used to examine loss in the domains of health, financial security, or work and career and self-esteem before and after the loss.Results: There was a small but significant decrease in self-esteem between Wave I and Wave II of the study. Loss in one of the domains explained less than 1% of the variance in self-esteem change.Discussion: The low incidence of loss and small change in high levels of self-esteem are further evidence of resilience in older adults’ psychological well-being. The implications for older adults’ use of cognitive strategies to manage losses and promote gains are discussed.

Pancreatic stellate cells (PSC) are a subset of pancreatic cancer-associated fibroblasts. These cells provide prosurvival signals to tumors; however, little is known regarding their interactions with immune cells within the tumor microenvironment. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes an immunosuppressive microenvironment. Primary PSC cell lines (n = 7) were generated from human specimens and phenotypically confirmed via expression of vimentin, α-smooth muscle actin (α-SMA), and glial fibrillary acidic protein (GFAP). Luminex analysis indicated that PSC but not human fetal primary pancreatic fibroblast cells (HPF; negative controls) produced MDSC-promoting cytokines [interleukin (IL-6), VEGF, macrophage colony-stimulating factor (M-CSF) ] and chemokines (SDF-1, MCP-1). Culture of peripheral blood mononuclear cells [peripheral blood mononuclear cell (PBMC), n = 3 donors] with PSC supernatants or IL-6/granulocyte macrophage colony-stimulating factor (GM-CSF; positive control) for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype and a subpopulation of polymorphonuclear CD11b+CD33+CD15+ cells. The resulting CD11b+CD33+ cells functionally suppressed autologous T-lymphocyte proliferation. In contrast, supernatants from HPF did not induce an MDSC phenotype in PBMCs. Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. IL-6 was an important mediator as its neutralization inhibited PSC supernatant-mediated STAT3 phosphorylation and MDSC differentiation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC supernatant-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent. These results identify a novel role for PSC in driving immune escape in pancreatic cancer and extend the evidence that STAT3 acts as a driver of stromal immunosuppression to enhance its interest as a therapeutic target. Cancer Res; 73(10); 3007–18. ©2013 AACR.

The construct validity of the Verbal Comprehension, Perceptual Organization, and Freedom from Distractibility factor scores was examined in a sample of school-aged referred children. Examination of correlations between factor scores and neuropsychological and achievement tests generally supported the construct validity of the factors. The Verbal Comprehension factor was associated with verbal, quantitative, and concept-formation abilities. The Perceptual Organization factor was related to nonverbal concept formation, tactual performance, and visual attention. The Freedom from Distractibility factor demonstrated a complex pattern of correlations and appeared to reflect a range of abilities including quantitative, language, attentional, and concept formation.

Background MicroRNAs (miRNAs) are small non‐coding genes which become dysregulated in cancer and may predict survival. The role of miRNAs in outcomes in cholangiocarcinoma (CC) has not been reported. Methods RNA was extracted from 32 resected CCs along with adjacent uninvolved bile duct epithelium. A total of 43 miRNAs were quantified using NanoString™. Clinicopathologic characteristics and outcomes were captured and compared. Overall survival curves were created using the Kaplan–Meier method; factors, including miRNA expression, were compared by log‐rank, chi‐squared or Cox regression analyses. Results Absolute expression of each miRNA was compared with overall survival after excluding perioperative deaths ( n = 3). One upregulated (miR‐151‐3p; P = 0.003) and one downregulated (miR‐126; P = 0.023) miRNA in resected CC relative to adjacent normal bile duct epithelium correlated with survival on univariate analysis. Clinical factors and these miRNAs were compared. Dysregulated miR‐151‐3p and miR‐126, respectively, were the only factors that correlated with improved overall survival [41.5 months vs. 12.3 months ( P = 0.002) and 21.9 months vs. 15.1 months ( P = 0.02), respectively]. In eight patients, both miRNAs were dysregulated. In the remainder, only one or neither showed dysregulation. Concomitant dysregulation correlated with the best overall survival (58.7 months vs. 15.1 months; P n = 8); clinicopathologic factors in these groups were otherwise similar. conclusions In resected CC, the concomitant dysregulation of both miR‐151‐3p and miR‐126 was the factor related to the greatest improvement in overall survival. Further analysis of the targets of these miRNAs may yield potential therapeutic targets or prognostic biomarkers.