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Proprotein convertase subtilisin/kexin type 9 (Pcsk9) binds to hepatic low‐density lipoprotein receptor (LDLR) and induces its internalization and degradation. Pcsk9 inhibition increases LDLR expression by hepatocytes, which causes increased uptake of circulating LDL, thereby reducing plasma LDL‐cholesterol. However, by increasing the uptake of LDL by the liver, Pcsk9 inhibition increases the exposure of the liver to cholesterol, which may result in higher risk of steatohepatitis and ever carcinogenesis. We compared Pcsk9‐/‐ knockout (KO) mice and appropriate wild‐type (WT) controls of the same strain assigned to a high‐fat (15%, wt/wt) diet for 9 months supplemented with 0.25%, 0.5%, or 0.75% dietary cholesterol. Pcsk9 KO mice on a high‐fat, high‐cholesterol diet exhibited higher levels of hepatic free cholesterol loading and hepatic cholesterol crystallization than their WT counterparts. Pcsk9 KO mice developed crown‐like structures of macrophages surrounding cholesterol crystal‐containing lipid droplets and hepatocytes, exhibited higher levels of apoptosis, and developed significantly more hepatic inflammation and fibrosis consistent with fibrosing steatohepatitis, including 5‐fold and 11‐fold more fibrosis at 0.5% and 0.75% dietary cholesterol, respectively. When injected with diethylnitrosamine, a hepatic carcinogen, early‐in‐life Pcsk9 KO mice were more likely to develop liver cancer than WT mice. Conclusion: Pcsk9 KO mice on high‐cholesterol diets developed increased hepatic free cholesterol and cholesterol crystals and fibrosing steatohepatitis with a higher predisposition to liver cancer compared with WT mice. Future studies should evaluate whether patients on long‐term treatment with anti‐PSCK9 monoclonal antibodies are at increased risk of hepatic steatosis, steatohepatitis or liver cancer, while accounting for concurrent use of statins.

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.

It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs. Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis (P

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

Gallstones, particularly cholesterol gallstones, are common in Western populations and may cause symptoms such as biliary colic or complications such as acute cholecystitis or gallstone pancreatitis. Recent studies have allowed for a better understanding of the risk of symptoms or complications in patients with gallstones. In addition, newer data suggest an association of gallstones with overall mortality, cardiovascular disease, gastrointestinal cancers, and non-alcoholic fatty liver disease. Knowledge of appropriate indications and timing of cholecystectomy, particularly for mild biliary pancreatitis, has gradually accumulated. Lastly, there are exciting possibilities for novel agents to treat or prevent cholesterol stone disease. This review covers new advances in our understanding of the natural history, clinical associations, and management of gallstone disease.

A variety of diseases are included under the umbrella term ‘cholangitis’, including hepatobiliary diseases with an autoimmune pathogenesis (such as primary sclerosing cholangitis, primary biliary cholangitis, and IgG4-associated sclerosing cholangitis) and disease processes associated with intraductal stones and infectious etiologies (such as ascending bacterial cholangitis, recurrent pyogenic cholangitis, and liver fluke-associated cholangitis). Recent advances in the pathophysiologic bases of these disorders, particularly with respect to the autoimmune variety, are allowing improved diagnosis and prognostication as well as providing the opportunity to refine and re-imagine treatment modalities. The aim of this review is to highlight selected advances in cholangitis research that point to novel insights into the pathophysiology, diagnosis, and treatment of this diverse array of disorders.

Fluorescence energy transfer from dehydroergosterol (DHE) to dansylated lecithin (DL) was used to characterize lecithin-cholesterol vesicles in the presence of the bile salt, sodium taurocholate. At lipid concentrations approximating physiological levels, exposure of fluorescently labeled vesicles to the bile salt led to a dose-dependent increase in the DHE-to-DL fluorescence ratio during the first 24 h after mixing. The initial changes in the fluorescence ratio correlated well with conventional turbidity measurements that quantify partial micellization of vesicles as a function of bile salt loading. In addition, fluorescence energy transfer from DHE to DL revealed cholesterol enrichment of vesicles and re-vesiculation of micelles at bile salt loadings for which vesicles and micelles coexisted. Samples containing the cholesterol-enriched vesicle fraction exhibited further increases in the DHE-to-DL fluorescence ratio during a 4-week observation period but only after a significant lag period of several days. The lag period decreased with cholesterol loading, and the increase in the fluorescence ratio always preceded the appearance of microscopic, birefringent, either needlelike or platelike, cholesterol crystals, in samples that were initially supersaturated with cholesterol. Cholesterol crystals were not observed, and the fluorescence ratio did not increase, for any sample that was undersaturated with cholesterol. Taken together, these results suggest that the latter changes in fluorescence are the result of cholesterol nucleation. Fluorescence energy transfer from DHE to DL is therefore a promising technique for the characterization of model bile and, possibly, provides a direct measurement of cholesterol nucleation.—Wrenn, S. P., M. Gudheti, A. N. Veleva, E. W. Kaler, and S. P. Lee. Characterization of model bile using fluorescence energy transfer from dehydroergosterol to dansylated lecithin.

We demonstrate Förster resonance energy transfer from dehydroergosterol to dansylated lecithin in lecithin-cholesterol vesicles and characterize the vesicles in the presence of the pro-nucleating enzyme, phospholipase C (PLC). Exposure to phospholipase C causes a temporary decrease in the dehydroergosterol to dansyl fluorescence ratio followed by an increase to and above the initial value. The temporary decrease in the fluorescence ratio results from an increase in the dansylated lecithin intensity that coincides with a dansyl blue shift. The extent of the blue shift correlates with the level of diacylglycerol generated in situ by PLC, suggesting an increased association between dansylated lecithin and cholesterol as membrane fluidity increases and membrane polarity decreases. The subsequent increase in the fluorescence ratio results from both an increase in the dehydroergsterol intensity and a concomitant decrease in the dansylated lecithin intensity of equal magnitude. This signifies a reduction in energy transfer from dehydroergosterol to dansylated lecithin and indicates an increased separation between the two fluorophores. The increase in the fluorescence ratio persists beyond the time scales for vesicle aggregation and fusion, as measured by turbidity, and precedes the onset of macroscopic cholesterol crystals observed with an optical microscope. Thus, the increased separation between dehydroergosterol and dansylated lecithin is consistent with a mechanism of cholesterol nucleation from the vesicles. Moreover, the onset and rate of increase in the fluorescence ratio correlate with the cholesterol:lecithin mole ratio of the vesicles. Fluorescence energy transfer from dehydroergosterol to dansylated lecithin therefore shows potential as a methodology for measuring cholesterol nucleation in model bile.—Wrenn, S. P., E. W. Kaler, and S. P. Lee. A fluorescence energy transfer study of lecithin-cholesterol vesicles in the presence of phospholipase C. J. Lipid Res. 1999. 40: 1483–1494.

The combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile are reported. The results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms.

Kimono (a traditional Japanese garment worn by women) has played an important role in Japanese indigenous cultural origins. Ecological and sustainable ideas have inherently existed in kimono culture within this lifestyle. Since the United Nations announced the Sustainable Development Goals (SDGs) in 2015, the Japanese government has focused on creating healthy spaces with a sustainable direction in mind. However, textile-related product recycling rates were relatively low in Japan at the time. This empirical study used SDGs as a conceptual framework to examine Japanese college students’ perceptions of kimono upcycling practices and challenges. A mixed method was used to analyze the data. An open-ended questionnaire was distributed to college students in June 2019 in Liberal Arts at a national women’s university (n=155). The findings showed that interest in kimono upcycling moderately correlated to those and an interest in western garments upcycling (.578; p

The COVID-19 pandemic complicates ingrained educational inequalities around the globe and foregrounds the pertaining challenges that teachers have encountered due to school closures and the shift to distance learning. This cross-border teacher education project intended to examine how academics and pre-service teachers in different geographic locales could collaboratively explore equitable learning opportunities for diverse learners through the use of critical media literacies to respond to interconnected global crises. In this six-week cross-border teacher education project, we recruited four Mandarin and English literacy teacher candidates in Hong Kong to interact with one another and one Canadian professor as part of the teacher preparation phase of a larger-scale cross-border research project that connects youth from Hong Kong and Canada in a social networking space. For the purposes of teacher professional development, the Hong Kong teacher candidates and Canadian researchers engaged in collective exploration of how instructional designs in literacy education could promote equitable learning opportunities for diverse learners. Findings show that the cross-border teacher education project supported teacher candidates’ development of pedagogical skills and espoused their agency in promoting educational equity and collective problem-solving through critical media literacies. Findings relate the teacher candidates’ shifted perspectives from focusing on students’ decontextualized language skills to nurturing critical media skills. Changing from a deficit-oriented view about what literacy learners could not do, the teacher candidates also adopted an asset-oriented view about the linguistic and cultural repertoires that diverse learners could bring to literacy classrooms.

We present tropospheric nitrogen dioxide (NO2) changes observed by the Canadian Pandora measurement program in the Greater Toronto Area (GTA), Canada, and compare the results with surface NO2 concentrations measured via in situ instruments to assess the local emission changes during the first two years of the COVID-19 pandemic. In the City of Toronto, the first lockdown period started on 15 March 2020, and continued until 24 June 2020. ECMWF Reanalysis v5 (ERA-5) wind information was used to facilitate the data analysis and reveal detailed local emission changes from different areas of the City of Toronto. Evaluating seven years of Pandora observations, a clear NO2 reduction was found, especially from the more polluted downtown Toronto and airport areas (e.g., declined by 35% to 40% in 2020 compared to the 5-year mean value from these areas) during the first two years of the pandemic. Compared to the sharp decline in NO2 emissions in 2020, the atmospheric NO2 levels in 2021 started to recover, but are still below the mean values in pre-pandemic time. For some sites, the pre-pandemic NO2 local morning rush hour peak has still not returned in 2021, indicating a change in local traffic and commuter patterns. The long-term (12 years) surface air quality record shows a statistically significant decline in NO2 with and without April to September 2020 observations (trend of −4.1%/yr and −3.9%/yr, respectively). Even considering this long-term negative trend in NO2, the observed NO2 reduction (from both Pandora and in situ) in the early stage of the pandemic is still statistically significant. By implementing the new wind-based validation method, the high-resolution satellite instrument (TROPOMI) can also capture the local NO2 emission pattern changes to a good level of agreement with the ground-based observations. The bias between ground-based and satellite observations during the pandemic was found to have a positive shift (5–12%) than the bias during the pre-pandemic period.

In Canada, perfluoroalkyl acids (PFAAs) have been the focus of several monitoring programs and research and surveillance studies. Here, we integrate recent data and perform a multi-media assessment to examine the current status and ongoing trends of PFAAs in Canada. Concentrations of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and other long-chain perfluorocarboxylates (PFCAs) in air, water, sediment, fish, and birds across Canada are generally related to urbanization, with elevated concentrations observed around cities, especially in southern Ontario. PFOS levels in water, fish tissue, and bird eggs were below their respective Draft Federal Environmental Quality Guidelines, suggesting there is low potential for adverse effects to the environment/organisms examined. However, PFOS in fish and bird eggs tended to exceed guidelines for the protection of mammalian and avian consumers, suggesting a potential risk to their wildlife predators, although wildlife population health assessments are needed to determine whether negative impacts are actually occurring. Long-term temporal trends of PFOS in suspended sediment, sediment cores, Lake Trout (Salvelinus namaycush), and Herring Gull (Larus argentatus) eggs collected from Lake Ontario increased consistently from the start of data collection until the 1990s. However, after this time, the trends varied by media, with concentrations stabilizing in Lake Trout and Herring Gull eggs, and decreasing and increasing in suspended sediment and the sediment cores, respectively. For PFCAs, concentrations in suspended sediment, sediment cores, and Herring Gulls generally increased from the start of data collection until present and concentrations in Lake Trout increased until the late 1990s and subsequently stabilized. A multimedia comparison of PFAA profiles provided evidence that unexpected patterns in biota of some of the lakes were due to unique source patterns rather than internal lake processes. High concentrations of PFAAs in the leachate and air of landfill sites, in the wastewater influent/effluent, biosolids, and air at wastewater treatment plants, and in indoor air and dust highlight the waste sector and current-use products (used primarily indoors) as ongoing sources of PFAAs to the Canadian environment. The results of this study demonstrate the utility of integrating data from different media. Simultaneous evaluation of spatial and temporal trends in multiple media allows inferences that would be impossible with data on only one medium. As such, more co-ordination among monitoring sites for different media is suggested for future sampling, especially at the northern sites. We emphasize the importance of continued monitoring of multiple-media for determining future responses of environmental PFAA concentrations to voluntary and regulatory actions. Keywords: Perfluoroalkyl acids, Canada, Multi-media analysis, Trends, Federal environmental quality guidelines, Potential sources

Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.