In many real world examples, the number of tuple fields produced by some generator can grow to be quite cumbersome.
Often, a subquery will only want to deal with a small subset of the fields produced. Cascalog provides a few methods that make it quite easy to deal with these wide taps.
This works, but is still a bit clunky. Next choice: select-fields.
If generator is some other subquery with named tuple fields, we can use select-fields to pull ?a and ?b without knowing their position.
Note that select-fields takes a generator and a sequence of (string representations of) variables, and returns a new generator that produces only those variables.
:#> sits to the right of our generator, and accepts two items: the total number of fields produced by the generator, and a map of variable names keyed by position within the generator. Variables names don't have to match the names on the original output tap, as position is already specified.
This documentation site is open source and we welcome pull requests.
Cascalog is an open source data processing and querying library for Clojure or Java licensed under Apache License, Version 2.0.


Just Windows and Doors Supply Company of St. Louis, MO supplies and installs new and replacement vinyl, wood, aluminum, and historical windows for residential and commercial clients. We also offer vinyl, steel, and fiberglass patio and exterior doors.We sell to builders building new homes and remodeling contractors for replacement windows.
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We have an excellent reputation and a high percentage of our business is from repeat customers and referrals. We have a long list of satisfied clients willing to provide a reference on our behalf.


Top Ten Reasons Why It Costs More than your own haircut to Get Your Canine Groomed.
3. Your stylist only washes and cuts the hair on your head.
4. Your haircut does not include a manicure or pedicure.
6. Your stylist does not remove the boogies from your eyes.
7. Your stylist does not clean your ears.
10. Your stylist does not wash and clean your groin.


Memphis native Bill Rhodes' path to becoming chairman, president and CEO of AutoZone began with the swing of a golf club.
As a member of the UT Martin Pacer golf team, Rhodes made the honorable mention All- America and Academic All-America teams during his senior year. That, combined with his pursuit of an MBA at the University of Memphis, landed him an interview with the head audit partner at Ernst & Whinney (now Ernst & Young).
"As it turned out, he wanted to hire me as an intern—not because of my audit skills—but because they had a golf challenge between the audit, tax and consulting departments at E&W, and he was tired of audit losing," Rhodes says.
Twists and turns along the way led him to AutoZone, where he ultimately climbed the ladder to become, at age 39, the youngest president and CEO of a Fortune 500 company.
Rhodes says his golf coach, Grover Page, however, was the most influential person from his days at UT Martin.
The skills he learned on campus have served him well in business, where he leads a team of more than 84,000 employees in 50 states. He says he lives "in the zone," and has no intention to slow down, which is par for the course.


Rue de Marli's No. 27 Bois Precieux candle is made from clean-burning soy wax and a unique blend of pure essential oils that call to mind the scents of Provencal forests. Their minimalist design is reminiscent of traditional apothecary products and pays homage to their all-natural crafting methods. Let these candles transform your home or office into a bright, warm, comforting space.


text:
AEW World Trios Championship – tytuł mistrzowski, który jest specjalistycznym mistrzostwem tag team, o które walczą drużyny składające się z trzech wrestlerów, promowany przez federację All Elite Wrestling (AEW). Inaguracyjnymi mistrzami byli The Elite (Kenny Omega, Matt Jackson i Nick Jackson).

Historia
27 lipca 2022 roku, na specjalnym odcinku Dynamite nazwanym Fight for the Fallen, All Elite Wrestling (AEW) ogłosiło turniej o inauguracyjny AEW World Trios Championship, którego kulminacja była na pay-per-view All Out 4 września 2022 roku. W przeciwieństwie do AEW World Tag Team Championship, który jest standardowym tytułem tag team, o które walczą drużyny składające się z dwóch wrestlerów, Trios Championship obejmuje drużyny składające się z trzech wrestlerów.

Inauguracyjny turniej

Panowania
Na stan z   , były trzy panowania między sześcioma indywidualnymi mistrzami i jednym wakatem. The Elite (Kenny Omega i The Young Bucks (Matt Jackson i Nick Jackson)) byli inauguracyjnymi mistrzami i obecnie mają najkrótsze panowanie, które trwało 3 dni, podczas gdy obecne panowanie Death Triangle (Pac, Penta El Zero M i Rey Fénix) jest najdłuższe i trwało 126 dni. Omega jest najstarszym mistrzem, zdobywając tytuł w wieku 38 lat, a Rey Fénix jest najmłodszym, po zdobyciu tytułu w wieku 31 lat.

Obecnymi mistrzami są The House of Black (Malakai Black, Brody King i Buddy Matthews), którzy są w swoim pierwszym panowaniu. Pokonali poprzednich mistrzów The Elite (Kenny'ego Omegę, Nicka Jacksona i Matta Jacksona) na Revolution, 5 marca 2023.

Łączna liczba panowań
Stan na

Drużynowo

Indywidualnie

Przypisy

All Elite Wrestling
Tytuły mistrzowskie w AEW

redpajama_set_name: RedPajamaWikipedia


Discussion in 'Grappling Technique' started by daninnashville, Mar 12, 2008.
Not sure if you guys have read this. Kenny Florian talks about his training background. I really like it because he takes the time to address the different aspects of MMA training (grappling especially).
I've found it to be helpful.
KFlo is a good fighter no doubt!


Bill Atkinson is one of the original designers of the Apple Macintosh. He talks with Leo Laporte about General Magic, the company that tried to invent the iPhone in the early 90s. They also talk about human conciousness, artificial intelligence, and the need for empathy.


I think I found the value of being at home.
The one thing I have learned during my journeys in the past is that no matter how good they were; there is no place like home.
I have always been a very enthusiastic traveller, even from a young age. I cannot remember ever being overly homesick or nervous before a trip, I always took every opportunity I could get for a travel, which was probably more stressful for my parents than for me a lot of times. My first trip alone was with my best friend at the time (who is still my best friend to date, by the way!) and we went to a horse ranch, about one and a half hours from home for a week. I must have been about eight years old back then and probably had a great time, since I went to visit a new friend I had made there about one day after I got home for three days. So you see: no problem with homesickness.
When I got older, the journeys got more daring and exotic. I went to a lot of new countries with a lot of different people, recently having been to Portugal with three of my best friends. But no matter where I go and no matter how much fun I have, there is this feeling of being home I just cannot miss and which will always have me seek my home. The sense of domesticy when you just drove across half of Europe back to your country and hear the people talking in your language for the first time in weeks is overwhelming and I also find myself just thinking 'mine' when I drive through my entirely boring yet entirely charming hometown for the first time after a holiday.
The strongest sense of domesticy, however, is understandably in my family house and even more in my bedroom. The feeling of lying in your own bed is the most relaxing thing in the world and really does not compare to any beaches you could ever lie on. So, I am definitely feeling my 'home, sweet home'.


Below is a definition for a store and combobox (don't comment on the style of the store definition this is generated by the designer). You can see that a the combobox is defined to use the 'value' field for it's value and the 'name' field as its display fields. However calls to comboBox.getValue(), comboxBox.getRawValue() and comboBox.getDisplayValue() all return the content of the 'name' field while I was expecting the getValue() and getRawValue() functions to return the value of the value field.
How should I be configuring the combobox so the getValue() function returns the value rather than the name?
I see the same behavior when using 4.0.7 and the nightly build from 0404.
My own fault. The combo box does not default to the first item so it has to be populated in code. That code does sets only a 'name' not a 'value'.


Since 1985, over 65 scholarships have been awarded which have not only given the recipients financial assistance, but have enabled them to broaden their network and strengthen their skills and knowledge. SCI Scholars receive access to publishing and mentoring opportunities and are given a platform to present their work amongst esteemed scientists and industrialists, thus raising their profile within the scientific community. In the past eight years alone, SCI has generously bequeathed nearly £115,000 of its charitable funds to SCI Scholars and the scientists of the future.
James Adams was awarded an SCI Scholarship in 2015. Below, he tells us about himself and his research project.
'My passion for science began whilst at Wolgarston High School, where I undertook my A-levels. This passion developed and focussed during my undergraduate studies at the University of Nottingham where I studied Chemistry (2009-2013). During this time I received numerous awards for academic achievement; BP Achievement Award 2012, Prof F S Kipping Scholarship 2011, GSK Scholarship 2010, University of Nottingham Higher Achievers Award 2009.
'Throughout my time as an undergraduate I had the opportunity to work on several summer research projects in the fields of both organic (Dr J Camp) and inorganic chemistry (Prof M George). As my knowledge developed and I began to specialise, it became apparent that my main research interest was in medicinal chemistry. Consequently, I chose to work on a collaborative Master's research project with GSK involving the design and synthesis of αvβ6 integrin inhibitors for the treatment of idiopathic pulmonary fibrosis, that resulted in two publications; MacDonald et al ACS Med. Chem. Lett. 2014, 5, 1207-1212 and ACS Med. Chem. Lett. 2015, 6, 221-224.
'In 2013, I graduated with a First Class MSci, this was accompanied by the award of the GSK prize for outstanding work in medicinal chemistry. I then took a year out before starting a BBSRC DTP PhD studentship under the supervision of Dr Lydia Tabernero and Prof David Procter at The University of Manchester in September 2014. I have also received support in the form of a Presidents Doctoral Scholar Award and a Sir Kenneth Murray Scholarship.
'The research that I am currently undertaking involves the utilisation of a novel strategy to obtain selective cell permeable PTP inhibitors. PTPs are an attractive therapeutic target; this enzyme class represents 6% of the druggable genome and may be key to treating a plethora of diverse diseases ranging from diabetes to cancer.


We share thought-provoking articles that show you the next step for your business, improve your process, and inspire your design. Brought to you by Flywheel.
Web designer and social media coordinator for Newport News Shipbuilding, a division of @hiindustries. Happily married, and soon to be a dad.


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Pathophysiology| November 19 2018
HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization
Qiuyue Kong;
Qiuyue Kong
1Department of Anesthesiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China
Nan Li;
Nan Li
Hao Cheng;
Hao Cheng
Xiaojin Zhang;
Xiaojin Zhang
2Department of Geriatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing, China
Xiaofei Cao;
Xiaofei Cao
Tao Qi;
Tao Qi
Leyang Dai;
Leyang Dai
Zhihong Zhang;
3Department of Pathology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China
Xuan Chen;
Xuan Chen
Chuanfu Li;
Chuanfu Li
4Department of Surgery, East Tennessee State University, Johnson City, TN
Yuehua Li;
Yuehua Li
5Department of Pathophysiology, Nanjing Medical University, Nanjing, China
Bin Xue;
Bin Xue
6State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Key Laboratory of Molecular Medicine and School of Medicine, Nanjing University, Nanjing, China
Lei Fang;
Lei Fang
7Medical School of Nanjing University, Nanjing, China
Li Liu;
Corresponding authors: Zhengnian Ding, zhengnianding@njmu.edu.cn, and Li Liu, liuli@njmu.edu.cn
Zhengnian Ding
Diabetes 2019;68(2):361–376
Qiuyue Kong, Nan Li, Hao Cheng, Xiaojin Zhang, Xiaofei Cao, Tao Qi, Leyang Dai, Zhihong Zhang, Xuan Chen, Chuanfu Li, Yuehua Li, Bin Xue, Lei Fang, Li Liu, Zhengnian Ding; HSPA12A Is a Novel Player in Nonalcoholic Steatohepatitis via Promoting Nuclear PKM2-Mediated M1 Macrophage Polarization. Diabetes 1 February 2019; 68 (2): 361–376. https://doi.org/10.2337/db18-0035
Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease worldwide. Macrophage-mediated inflammation plays a critical role in NASH pathogenesis; however, optimum therapies for macrophage activation and NASH remain elusive. HSPA12A encodes a novel member of the HSP70 family. Here, we report that NASH patients showed increased hepatic HSPA12A expression and serum HSPA12A contents. Intriguingly, knockout of HSPA12A (Hspa12a−/−) in mice attenuated high-fat diet (HFD)–induced hepatic steatosis and injury. HFD-induced macrophage polarization toward an M1 phenotype and inflammatory responses in the liver of Hspa12a−/− mice were also attenuated. Loss- and gain-of-function studies revealed that the de novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A rather than by hepatocyte HSPA12A. In-depth molecular analysis revealed that HSPA12A interacted with the M2 isoform of pyruvate kinase (PKM2) in macrophages and increased its nuclear translocation, thereby promoting M1 polarization and secretion of proinflammatory M1 cytokines; this led, ultimately, to hepatocyte steatosis via paracrine effects. Taken together, these findings show that HSPA12A acts as a novel regulator of M1 macrophage polarization and NASH pathogenesis by increasing nuclear PKM2. Strategies that inhibit macrophage HSPA12A might be a potential therapeutic intervention for NASH.
Nonalcoholic fatty liver disease (NAFLD) affects ∼22–28% of the global population (1). The spectrum of NAFLD begins with isolated steatosis, with subsequent development of steatohepatitis (NASH) and steatofibrosis, eventually leading to cirrhosis and hepatocellular carcinoma. Patients with NAFLD, particularly NASH, show reduced survival, primarily due to cardiovascular and liver-related diseases (2). NASH livers are more injured than livers with isolated steatosis, which more likely leads to progressive fibrosis and eventual liver-associated illness and death (3). The molecular players that modulate disease pathogenesis are starting to be identified, enabling translation of research findings to clinical trials (4,5). However, no optimum therapy is yet available, suggesting that a more comprehensive understanding of the onset and progression of NAFLD is needed.
The shift from isolated steatosis to NASH is defined histologically as emergence of liver inflammation and hepatocellular injury. The most common theory explaining this shift is the "double-hit hypothesis" (3,5,6). The first hit consists mainly of lipid accumulation, whereas the second comprises sequential innate immune responses (6–8). Compelling evidence indicates that liver macrophages play a key role in regulating the second hit. Macrophages are a highly heterogeneous, plastic cell population that undergoes pleiotropic and coordinated responses to the immunological environment; two common phenotypes are "proinflammatory" M1 and "immunoregulatory" M2 macrophages (7,9,10). Polarization of macrophages toward the M1 phenotype plays a critical role in pathogenesis of several chronic inflammatory disorders, including NASH. By contrast, M2 macrophages promote resolution of inflammation (10). Indeed, suppressing M1 polarization alleviates diet-induced NASH (7,10–12).
The M2 isoform of pyruvate kinase (PKM2) is a rate-limiting enzyme of glycolysis. Cis-trans isomerization and conversion of PKM2 from a tetramer to a dimer or monomer lead to nuclear translocation and regulate expression of target genes that play roles in the Warburg effect and cell cycle progression (13,14). Intriguingly, nuclear PKM2 is a critical determinant of macrophage M1 activation and is involved in inflammatory diseases such as coronary artery disease (13,15,16). Nuclear PKM2 increases macrophage M1 activation in response to lipopolysaccharides (LPS), mainly through TLR4 signaling (13,15,17,18). A recent study shows that PKM2 expression is associated with liver mass (19); however, the role of PKM2 in NASH pathogenesis is largely unknown.
HSPA12A is a novel and atypical member of the HSP70 family (20). Hspa12a mRNA is expressed at high levels in the brain of humans and mice under normal conditions but is decreased in the prefrontal cortex of patients with schizophrenia (20,21). Of particular interest, expression of HSPA12A is associated with shortened survival of patients with hepatocellular carcinoma (22), suggesting possible involvement of HSPA12A in modulating hepatic homeostasis. However, the role of HSPA12A in any disorders, including liver disease, is unknown.
Here, we report that NASH patients showed increased hepatic HSPA12A expression and serum HSPA12A contents. Intriguingly, knockout of HSPA12A (Hspa12a−/−) in mice attenuated high-fat diet (HFD)–induced NASH pathogenesis and M1 macrophage activation. Further molecular analyses revealed that regulation of de novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A via promotion of nuclear PKM2-mediated M1 polarization. Taken together, these findings suggest that HSPA12A acts as a novel regulator of macrophage polarization and NASH pathogenesis. Strategies designed to inhibit macrophage-specific expression of HSPA12A might have therapeutic potential for patients with NASH.
Human Samples
Liver Specimens
Liver specimens were collected from patients who underwent hepatic hemangioma surgery at the First Affiliated Hospital of Nanjing Medical University. Patients provided informed consent at the time of recruitment. Normal or NASH livers were identified using hematoxylin-eosin (H-E) staining, Oil Red O (ORO) staining, and serum alanine aminotransferase (ALT) and AST activity measurements, according to the NASH Clinical Research Network–modified Brunt methodology and other studies (3,23). Livers with bridging fibrosis or cirrhosis were excluded using Masson's staining (Supplementary Fig. 1A and B and Supplementary Table 1A and B).
Blood Samples
Human blood samples were collected from bariatric surgery patients with NASH diagnosed after clinic and B-type ultrasonographic assessment (Supplementary Table 2A and B). Patients were fasted for 12 h before blood sampling. The study was approved by the Ethical Board of the First Affiliated Hospital of Nanjing Medical University (2016-SR-122 and 2016-SR-123). All human studies were conducted according to the World Medical Association Declaration of Helsinki.
Hspa12a Knockout Mice
Conditional Hspa12a knockout mice were generated using the loxP and Cre recombinant system. The Hspa12a targeting vector was constructed using the bacterial artificial chromosome (BAC) retrieval method. In brief, the region of the Hspa12a gene containing exons 2–4 was retrieved from a 129/sv BAC clone (BACPAC Resources Center, Oakland, CA) using a retrieval vector containing two homologous arms. Exons 2 and 3 were replaced by loxP sites flanking a PGK-neo cassette (a positive selection marker) (Supplementary Fig. 2A). The breeding strategy to generate wild-type (WT) (Hspa12aflox/flox) and Hspa12a−/− (Hspa12aflox/flox, CreTg) mice is shown in Supplementary Fig. 2B. Mice were bred at the Model Animal Research Center of Nanjing University and maintained on a 12-h light/dark cycle at 23 ± 1°C with access to food and water ad libitum. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (8th edition, 2011). The animal care and experimental protocols were approved by the Committee on Animal Care at Nanjing University.
HFD Feeding Protocol
For the HFD experiments, mice were fed an HFD (60% of kcal from fat, D-12492; Research Diets, New Brunswick, NJ) for 14 weeks beginning at age 5 weeks. Mice that were fed a normal chow diet and received only 6% of kcal from fat served as controls.
Immunoblotting and Immunoprecipitation-Immunoblotting
Cytosolic and nuclear proteins were prepared from livers or cells using the NucBuster protein extraction kit according to the manufacturer's instructions (Novagen, Darmstadt, Germany). Western blotting was performed according to our previous methods (24,25). To control for lane loading, the membranes were probed with anti-GAPDH or anti–α-tubulin antibodies for cytosolic proteins and anti-histone H3 or anti-lamin A/C antibodies for nuclear proteins.
For analyzing the interaction between HSPA12A and PKM2 by immunoprecipitation-immunoblotting, Raw264.7 macrophages were overexpressed with flag-tagged HSPA12A. After challenge with LPS or vehicle for 16 h, cells were collected for protein extraction. Aliquots of equal volume and protein content were precipitated with anti-flag antibodies, followed by Western blotting with anti-PKM2 and anti-HSPA12A antibodies, as described previously (26). The antibodies used in the experiments are listed in Supplementary Table 3.
Quantitative Real-time PCR
Quantitative real-time PCR was performed as described previously (27). In brief, total RNA was extracted for cDNA synthesis using the oligo (dT) primer. After cDNA synthesis, the expressions of indicated genes were estimated by real-time PCR using the SYBR Green master mix (Roche, Indianapolis, IN). The PCR results of β-actin served as internal controls. The primers used for PCR are listed in Supplementary Table 4.
Biochemical Analysis
Levels of HSPA12A in human serum and insulin in mouse serum and secretion of HSPA12A, IL-1β, IL-6, IL-12, and TNF-α from macrophages were detected using ELISA kits (Supplementary Table 5) according to the manufacturer's instructions. Serum ALT and AST activity, glucose, LDL cholesterol, HDL cholesterol, total cholesterol, and triglyceride (TAG) were measured using a Beckman Coulter AU5800 Chemistry System analyzer (Brea, CA).
Measurement of Lipid Synthesis
Lipid content was evaluated using both ORO (Sigma-Aldrich, St. Louis, MO) staining and a TAG assay. ORO staining was performed on 4% paraformaldehyde-fixed frozen liver sections or hepatocyte cultures. Quantification was performed using spectrophotometry at a wavelength of 510 nm after extraction of the stained ORO in cell cultures. TAG was evaluated in liver tissues or hepatocyte cultures using a TAG assay kit according to the manufacturer's instructions (Jiancheng Biotech, Nanjing, China).
Histological Analysis, Masson's Staining, and Immunofluorescence Staining
Paraffin-embedded liver sections were stained with H-E to evaluate the histological changes and averaged hepatocyte areas. Masson's staining was also performed to indicate fibrosis, as described previously (24). Immunofluorescence staining of frozen liver sections or Raw264.7 macrophages was performed as described previously (24,25) using appropriate antibodies (Supplementary Table 3). In brief, after incubation with the indicated primary antibodies overnight at 4°C, Cy3- or FITC-conjugated appropriate secondary antibodies were added to the sections to visualize the staining. Hoechst 33342 reagent was used to counterstain the nuclei. The staining was observed and quantified in 7–10 randomly selected areas of each sample using a fluorescence microscope with cellSens Dimension 1.15 software (Olympus, Tokyo, Japan).
Adenovirus Construction
The adenoviral vector containing a three flags–tagged coding region of mouse Hspa12a (NM_175199) was generated by GeneChem (Shanghai, China). A schematic overview of virus construction is shown in Supplementary Fig. 3.
Cell Culture and Treatment
Primary hepatocytes and liver macrophages (Kupffer cells) were isolated from 7- to 10-week-old mice by digestion with 0.06% collagenase type IV, followed by centrifugation on a 25–50% Percoll gradient, as described previously (28). Primary hepatocytes were grown in DMEM supplemented with 10% FBS and 0.01 mmol/L dexamethasone. Mouse Raw264.7 macrophages and human hepatocellular carcinoma HepG2 cells were grown in DMEM supplemented with 10% FBS. Mouse AML-12 hepatocytes were cultured in DMEM/F12 supplemented with 10% FBS, ITS (5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenium), and 40 ng/mL dexamethasone.
Overexpression of HSPA12A (Hspa12ao/e)
Cells were infected for 48 h with adenovirus carrying the Hspa12a expression sequence. Cells infected with empty adenovirus served as normal expression controls (Hspa12an/e).
Effect of HSPA12A on Hepatocyte Steatosis
Hspa12ao/e or Hspa12a−/− hepatocytes were incubated with 200 μmol/L oleic acid (OA) for 24 h.
Collection of Conditioned Medium from Macrophages
Hspa12ao/e or Hspa12an/e Raw264.7 macrophages were treated for 16 h with LPS (500 ng/mL) to induce M1 polarization. The medium was then collected (referred to here as Hspa12ao/e conditioned medium [CM] or Hspa12an/e CM).
Paracrine Effects of Macrophage HSPA12A on Hepatocyte Steatosis
AML-12 hepatocytes were incubated for 24 h with Hspa12ao/e CM or Hspa12an/e CM in the presence of OA (200 μmol/L).
Activation of Kupffer Cells
After they were grown for 48 h, WT and Hspa12a−/− Kupffer cells were incubated with LPS (200 ng/mL) for 16 h. Kupffer cells were harvested for immunoblotting analysis, and the medium were collected and used as WT CM and Hspa12a−/− CM, respectively.
RAW 264.7 macrophages were treated with 500 ng/mL LPS for 16 h. Cross-linking was performed using 500 μmol/L disuccinimidyl suberate (Thermo Fisher Scientific, Waltham, MA) for 30 min. Lysates were analyzed by Western blot as previously described (15).
To examine the effects of macrophage HSPA12A on HFD-induced NASH, we transplanted the bone marrows of Hspa12a−/− mice to WT mice according to previously described methods (29). In brief, the recipient C57BL/6 WT mice (6 weeks old) were given acidified and antibiotic water (100 mg/L neomycin and 10 mg/L polymyxin) for 1 week before irradiation. At the day of transplantation, recipient mice were given a lethal irradiation (9 Gy). Four hours after irradiation, a total of 5 × 106 bone marrow cells isolated from donor WT or Hspa12a−/− mice (4 weeks old) were intravenously injected into a recipient WT mouse via tail vein, referred to as Hspa12a−/−/WT and WT/WT mice, respectively. After receiving acidified and antibiotic water for 4 weeks after transplantation, the bone marrow transplantation (BMT) mice were fed with HFD for 14 weeks.
Liquid Chromatography–Tandem Mass Spectrometry Analysis
Primary mouse adipocytes differentiated from preadipocytes were used in the experiments. The primary mouse preadipocytes were used as controls. The proteins that specifically interact with HSPA12A were pulled down by HSPA12A antibody and were separated by SDS-PAGE followed by Coomassie blue staining, digestion in gel with trypsin, and analysis by liquid chromatography–tandem mass spectrometry. In brief, peptides were dissolved in solvent A (2% formic acid in 3% acetonitrile) and loaded directly onto a reversed-phase trap column (Chrom XP C18-CL-3m 120A; Eksigent). Peptide separation was performed using a reversed-phase analytical column (3 μm, 120A) (3C18-CL-120; Eksigent). Eluting peptides from the column were analyzed using an AB Sciex 5600+ TripleTOF system. Tandem mass spectrometry data were processed using ProteinPilot Software 4.5 (AB Sciex). Tandem mass spectra were searched against the UniProt mouse database (16,923 sequences) concatenated with a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, allowing up to three missing cleavages, four modifications per peptide, and five charges.
Data are expressed as the mean ± SD. Groups were compared using an unpaired, two-tailed Student t test or using one-way or two-way ANOVA followed by Tukey post hoc test. A P value of <0.05 was considered significant.
Liver Macrophages Express High Levels of HSPA12A
The expression profile of HSPA12A protein in liver and cells is unclear. Western blotting revealed low-level expression of HSPA12A in mouse liver (Supplementary Fig. 4). In mouse liver, expression of HSPA12A by macrophages was 17.2-fold higher than that in hepatocytes (Fig. 1A). Macrophages in human livers also showed abundant expression of HSPA12A, whereas expression by hepatocytes was barely detectable (Fig. 1B and Supplementary Fig. 5).
HSPA12A upregulation was positively correlated with human NASH. A: HSPA12A expression was examined in primary hepatocytes and macrophages. Primary cells were isolated from mice for immunoblotting analysis. **P < 0.01; mean ± SD; n = 3 mice/group. Data were analyzed by unpaired, two-tailed Student t test. B: HSPA12A expression was examined in human normal livers. Immunofluorescence staining of HSPA12A, CD68 (macrophage), and HEP1 (hepatocyte) was performed on frozen liver sections. Hoechst 33342 was used to counterstain the nuclei. Note that HSPA12A, indicated by arrows in images, was preferably expressed in macrophages (right panel) compared with hepatocytes (left panel). Scale bar = 5 μm. **P < 0.01; mean ± SD; n = 3 human subjects/group. C: Increased Hspa12a mRNA expression in livers of NASH patients. **P < 0.01; mean ± SD; n = 7 for the control and n = 12 for NASH group. Data were analyzed by unpaired, two-tailed Student t test. D: Increased circulating HSPA12A content correlated with hepatic injury in NASH patients. Hepatic injury was reflected by abnormal elevated activities of ALT (males >50 and females >40 units/L) and AST (males >40 and females >35 units/L). *P < 0.05 and **P < 0.01; mean ± SD; n = 17 for the normal ALT group and n = 11 for elevated ALT group, and n = 22 for the normal AST group and n = 6 for elevated AST group. Data were analyzed by unpaired, two-tailed Student t test. E: Nuclear translocation of HSPA12A in livers of human NASH patients. Cytosolic and nuclear protein extracts were prepared from human livers with or without NASH. HSPA12A expression was analyzed by immunoblotting analysis. Blots for GAPDH or histone H3 served as loading controls. **P < 0.01; mean ± SD; n = 3 human individuals/group. Data were analyzed by unpaired, two-tailed Student t test. F: Nuclear translocation of HSPA12A in livers of NASH mice. WT mice were fed with HFD for 14 weeks to induce NASH. Mice fed with normal chow served as controls. Cytosolic and nuclear protein extracts were prepared for immunoblotting against HSPA12A. *P < 0.05 and **P < 0.01; mean ± SD; n = 4 mice/group. Data were analyzed by unpaired, two-tailed Student t test.
Upregulation of HSPA12A in Human Patients Shows a Positive Correlation with NASH
To investigate the clinical significance of HSPA12A in NASH, we evaluated expression of HSPA12A in NASH patients. Expression of Hspa12a mRNA in NASH livers was significantly higher than that in non-NASH controls (Fig. 1C). Moreover, we observed a positive correlation between circulating HSPA12A levels and ALT and AST activity in NASH patients (Fig. 1D and Supplementary Fig. 6). Unexpectedly, expression of HSPA12A decreased in the cytosolic fraction but increased in the nuclear fraction, suggesting nuclear translocation of HSPA12A in the human NASH liver (Fig. 1E). Similar distribution of HSPA12A was observed in the livers of mice fed an HFD (Fig. 1F). Therefore, NASH is associated with upregulated expression of HSPA12A.
HSPA12A Deficiency Ameliorates HFD-Induced NASH
Because expression of HSPA12A was upregulated in NASH livers, it is conceivable that HSPA12A knockout may have therapeutic potential. Therefore, we generated HSPA12A knockout mice (Hspa12a−/−) using the Cre-loxP recombinant system (Supplementary Fig. 2A and B). Successful knockout of HSPA12A in the liver was verified by protein analysis (Fig. 2A). The body weight of adult Hspa12a−/− mice, along with serum levels of glucose and lipids, was similar to that in WT mice with access to food and water ad libitum, as were fasting serum insulin levels (Supplementary Fig. 7A–C). Liver weight, hepatocyte size, and lipid amount were similar in Hspa12a−/− and WT mice fed a normal chow diet (Fig. 2B–D). However, HFD-induced increases in liver weight and hepatocyte size were decreased in Hspa12a−/− compared with WT mice (Fig. 2B and C). Furthermore, HFD-induced steatosis was less marked in Hspa12a−/− mice, as indicated by reduced TAG content, histological alteration, and ORO staining (Fig. 2C and D). Notably, HFD-induced liver injury, as indicated by elevated serum ALT and AST activities, was abolished in Hspa12a−/− mice (Fig. 2E). No significant fibrosis, indicated by Masson's staining, was observed in both genotypes after HFD feeding for 14 weeks (Supplementary Fig. 8). Taken together, these findings strongly suggest that HSPA12A deficiency prevents progression of NAFLD to NASH.
Deficiency of HSPA12A attenuated HFD-induced hepatic steatosis and injury. A: HSPA12A expression was absent in livers of Hspa12a−/− mice that were analyzed by immunoblotting. n = 10 mice/group. B: HFD-induced hepatomegaly was attenuated in Hspa12a−/− mice. Mice aged 5 weeks were fed with HFD or normal chow diet for 14 weeks. Livers were removed and weighed. The liver weight (LW) was normalized with body length (BL). **P < 0.01; mean ± SD; sample numbers are indicated. Data were analyzed by two-way ANOVA analysis followed by Tukey test. C: Hepatic histology. After feeding with HFD or chow for 14 weeks, livers were removed and analyzed by H-E staining on paraffin sections. Hepatocyte areas were measured. Arrows indicate the lipid droplet cavitation. **P < 0.01; mean ± SD; n = 7 for HFD-WT group and n = 5 for other groups. Data were analyzed by two-way ANOVA analysis followed by Tukey test. Scale bar = 20 μm. D: Lipoid accumulation. Top panel: after feeding with HFD or chow for 14 weeks, frozen liver sections were prepared for ORO staining to indicate lipid deposition. n = 4 for HFD-WT group and n = 5 for other groups. Bottom panel: after feeding with HFD for 14 weeks, livers were collected for TAG content analysis. *P < 0.05; mean ± SD; n = 4 for WT group and n = 5 for Hspa12a−/− group. Data were analyzed by unpaired, two-tailed Student t test. Scale bar = 20 μm. E: Hepatic function. Serum samples were collected from mice fed with HFD or chow diet for 14 weeks for ALT and AST evaluation. **P < 0.01; mean ± SD; n = 9/group. Data were analyzed by two-way ANOVA analysis followed by Tukey test. F: mRNA levels. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated genes' expression. *P < 0.05 and **P < 0.01; mean ± SD; n = 6 for WT group and n = 5 for Hspa12a−/− group. Data were analyzed by unpaired, two-tailed Student t test.
Hspa12a−/− Mice Show Decreased Expression of Genes Involved in De Novo Fat Synthesis Upon HFD Feeding
To gain insight into the mechanisms underlying HSPA12A deficiency–mediated inhibition of NAFLD progression, we examined expression of genes linked to lipid metabolism in the livers of HFD-fed mice. We noted a significant decrease in transcription of lipogenic transcription factors (Chrebp, Srebp-1c, Ppara, and Pparg) in Hspa12a−/− livers compared with WT livers (Fig. 2F). In agreement, the expression of target genes, including Gck, Lpk, Acc, Fas, Scd1, Gpat, Dgat2, and Elovl6, involved in de novo fat synthesis were reduced in Hspa12a−/− livers compared with WT livers (Fig. 2F). We also noticed that genes regulating lipid storage and transportation (Cidea, Plin1, and Apob) and lipolysis (Hsl and Cgi58) were downregulated in Hspa12a−/− livers (Supplementary Fig. 9A and B). As the most prominent changes in expression were observed for transcription factors and target genes involved in de novo fat synthesis, we examined expression of PPARγ and SCD1 proteins. In line with the aforementioned results, expression of both proteins was reduced in the liver of Hspa12a−/− compared with WT controls upon HFD feeding (Supplementary Fig. 9C). The findings suggest that HSPA12A regulates de novo fat synthesis and other lipid metabolic signaling in the liver upon HFD feeding.
Lipid Accumulation in Hepatocytes Is Regulated by the Paracrine Effects of Macrophage HSPA12A
To investigate how HSPA12A regulates accumulation of lipids in hepatocytes, we examined the effects of loss- and gain-of-HSPA12A function on lipid deposition using three types of hepatocyte culture: primary mouse hepatocytes, mouse hepatocyte AML-12 cells, and human hepatocellular carcinoma HepG2 cells. Unexpectedly, we found that OA-induced lipid deposition in primary hepatocytes was not affected by either Hspa12a deficiency or overexpression (Fig. 3A and B). Also, overexpression of HSPA12A did not affect OA-induced lipid deposition in either AML-12 or HepG2 hepatocytes (Fig. 3B).
Hepatocyte steatosis was regulated by paracrine effects of macrophage HSPA12A. A: No effects of HSPA12A deficiency on hepatocyte steatosis. Primary hepatocytes isolated from WT and Hspa12a−/− mice were exposed to OA for 24 h. Vehicle-treated cells served as controls. TAG contents were examined. *P < 0.05 and **P < 0.01; mean ± SD. Sample numbers are indicated. Data were analyzed by two-way ANOVA analysis followed by Tukey test. N.S., not significant; prot, protein. B: No effects of HSPA12A overexpression on hepatocyte steatosis. Three types of hepatocytes with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with OA or vehicle (control) for 24 h. Lipid accumulation was examined by ORO staining. **P < 0.01; mean ± SD. Scale bar = 50 μm. Sample numbers are indicated in the figures. Data were analyzed by two-way ANOVA analysis followed by Tukey test. C: Paracrine effects of Raw264.7 macrophage HSPA12A on hepatocyte steatosis. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were challenged with LPS for 16 h. The CM of macrophage cultures was collected and applied to AML-12 hepatocytes for 24 h. Lipid deposition in AML-12 hepatocytes was then evaluated by ORO staining. **P < 0.01; mean ± SD. Scale bar = 50 μm. Sample numbers are indicated. Data were analyzed by unpaired, two-tailed Student t test. D: Paracrine effects of Primary Kupffer cell HSPA12A on hepatocyte steatosis. Primary Kupffer cells isolated from WT and Hspa12a−/− mice were challenged with LPS (200 ng/mL) for 16 h. The CMs were collected and applied to AML-12 hepatocytes for 24 h. Lipid deposition in AML-12 hepatocytes was then evaluated by ORO staining. **P < 0.01; mean ± SD. Scale bar = 50 μm. n = 8/group. Data were analyzed by unpaired, two-tailed Student t test.
Because hepatic macrophages showed higher HSPA12A expression than hepatocytes (Fig. 1A and B), we designed culture experiments to evaluate the interaction between macrophage HSPA12A and hepatocyte lipid deposition. To do this, we overexpressed HSPA12A (Hspa12ao/e) in Raw264.7 macrophages by infection with the adenovirus carrying the Hspa12a expression sequence; macrophages infected with empty virus served as normal expression controls (Hspa12an/e). The CM collected from these macrophage cultures were referred to as Hspa12ao/e CM and Hspa12an/e CM. Notably, Hspa12ao/e CM increased the OA-induced lipid deposition in AML-12 hepatocytes (Fig. 3C). The regulation of primary Kupffer cell HSPA12A on hepatocyte lipid deposition was also examined. The CM was collected from LPS-treated primary Hspa12a−/− and WT Kupffer cell cultures, referred to as Hspa12a−/− CM and WT CM, respectively. AML-12 hepatocytes incubated with Hspa12a−/− CM accumulated significantly less lipid than cells treated with WT CM in the presence of OA (Fig. 3D). Taken together, these data indicate that macrophage HSPA12A regulates hepatocyte steatosis through paracrine effects.
HSPA12A Deficiency Reduces Both Macrophage Recruitment and M1 Polarization in the Liver of HFD-Fed Mice
The effects of macrophage HSPA12A on hepatocyte lipid deposition prompted us to focus on macrophage activation and inflammatory responses because they play critical roles in NASH pathogenesis (7,10). Indeed, upon HFD feeding, we found a significant reduction in the number of F4/80-positive cells in the liver of Hspa12a−/− mice compared with that of WT mice (Fig. 4A). Accordingly, analysis of mRNA levels revealed markedly lower expression of proinflammatory mediators and M1 markers, including Il-1β, Il-6, Tnfα, Nfκb, Tlr4, Inos, Mcp1, Ifnγ, Il-12, Cd86, and Cd68, in livers of Hspa12a−/− mice compared with WT controls upon HFD (Fig. 4B and C). By contrast, the livers of Hspa12a−/− mice showed markedly higher expression of mRNA encoding M2 markers, including Cd163, Arg1, and Cd206, than that of WT mice fed an HFD (Fig. 4C). In agreement, protein analysis confirmed reduced expression of iNOS (an M1 marker) and increased expression of CD206 (an M2 marker) in livers from HFD-fed Hspa12a−/− mice (Fig. 4D). Moreover, livers from Hspa12a−/− mice showed lower expression of TLR4, MyD88, and phosphorylated (p-) NF-κB (p65) than those from WT controls upon HFD (Fig. 4D). Collectively, these data indicate that HSPA12A deficiency attenuates both macrophage recruitment and M1 polarization in the liver of HFD-fed mice.
Deficiency of HSPA12A reduced M1 macrophage polarization and inflammatory responses in mouse NASH livers. A: Macrophage recruitment. Liver tissues were collected from mice fed with HFD or chow diet for 14 weeks. Frozen sections were immunostained with F4/80 to visualize macrophages. Hoechst 33342 was used to counterstain nuclei. Macrophage numbers were expressed as a percentage of total liver cells. Scale bar = 50 μm. **P < 0.01; mean ± SD; n = 5 for HFD-WT group and n = 4 for all the other groups. Data were analyzed by two-way ANOVA analysis followed by Tukey test. B: mRNA levels of inflammatory mediators. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated gene expression. *P < 0.05 and **P < 0.01; mean ± SD; n = 6 for WT group and n = 5 for Hspa12a−/− group. Data were analyzed by unpaired, two-tailed Student t test. C: mRNA levels of M1 and M2 markers. Livers were collected from mice fed with HFD for 14 weeks. Real-time PCR was performed to evaluate the indicated gene expression. *P < 0.05 and **P < 0.01; mean ± SD; n = 6 for WT group and and n = 5 for Hspa12a−/− group. Data were analyzed by unpaired, two-tailed Student t test. D: Protein expression. Livers were collected from mice fed with HFD for 14 weeks. Immunoblotting for the indicated proteins was performed. **P < 0.01; mean ± SD; n = 4 for CD206 and p-NF-κB groups and n = 5 for the other groups. Data were analyzed by unpaired, two-tailed Student t test.
Overexpression of HSPA12A Promotes M1 Macrophage Polarization by Increasing Nuclear Translocation of PKM2
HSPA12A Is Upregulated During M1 Macrophage Polarization
To investigate how HSPA12A regulates macrophage polarization, we first examined whether HSPA12A expression is altered upon activation of M1 macrophages. Liver macrophages from NASH patients showed markedly higher protein expression and nuclear translocation of HSPA12A than controls, as illustrated by immunostaining (Fig. 5A and Supplementary Fig. 10). The same pattern of HSPA12A expression and distribution was observed in Raw264.7 macrophages after LPS treatment, as indicated by immunoblotting and immunostaining (Fig. 5B and C). We also observed increased expression of HSPA12A in Raw264.7 macrophages exposed to OA (Supplementary Fig. 11). These findings suggest that HSPA12A in macrophages is upregulated and nuclear translocated during an M1 response.
HSPA12A promoted M1 macrophage polarization. A: Human NASH livers demonstrated an increase of HSPA12A nuclear translocation in macrophages. Immunostaining against HSPA12A and CD68 (macrophage) was performed on frozen sections of human livers. Hoechst 33342 was used to counterstain nuclei. Scale bar = 20 μm. **P < 0.01; mean ± SD; n = 3 human subjects/group. B and C: HSPA12A nuclear translocation was increased in macrophages by LPS. Raw264.7 macrophages were stimulated with LPS for 16 h. Cells were then subjected to the analysis of HSPA12A expression in cytosolic and nuclear fractions by immunoblotting (B) and immunofluorescence staining (C). Scale bar = 2 μm. *P < 0.05; **P < 0.01; mean ± SD; n = 3/group. Data were analyzed by unpaired, two-tailed Student t test. D: HSPA12A increased mRNA expression of M1 markers in LPS-treated macrophages. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cells were then collected for analyzing the indicated M1 markers by real-time PCR. *P < 0.05 and **P < 0.01; mean ± SD; n = 6/group. Data were analyzed by unpaired, two-tailed Student t test. E: HSPA12A increased protein expression of M1 markers and the TLR4/NF-κB pathway in LPS-treated macrophages. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with LPS for 16 h. Cells were then collected for analyzing the indicated proteins by immunoblotting. *P < 0.05 and **P < 0.01; mean ± SD; n = 3/group. Data were analyzed by unpaired, two-tailed Student t test.
HSPA12A Promotes M1 Macrophage Polarization
Next, we examined the direct effects of HSPA12A on the M1 macrophage response. Overexpression of HSPA12A increased mRNA expression of M1 markers (Inos, Il-1β, Tnfα, Mcp1, Ccl3, and Ccl4) in LPS-treated Raw264.7 macrophages (Fig. 5D). In agreement, expression of iNOS and TNF-α protein in Hspa12ao/e macrophages was higher than that in Hspa12an/e macrophages in the presence of LPS (Fig. 5E). Expression of TLR4 and p-NF-κB, both critical for M1 macrophage activation, also increased in Hspa12ao/e macrophages (Fig. 5E). By contrast, primary Kupffer cells from Hspa12a−/− mice showed decreased expression of iNOS, TLR4 MyD88, and p-NF-κB protein compared with WT Kupffer cells after LPS stimulation (Supplementary Fig. 12). These findings confirm the aforementioned in vivo results showing that HSPA12A deficiency attenuates M1 macrophage polarization in the liver of HFD-fed mice (Fig. 4B–D).
HSPA12A Increases Monomeric and Dimeric PKM2 Levels and Nuclear Translocation of PKM2 in Macrophages
Nuclear PKM2 promotes M1 macrophage polarization (15). Because mass spectrometry analysis revealed an interaction between PKM and HSPA12A in differentiated adipocytes (Supplementary Fig. 13A and B), we examined whether PKM2 mediates HSPA12A-induced M1 macrophage polarization. PKM2 expression and nuclear translocation was increased in Raw264.7 macrophages in response to LPS stimulation (Supplementary Fig. 14). Liver macrophages of NASH patients and NASH mice demonstrated marked increases in PKM2 expression and nuclear translocation (Fig. 6A and B and Supplementary Fig. 15A–C). However, HSPA12A deficiency decreased Pkm2 mRNA expression in liver and PKM2 nuclear translocation in liver macrophages of HFD-fed mice (Fig. 6C and D and Supplementary Fig. 16). By contrast, HSPA12A overexpression increased PKM2 expression and nuclear translocation in Raw264.7 macrophages (Fig. 7A and Supplementary Fig. 17). In line with this, Hspa12ao/e Raw264.7 macrophages expressed higher levels of monomeric and dimeric PKM2 than Hspa12an/e control cells in the presence of LPS (Supplementary Fig. 18). Collectively, these data suggest that HSPA12A regulates PKM2 expression and nuclear translocation during M1 macrophage polarization.
Increased nuclear PKM2 in macrophages of NASH livers and the inhibitory effect of HSPA12A deficiency. A: Human NASH livers demonstrated increased PKM2 nuclear translocation in macrophages. Immunostaining against PKM2 and CD68 (macrophage) was performed on frozen sections of human livers. Hoechst 33342 was used to counterstain nuclei. Scale bar = 50 μm. **P < 0.01; mean ± SD; n = 3 human subjects/group. B: Mouse NASH livers demonstrated increased PKM2 nuclear translocation in macrophages. Immunostaining against PKM2 and F4/80 (macrophage) was performed on frozen liver sections of WT mice fed with HFD or chow diet for 14 weeks. Hoechst 33342 was used to counterstain nuclei. Scale bar = 20 μm. **P < 0.01; mean ± SD; n = 4 mice/group. C and D: HSPA12A deficiency suppressed PKM2 nuclear translocation in macrophages of NASH livers. Livers were collected from mice fed with HFD for 14 weeks. PKM2 expression and nuclear translocation in macrophages were examined by immunoblotting against PKM2 in cytosolic and nuclear protein extracts (C) and immunostaining against PKM2 and F4/80 on frozen sections (D). Scale bar = 20 μm. *P < 0.05; **P < 0.01; mean ± SD; n = 6/group (C) and n = 4/group (D). Data were analyzed by unpaired, two-tailed Student t test.
Inhibition of PKM2 nuclear translocation reversed the HSPA12A-induced promotion of M1 macrophage polarization. DASA-58 reversed the HSPA12A-induced promotion of M1 macrophage polarization. Raw264.7 macrophages with HSPA12A overexpression (Hspa12ao/e) or normal expression (Hspa12an/e) were treated with DASA-58 or vehicle control in the presence of LPS for 16 h. Cytosolic and nuclear protein fractions were immunoblotted with the indicated antibodies (A). Levels of Mcp-1 and Tnfα mRNA were also analyzed (B). *P < 0.05 and **P < 0.01; mean ± SD; n = 3/group (A) and n = 6/group (B). Data were analyzed by one-way ANOVA followed by Tukey test.
Nuclear PKM2 Mediates the Effects of HSPA12A on M1 Macrophage Activation
To determine the role of PKM2 during HSPA12A-mediated M1 macrophage polarization in response to LPS, Hspa12ao/e Raw264.7 macrophages were treated with a small molecule, DASA-58. DASA-58 is a specific enzyme activator of PKM2, which can prevent PKM2 nuclear translocation (15,30,31). Notably, DASA-58 reversed the HSPA12A overexpression-induced nuclear translocation of PKM2 and expression of M1 markers (iNOS, MCP-1, and TNF-α) in LPS-treated macrophages (Fig. 7A and B). Taken together, these findings suggest that nuclear PKM2 mediates the HSPA12A-induced M1 macrophage polarization.
Nuclear PKM2 Mediates the Paracrine Effects of Macrophage HSPA12A on Hepatocyte Steatosis
Next, we asked whether nuclear PKM2 mediates the paracrine effects of macrophage HSPA12A on hepatocyte steatosis. We found that overexpression of HSPA12A led to increased secretion of IL-1β, IL-6, TNF-α, and IL-12 by LPS-treated Raw264.7 macrophages; however, this increase was reversed by DASA-58 (Fig. 8A and Supplementary Fig. 19). ELISA assay detected no difference in HSPA12A levels in medium from Hspa12ao/e and Hspa12an/e Raw264.7 macrophages (Supplementary Fig. 20).
Nuclear PKM2 mediated the paracrine effect of macrophage HSPA12A on hepatocyte steatosis. A: DASA-58 reversed the HSPA12A-promoted secretion of cytokines from macrophages. Raw264.7 macrophages were challenged with LPS (500 ng/mL) for 16 h in the presence or absence of DASA-58 (50 μmol/L). Culture medium was collected to analyze IL-1β and IL-6 contents by ELISA assay. *P < 0.05 and **P < 0.01; mean ± SD; n = 4/group for IL-6 analysis and n = 5/group for IL-1β analysis. Data were analyzed by one-way ANOVA followed by Tukey test. B and C: DASA-58 blocked the paracrine effects of macrophage HSPA12A on hepatocyte steatosis. CM was collected from the LPS-treated Raw264.7 macrophages in the presence or absence of DASA-58 for 16 h. The CM was then applied to the OA-treated AML-12 hepatocytes for 24 h. Hepatocytes were collected for lipid analysis by ORO staining (B) (scale bar = 50 μm) and the indicated gene expression by real-time PCR (C). *P < 0.05 and **P < 0.01; mean ± SD; n = 8/group (B) and n = 4/group (C). Data were analyzed by one-way ANOVA followed by Tukey test. D: Interaction between HSPA12A and PKM2 in macrophages. RAW264.7 macrophages overexpressing the flag-tagged HSPA12A were challenged with or without LPS for 16 h. Cellular protein extracts were immunoprecipitated (IP) with primary antibody for flag. The immunoprecipitates were blotted (IB) with PKM2 and HSPA12A. Protein extracts without immunoprecipitation (input) served as positive controls, and immunoprecipitates from IgG incubation served as negative controls. E: Schematic represents the regulation of HSPA12A on M1 macrophage polarization and NASH pathogenesis. Macrophage HSPA12A is upregulated by LPS or other stimuli. By directly interacting with PKM2, HSPA12A promotes PKM2 translocation to nuclei, thereby promoting M1 macrophage polarization and secretion of proinflammatory M1 cytokines, and ultimately leads to hepatocyte steatosis through paracrine effects. Both the M1-polarized macrophage-mediated inflammation and hepatocyte steatosis are essential for the pathogenesis of NASH. FFA, free fatty acid.
CM collected from DASA-58–treated Hspa12ao/e macrophage cultures was termed DASA-58 Hspa12ao/e CM. Lipid deposition in AML-12 hepatocytes treated with DASA-58 Hspa12ao/e CM was significantly lower than that in AML-12 hepatocytes treated with Hspa12ao/e CM (Fig. 8B). In agreement, we noted a marked reduction in the expression of genes associated with de novo fat synthesis and lipolysis in AML-12 hepatocytes treated with DASA-58 Hspa12ao/e CM compared with AML-12 hepatocytes treated with Hspa12ao/e CM (Fig. 8C).
HSPA12A Forms Complex with PKM2
Simultaneous nuclear translocation of HSPA12A and PKM2 during M1 macrophage activation motivated us to investigate whether there is an interaction between them. Flag-tagged HSPA12A was immunoprecipitated from Hspa12ao/e Raw264.7 macrophages treated with LPS or vehicle for 16 h. Precipitates were immunoblotted with antibodies specific for PKM2 and HSPA12A. PKM2 protein was recovered from the flag-tagged HSPA12A immunocomplexes, with more PKM2 recovered after LPS treatment (Fig. 8D). These data suggest that HSPA12A formed a complex with PKM2, and the formation of the HSPA12A-PKM2 complex was increased during macrophage polarization to an M1 phenotype.
Macrophage HSPA12A Regulates HFD-Induced Hepatic Steatosis in Mice
To examine whether macrophage HSPA12A affects HFD-induced hepatic steatosis, we transplanted bone marrow from Hspa12a−/− mice into recipient WT mice (Hspa12a−/−/WT). Recipient WT mice transplanted with WT bone marrow served as controls (WT/WT). After a lethal irradiation, all the mice (15/15) without BMT died within 15 days; however, none of the BMT mice died (Supplementary Fig. 21A). HSPA12A expression in white blood cells from Hspa12a−/−/WT mice was hardly detectable compared with that in WT/WT controls (Supplementary Fig. 21B and C). After an HFD for 14 weeks, liver weight, hepatocyte area, and lipid accumulation (as indicated by ORO staining and TAG content) in Hspa12a−/−/WT mice were reduced compared with WT/WT mice (Supplementary Fig. 21D–F). In addition, expression of iNOS, TNF-α, TLR4, and MyD88 protein, along with NF-κB phosphorylation, in the livers of Hspa12a−/−/WT mice was lower than in WT/WT mice fed an HFD (Supplementary Fig. 22).
Here, we identified macrophage HSPA12A as a novel regulator of hepatic inflammation and NASH pathogenesis. Thus, strategies aimed at inhibiting macrophage-specific HSPA12A may have potential as therapeutic interventions for NASH patients.
HSPA12A was first cloned from atherosclerotic lesions, and its cerebral expression is decreased in patients with schizophrenia (20,21,32). Recently, a study identified a correlation between increased HSPA12A expression and shortened survival of patients with hepatocellular carcinoma (22). However, there is no evidence that HSPA12A is the direct cause of any pathophysiological event. Here, we used human samples, mouse models, and cell culture models to show the following: 1) NASH patients display increased hepatic expression of HSPA12A and higher levels of circulating HSPA12A, which are positively associated with hepatic steatosis and injury, respectively; 2) HSPA12A is expressed preferably in liver macrophages rather than hepatocytes; 3) HSPA12A deficiency either globally or in macrophages attenuates HFD-induced hepatic steatosis, liver injury, and inflammatory M1 macrophage responses in mice; and 4) hepatocyte steatosis is regulated by the paracrine effects of macrophage HSPA12A, which are mediated, at least in part, by nuclear PKM2-dependent modulation of M1 macrophage polarization. Taken together, these results provide evidence that the paracrine effects of macrophage HSPA12A regulate hepatocyte steatosis and injury.
Activation of liver macrophages, and subsequent secretion of proinflammatory mediators, is a key event for onset and progression of NAFLD (9,10). Hepatic macrophages comprise resident (Kupffer cells) and recruited macrophages, all of which express macrophage markers such as CD68, F4/80, and CD11b. Macrophages are activated by LPS or free fatty acids, which induce an M1 phenotype and expression of cytokines, chemokines, and signaling molecules through TLR4 signaling (7,33,34). Subsequently, chemokines attract blood-derived monocytes/macrophages to the liver; these cells then release cytokines that drive NASH progression, which is characterized by increased hepatocellular lipid accumulation and damage (6,34). Indeed, diet-induced NASH is alleviated by depleting Kupffer cells and knockout of IL-1β and TLR4 (11,35,36). Thus, limiting polarization of macrophages toward an M1 phenotype is an attractive strategy for preventing progression of NAFLD (9,10). Here, we observed that deficiency of HSPA12A alleviated HFD-evoked onset and progression of NAFLD, which were concomitant with reduced macrophage recruitment and M1 polarization; HSPA12A deficiency also reduced expression of chemokines and proinflammatory cytokines in HFD-fed mice. By contrast, overexpression of HSPA12A increased M1 macrophage polarization in response to LPS, as indicated by increased expression of iNOS, IL-1β, TNF-α, CCL3, CCL4, and TLR4, and by secretion of IL-1β, IL-6, IL-12, and TNF-α. More importantly, CM from HSPA12A-overexpressing macrophages led to a marked increase in lipid deposition in hepatocytes. However, we observed no change in HSPA12A secretion by macrophages after LPS treatment, suggesting that elevated serum levels of HSPA12A in NASH patients may be due to secretion by other cell types. Considering that hepatocellular HSPA12A had no effect on its own lipid deposition, our results suggest that hepatocyte steatosis is regulated by the paracrine effects of macrophage HSPA12A. We detected only a modest increase in secretion of TNF-α, IL-1β, IL-6, and IL-12 by HSPA12A-overexpressing macrophages, suggesting that the relationship between macrophage HSPA12A and hepatocyte lipid deposition is regulated by the synergistic actions of a group of secreted cytokines and/or other as yet undetermined factors.
Next, we sought answers to the following two questions: does M1 polarization mediate the paracrine effects of macrophage HSPA12A on hepatocyte steatosis, and how does HSPA12A modulate macrophage polarization? Recent studies show that PKM2 is a critical determinant of macrophage activation and subsequent inflammatory responses. The PKM2 tetramer acts as a powerful glycolytic enzyme that regulates glycolysis in the cytosol. However, PKM monomers or dimers are enzymatically inactive and can translocate to the nucleus, where they act as cofactors to activate expression of IL-1β et al., ultimately leading to activation of M1 macrophages (15–17). Induction of PKM2 expression by LPS is mediated by the transcription factors NF-κB and PPARγ (14,15,19). Interestingly, we observed that PKM2 expression in the liver of NASH patients was upregulated and that this upregulation was more prominent in liver macrophages showing notable levels of nuclear translocation. The livers of HFD-fed mice also showed more nuclear translocation of PKM2, which was attenuated by HSPA12A deficiency, suggesting a regulation of HSPA12A in PKM2 nuclear translocation. Importantly, preventing nuclear translocation of PKM2 in macrophages reversed HSPA12A-induced M1 polarization and blocked the paracrine effects of macrophage HSPA12A on hepatocellular steatosis. Immunoprecipitation-Western blot analyses revealed that HSPA12A forms a complex with PKM2 in macrophages. Collectively, these results indicate that HSPA12A interacts with PKM2 and increases its nuclear translocation, thereby inducing M1 macrophage polarization and secretion of proinflammatory M1 cytokines; ultimately, this leads to fat accumulation in hepatocytes via paracrine effects (Fig. 8E). However, it is unknown how HSPA12A regulates PKM2 expression and how HSPA12A interacts with PKM2. Further studies need to address these issues.
In conclusion, we show that deficiency of HSPA12A has a beneficial effect on hepatic steatosis and injury in mice with HFD-induced NASH. The underlying mechanism involves paracrine interactions between macrophages and hepatocytes. The data suggest that inhibitors of macrophage-specific HSPA12A may improve treatment and management of nonalcoholic liver disease.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0035/-/DC1.
Acknowledgments. The authors thank the Translational Medicine Core Facilities, Medical School of Nanjing University, for generous help in mass spectrum analysis.
Funding. This work was supported by the National Natural Science Foundation of China (81870234, 81770854, 81571378, 81571290, 81370260, and 81371450), Jiangsu Province's Outstanding Medical Academic Leaders program (15), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, and Jiangsu Provincial Key Discipline of Medicine (ZDXKA2016003).
Author Contributions. Q.K., N.L., H.C., L.D., B.X., and L.F. performed all animal study procedures and most of the in vitro experiments. X.Z., X.Ca., T.Q., and X.Ch. collected and analyzed human samples. Z.Z. scored livers for NASH features in humans and mice. C.L. developed the study concept and experimental design. Y.L. interpreted the data and wrote the manuscript. L.L. and Z.D. developed the study concept and experimental design, interpreted the data, and wrote the manuscript. Z.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis
Nat Commun
Nuclear PKM2 regulates the Warburg effect
Palsson-McDermott
Goel
Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages
[published correction appears in Cell Metab 2015;21:347].
Cell Metab
Shirai
Nazarewicz
The glycolytic enzyme PKM2 bridges metabolic and inflammatory dysfunction in coronary artery disease
PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation
Weinlich
Bortoluci
Chehab
TLR4/MYD88-dependent, LPS-induced synthesis of PGE2 by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells
Cell Death Differ
Panasyuk
Espeillac
PPARγ contributes to PKM2 and HK2 expression in fatty liver
Truong
Breslow
Two Hsp70 family members expressed in atherosclerotic lesions
Pongrac
Mirnics
Heat shock protein 12A shows reduced expression in the prefrontal cortex of subjects with schizophrenia
Szatmary
Upregulation of heat shock proteins (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) in tumour tissues is associated with poor outcomes from HBV-related early-stage hepatocellular carcinoma
Int J Med Sci
Van Natta
, et al.;
Nonalcoholic Steatohepatitis Clinical Research Network
Design and validation of a histological scoring system for nonalcoholic fatty liver disease
HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism
Cardiovasc Res
Qian
Attenuation of cardiac dysfunction by HSPA12B in endotoxin-induced sepsis in mice through a PI3K-dependent mechanism
Inagaki
Kushida
Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice
HSPA12B attenuated acute myocardial ischemia/reperfusion injury via maintaining endothelial integrity in a PI3K/Akt/mTOR-dependent mechanism
22-S-hydroxycholesterol protects against ethanol-induced liver injury by blocking the auto/paracrine activation of MCP-1 mediated by LXRα
J Pathol
Lipocalin-2 mediates non-alcoholic steatohepatitis by promoting neutrophil-macrophage crosstalk via the induction of CXCR2
HIF1α and metabolic reprogramming in inflammation
J Clin Invest
Giannoni
Taddei
Targeting stromal-induced pyruvate kinase M2 nuclear translocation impairs oxphos and prostate cancer metastatic spread
Transcriptome profiling of hippocampal CA1 after early-life seizure-induced preconditioning may elucidate new genetic therapies for epilepsy
Baffy
Kupffer cells in non-alcoholic fatty liver disease: the emerging view
Koyama
Liver inflammation and fibrosis
Shaish
Lack of interleukin-1α or interleukin-1β inhibits transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice
Csak
Velayudham
Hritz
Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice
Am J Physiol Gastrointest Liver Physiol

redpajama_set_name: RedPajamaCommonCrawl


text:
Back to all posts — Literature Essays
by Expert Sweet Alicia PhD | 23 Feb 2017
Edgar Allan Poe's "Fall of the House of Usher" is narrated in the first person by one of the active participants in the story. The unnamed narrator relates his experience in the house of Roderick Usher who is a descendant of an old family and who believes that the house is contributing to his mental deterioration which is also affected by the eventual decease of his twin sister Madeline. Characteristic of Poe's writing, we once again go through the experiences of a person's mind and thought. The difference with the "Fall of the House of Usher" is that we are seeing two minds working at the same time – the mind of the narrator and the mind of Roderick Usher from the perspective of the narrator which may or may not be biased.
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What makes this story more interesting is the participation of two passive characters – the sister of Usher Madeline and the house itself – and it is in the interaction of all four characters that critics have focused their interpretations, critiques and reviews.
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This story has been interpreted by literary critics numerous times and the interpretation of each critic varies from religion, to philosophy to sociology and to psychology.
Concepts of Freudian sexuality and psychoanalysis have been associated to this story as well as well as the philosophical concepts of Locke, Kant and Hegel. It is specifically in the philosophical concepts of empiricism and transcendentalism that this paper will focus on to get to what Poe is trying to relay in his creation of this story. As the story goes, we wonder whether or not what is happening to Usher is as the narrator describes as hypochondria and madness or is what Usher is uttering the actual truth.
Beverly Voloshin claims that the "Fall of the House of Usher" is heavily influenced by the transition from the age of Enlightenment to the age of Romanticism in literary art. According to her, writers at the time were moving from empiricism towards transcendentalism: "When the writers of the 1830s and 1840s tried to get out of the block universe or dead level of eighteenth-century empiricism, they produced curious and paradoxical figures of transcendence." (Voloshin 19)
To better understand what Voloshin is referring to it is best to differentiate empiricism and transcendence. Empiricism posits that knowledge can only be acquired through sensual experience while Transcendence claims that there is a consciousness outside the realm of the world that moves the world – in other words a God.
Therefore, Voloshin is claiming that from a sensual experience that literature took in the Age of Enlightenment writers are moving to a more romantic inclination towards transcendence as best represented, according to her, by Ralph Waldo Emerson's "Nature." (19) She compares Emerson's Nature which reaches up to the divine to Poe's "Usher" which she avers to have a downward movement of transcendence: "Poe presents transcendental projects which threaten to proceed downward rather than upward. The tales have a paradoxical structure in which transcendence is figured as an outward or downward movement, as the method for going beyond the universe of Lockean empiricism is to go through it." (19) She is basing this downward movement on the depression of the house and it's invertedness in the tarn claiming that somehow the story gives the impression that the divine experience has been finished and the house and the characters are now in "the after-dream of the reveller upon opium."(Poe 7.)
John Timmerman also acknowledges the fact that "The Fall of the House of Usher" is heavily influenced by Poe's movement from Enlightenment to Romanticism, however, he adds one more factor to the equation – Poe's cosmology. (Timmerman 228) Timmerman claims that "The Fall of the House of Usher" fully utilizes Poe's spiritual beliefs which Poe indicated in his essay "Eureka": "we can observe the theory for unity, symmetry, and harmony emerging from Eureka, the aesthetic principles of the theory in his essays, and the application of those principles in a study of the conflict between Romanticism and Enlightenment in "The Fall of the House of Usher." (228)
In Timmerman's article, he enumerates the different spiritual beliefs of Poe which are contained in Poe's "Eureka". Poe apparently believes in the existence of a Prime Mover as the source of all - "'In the beginning' we can admit-indeed, we can comprehend, but one First Cause, the truly ultimate Principle, the Volition of God" (Poe qtd. in Timmerman 229) - but at the same time Poe believes that this first cause is represented by unity. (229) Timmerman continues on to discuss Poe's "Poetic Principle" which states that "that the task of the poet is 'to apprehend the supernal loveliness' (Essays 77) of God's order and that the best way to do so is through sadness. Poe reflects 'that (how or why we know not) this certain taint of sadness is inseparably connected with all the higher manifestations of true Beauty' (Essays 81)." (230) This discussion eventually leads to Poe's sentience theory wherein "If indeed all things are willed into being ex nihilo, then not only all humanity but also all matter is part and parcel with God." (230)
All of these, Timmerman puts together to explain how he believes Poe creates an "architecture of mirrors" to demonstrate his cosmological and philosophical beliefs. (231) Each an every character –from Roderick to Madeline to the Narrator and the house – represent opposite forces of the scale like irrationality/rationality, psychological disease/physical disease, order/disorder, and harmony/chaos. Timmerman continues that these mirror images ultimately represent the move from enlightenment to romanticism: "In this story, however, the balance between Enlightenment and Romantic itself is situated at the heart of the story. Roderick himself is emblematic of Romantic passion, while Madeline is emblematic of Enlightenment. Their genesis, as fraternal twins, is unified-a perfectly mirrored complementarity-but the story unveils their splitting to mutual destruction." (237) Timmerman, however, does not touch on empiricism which is a highlight during Enlightenment and transcendentalism.
I believe that both articles, combined, fully interpret the story of "The Fall of the House of Usher." However, I believe Voloshin made the mistake of confusing transcendence with transcendentalism. Transcendentalism, which states that knowledge can be formed a priori even without sensual experience, is the reaction towards empiricism, not transcendence. Transcendence is more Aristotelian, nevertheless, transcendentalism complements transcendence in the sense that the a priori knowledge which humans supposedly have comes from a First Cause or Prime Mover. Thus I do not agree that the transcendence in "The Fall of the House Of Usher" has a downward movement. I believe that with the sentience theory and cosmological belief of Poe, empiricism and transcendentalism in the story eventually resulted in transcendence proving that the events in the house are real.
The narrator represents empiricism wherein all his knowledge and judgments are based on his sensual experiences, from the sight of the house inverted in the tarn to the density of the air on top of the house. He believed that his experience can easily be changed if that which appeals to his senses are changed a s well: "It was possible, I reflected, that a mere different arrangement of the particulars of the scene, of the details of the picture, would be sufficient to modify, or perhaps to annihilate its capacity for sorrowful impression"( Poe 7) Roderick represents transcendentalism because even without sensual experience he already has knowledge of what will occur and of the sentience of the house. The house, although inanimate, represents the supernatural force.
Remember that unity is representative of the divine, as far as Poe is concerned, and the house in the manner in which it was constructed provided order at the same time a union that lasted through time despite an existing crack in the wall. This is supported when the narrator said, "There was an iciness, a sinking, a sickening of the heart—an unredeemed dreariness of thought which no goading of the imagination could torture into aught of the sublime." (7) Although the author claims that the heaviness cannot be attributed to the divine, knowing Poe's cosmological belief, we know that that depression which the house permeated is part and parcel of the Beauty that can only be divine. Therefore, order, unity and sadness are existent in the description of the house.
Madeline is the anti thesis of all – transcendence, empiricism, and transcendentalism. She represents the mundane world, mortality and the frailty of man. She does not feel for her senses have already failed her ("a partially cataleptical
Character" (11)), she does not show any knowledge of anything whether sensual or a priori and she is sick showing the weakness of man and man's eventual death. However, Madeline remains the catalyst of the story which brings both characters – the narrator and Roderick – towards transcendence.
Initially, the narrator tries to remains objective giving natural explanations to the occurrences that is happening in the house (i.e. calling Roderick a hypochondriac, reading the Mad Trist of Sir Lancelot to appease the fear felt both by himself and Roderick due to the storm.). His knowledge of reality is based on what he feels and what he perceives, anything beyond this means it was not real thus he made the conclusion that Roderick is mad. On the other hand, Roderick who has a priori knowledge believes that he is falling into a mental illness. He believes that his senses are acute, so acute it can be considered "godly" or insane to the narrator. Without any sensual experience or rational proof, he knows for a fact that the house is "alive". Nonetheless, Roderick feels that he is losing any form of order and unity. It is when Madeline jumps on Roderick, killing him, that Roderick's transcendentalism reaches transcendence through death. Roderick, through death has overcome his fear and has reached the next spiritual dimension. On the other hand, the narrator finally reaches and believes transcendence through his sensual experience. He experiences, first hand, the re emergence of Madeline and the force that seems to control the events in the house. He has sensual proof that the house is alive. And finally the shattering of the house, is the destruction of the physicality of God and the transcending from the senses to the spirit because now, even without the house, which can be perceived and sensed, the narrator knows, through his experience that there is a supernatural force there. In the end, both empiricism and transcendentalism resulted in proving transcendence.
In conclusion, during the time that "The Fall of the House of Usher was written, society was going through a transition. Initially, the existence of God and anything supernatural was without question and then the Age of Enlightenment had arrived and it gave the whole of society a different view of God, the supernatural and religion. Empiricism was one of the intellectual concepts that questioned the existence of any supernatural force. However, the dawn of Romanticism came the Transcendental philosophies defending the existence of God, and posits that man's intelligence came from this transcendent being. Poe was in the middle of this transition. He also had his own convictions about religion and he definitely believed that there is a God, if not, a Prime force. All of these – from the changing philosophies and intellectual concepts to his own beliefs – he portrayed best in "The Fall of the House of Usher". He used his genius in manipulating his characters and allowing the reader to go into the mind, of not just one, but two of his characters to better demonstrate the two different philosophies and how the concepts eventually intersect into his own cosmological beliefs with the presence of the third character Madeline.
It may be so that this story can be interpreted in numerous ways, but looking at it through empiricism and transcendentalism, Poe was able to portray the prominent ideas during his time and he was able to synchronize two supposedly opposing philosophical concepts into one story – a feat most writers tried to do during the transition, but that which Poe was able to do masterfully.
"Rationalism vs. Empiricism." Stanford University of Philosophy. 19 August 2004. 24 October 2007.
Irani, Tushar. "PHILOSOPHY AS IDEALISM: DEFINITION AND TRANSCENDENCE IN HEGEL'S THEORY OF FREEDOM." 2 October 2002.
Poe, Edgar Allan. "The Fall of the House of Usher". The Harvard Classics Shelf of Fiction, 10.3. Comp. and Ed. Charles William Eliot. New York: P.F. Collier & Son, 1917: 7-18. Bartleby.com, 2001. 24 October 2007
Robinson, E. Arthur. "Order and Sentience in "The Fall of the House of Usher." PMLA, Vol. 76.1. (1961): 68-81.
Timmerman, John H. "House of mirrors: Edgar Allan Poe's "The Fall of the House of Usher." Papers on Language & Literature, Vol. 39.3 (2003): 227-244
Voloshin, Beverly. "Transcendence Downward: An Essay on 'Usher' and 'Ligeia.'" Modern Language Studies 18.3 (1988): 18-29.
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Thai Night returns to Hong Kong FILMART in 2019
By Miscw.com
KONG, CHINA - Media OutReach - 19 March 2019 - The Department of International Trade Promotion (DITP), Ministry of
Commerce, Royal Thai Government will host Thai Night at the Grand Hyatt, Hong
Kong, on the evening of the 20th March, 2019.
Her Royal Highness
Princess Ubolratana Rajakanya Sirivadhana Barnavadi
will preside over Thai Night: 'Thailand -- Where Films Come Alive', which this
year celebrates the achievements of 'Creative Thai' talent. Her Royal Highness
has long been a keen supporter of Thailand's
and has starred in a number of films and television series.
Thai Night is presented as part of the
program by the Department of International Trade Promotion (DITP) to support and
promote Thailand's film and television
industry in international markets. This year, we celebrate 'Creative Thai'
achievements; the films that have attained international recognition, the
companies who continue to expand their reach in global markets, and Thai
creative individuals who have earned a strong international reputation.
Twenty-two Thai companies will be represented
at the Thai Pavilion. These include eight production and post-production
services and facility companies, seven film production and distribution
companies, four television production and licensing companies and three
documentary production companies
Thai Night will
also include special appearances by top film and television stars.
guest Ray Lui Leung Wai is best known for his role as "Ting Lik" in
the 1980 Hong Kong television series The Bund and its two sequels which is
praised as "The Godfather of the East". Ray Lui also starred in
Transformers 4 and works on films and Chinese television series. He will be
accompanied by distinguished corporate executives H.E. Tun Sri Brandon Toh, Partner
of P+ Capital and Sir Eldee Tang, CEO & Founder of Noble Vici Group, Inc
(NVGI:OTC).
guest Nawat Kulrattanarak rose to stardom with his role in 'Battle of Angels',
which was not only a hit in Thailand, but also became popular throughout the
region and in China. Nawat is the perfect example of a Thai star whose
popularity has expanded both at home and internationally. While he has won
awards for his role in 'Pitsawat' in Thailand, he has also starred in two
Chinese television series, 'Love Jewelry' and 'Revive'.
guest Puttichai Kasetsin's charm and charisma have won him such awards as 'Most
Charming Guy', 'Heartthrob Award', and 'Hottie Male', but his dramatic
performances in Thai series such as 'Leh Nang Fah' and 'Ugly Duckling', earned
him awards for his acting. He was casted as one of the lead characters in
Chinese television drama 'Stairway to Stardom'.
recent years, Thailand has benefited from the popularity in the Chinese film
number of films from China and Hong Kong have come to film in Thailand
including 'Battle of Memories', 'Operation Mekong', Project Gutenberg', and
'Paradox'.
by the success of the Thai film drama 'Bad Genius' which returned over $40m at
the Chinese box office, the Chinese market has opened up to Thai feature films
and television series.
As a display of such success of
Creative Thai talent, the Department of International Trade Promotion (DITP) is
hosting Thai Night at the Grand Hyatt Ballroom, Hong Kong, on 20th March, to coincide with the Hong Kong
International Film and TV Market (FILMART) as part of its on-going campaign for
the promotion of the Thai film and television industry which in 2017, grossed
over $2.89 billion from film, animation and television industries.
This campaign focuses on key events in
the annual film industry calendar, including the Cannes Marché du Film, the
American Film Market, as well as the Hong Kong FILMART.
The Thai Pavilion brings Thai companies
and government representative booths together in a single, co-ordinated and
branded exhibition space at FILMART in Hall 1B,
between rows A and B.
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