Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on LDs and is phosphorylated by AMPK, thereby enhancing its affinity for LC3/GABARAPs. Deletion of ORP8 or interruption of ORP8-LC3/GABARAP interaction results in accumulation of LDs and increased intracellular triglyceride. Overexpression of ORP8 alleviates LD and triglyceride deposition in the liver of ob/ob mice, and Osbpl8-/- mice exhibit liver lipid clearance defects. Our results suggest that ORP8 is a lipophagy receptor that plays a key role in cellular lipid metabolism.
Animals ; Mice ; Lipid Droplets ; Autophagy ; Autophagosomes ; Homeostasis ; Triglycerides

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Animals ; Cell Differentiation ; Embryo, Mammalian/metabolism* ; Embryonic Development ; Mice ; Pluripotent Stem Cells/metabolism* ; RNA, Messenger/genetics* ; RNA, Ribosomal ; RNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism* ; Zygote/metabolism*

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

Objective:To investigate the diagnostic value of 11C-Pittsburgh compound B (PIB) in patients with mild cognitive impairment (MCI) and Alzheimer′s disease (AD) and explore the factors that may affect the binding of 11C-PIB. Methods:From January 2017 to December 2019, the 11C-PIB uptake of 6 patients with normal cognitive (NC; 3 males, 3 females, age: (64.5±12.3) years), 11 patients with MCI (4 males, 7 females, age: (64.5±9.8) years) and 21 patients with AD (7 males, 14 females, age: (68.1±9.1) years) from Daping Hospital, Army Medical University were retrospectively analyzed. Regional 11C-PIB binding was assessed by using standardized uptake value ratio (SUVR) and visual reading of 11C-PIB scan. Clinical data, including age, gender, education level, cognitive impairment, neuropsychological scale score, vascular risk factors (VRF), apolipoprotein E (ApoE) gene, were collected and differences among groups were analyzed by using one-way analysis of variance, least significant difference t test or Fisher exact test. Factors that affected the 11C-PIB binding were analyzed by multiple linear regression. Results:SUVR of cerebral lobe among NC, MCI and AD groups were significantly different (range of mean SUVR: 1.16-1.26, 1.19-1.35 and 1.40-1.61; F values: 5.331-9.279, all P<0.05). For positive PIB patients, SUVR of posterior cingulate and precuneus were increased in MCI group compared with NC group (1.20±0.15 vs 1.50±0.12, 1.18±0.15 vs 1.59±0.13; F values: 6.389 and 10.668, t values: -2.33 and -3.10, both P<0.05), and there were no significant differences in all lobes between MCI and AD group ( t values: from -1.29 to -0.51, all P>0.05). Visual analysis showed that the positive rates of PIB in frontal lobe (85.7%(18/21)), posterior cingulate (85.7%(18/21)), precuneus (81.0%(17/21)), temporal lobe (81.0%(17/21)) and occipital lobe (47.6%(10/21)) in AD were higher than those in MCI (4/11, 4/11, 4/11, 3/11 and 1/11, respectively; all P<0.05). Multiple linear regression showed that the degree of cognitive impairment were independent risk factors for SUVR of all lobes ( b values: 0.377-0.536, all P<0.05). The ApoE ε4 gene was independent risk factor for SUVR of precuneus ( b=0.290, P<0.05). Conclusion:11C-PIB is helpful for clinical diagnosis of MCI and AD patients and the degree of cognitive impairment and ApoE ε4 gene may be independent risk factors for increasing 11C-PIB binding.

Objective: To explore the clinical features and risk factors of hepatic injury due to immune checkpoint inhibitors (CPI) therapy in malignant tumor. Methods: Data of 112 patients (64 men and 48 women) who received CPI between January 2016 and March 2019 in Chinese Academy of Medical Sciences and Peking Union Medical College Shenzhen Hospital, and Huazhong University of Science and Techology Union Shenzhen Hospital were retrospectively collected. The median age of these patients was 60 years. Results: Hepatic adverse events were observed in 30 patients out of 112 patients (26.8%). Among them, the incidence of grade 3-5 hepatic adverse events were 7.14% (8/112). The median time of hepatic adverse event occurrence was 3 weeks (2-30) after undergoing therapy. The results of univariate and multivariate analyses showed that liver cancer was attributed to the CPI induced hepatitis (P<0.05). Patients with severe hepatic injury got almost complete resolution after receiving methlprednisolone for 4 to 6 weeks. Conclusion Live cancer is the risk factor of CPI-related hepatic adverse events.

Objective: To detect norovirus (NoV) GⅠ.1- and GⅡ.4-specific IgG, IgA and histo-blood group antigen (HBGA)-blocking antibodies in healthy populations of all age groups in China for better understanding the epidemiological features of norovirus in China from a serological point of view and providing basic data for vaccine development and clinical trial design. Methods: Indirect ELISA and HBGA-blocking assay were used to detect NoV-specific IgG, IgA and HBGA-blocking antibodies in serum samples collected from healthy natural populations (n=839, aged from six months to 88 years old) in Guangzhou, Fuyang and Yantai. The results were statistically analyzed. Results: The total positive rates of NoV GⅠ.1- and GⅡ.4-specific IgG antibodies were 91.9% and 93.0%. The positive rates of GⅠ.1- and GⅡ.4-specific IgA antibodies were 48.6% and 75.6%, and the titers of HBGA-blocking antibodies to GⅠ.1 and GⅡ.4 norovirus were 5.04 (95%CI: 4.63-5.49) and 18.15 (95%CI: 16.11-20.44). The positive rates of IgG and IgA antibodies generally showed an increasing trend with age. The positive rates of GⅠ.1- and GⅡ.4-specific IgG antibodies ranged from 79.2% to 100.0% and 76.7% to 100.0% in different age groups. They were 81.7% and 85.0% in the age group of 0.5-<1 year, 79.2% and 76.7% in the age group of 1-<2 years, and 98.1% and 96.3% in the age group of 12-<18 years, and maintained at 96% and 98% in the older age groups. The positive rates of GⅠ.1-specific IgA antibody ranged from 11.7% to 93.8% in different age groups and rapidly increased with age. It was 11.7% in the age group of 0.5-<1 year, and reached 93.3% in people aged 45-<60 years and 93.8% in people aged ≥60 years. The positive rates of GⅡ.4-specific IgA antibody ranged from 50.8% to 88.8% in different age groups with 50.8% in people aged 0.5-<1 year, and 86.7%-90.7% in people aged 12-<18 years and older. The titer of GⅠ.1 HBGA-blocking antibody generally increased with age. The antibody titer in populations aged 0.5-<12 years old was lower than that in those aged 18 years and above (GMT: 2.98-4.07 vs 8.21-11.62, P<0.001), and the titer in people of 12-<18 years old was lower than that in those of 45 years old and above (GMT: 5.21 vs 11.03-11.62, P<0.05). No obvious change with age was observed in the titer of GⅡ.4 HBGA-blocking antibody excepting the significant difference between populations of 2-<5 and 22-<45 years old (GMT: 26.73 vs 11.87, P<0.01). Conclusions This study revealed the characteristics of serum NoV GⅠ.1- and GⅡ.4-specific IgG, IgA and HBGA blocking antibodies in populations of different age groups in central and eastern China through analyzing their positive rates and titers and provided preliminary seroepidemiological data for the development of NoV vaccines in China.

Hyaluronic acid (HA) is a natural ligand of tumor-targeted drug delivery systems (DDS) due to the relevant CD44 receptor overexpressed on tumor cell membranes. However, other HA receptors (HARE and LYVE-1) are also overexpressing in the reticuloendothelial system (RES). Therefore, polyethylene glycol (PEG) modification of HA-based DDS is necessary to reduce RES capture. Unfortunately, pegylation remarkably inhibits tumor cellular uptake and endosomal escapement, significantly compromising the antitumor efficacy. Herein, we developed a Dox-loaded HA-based transformable supramolecular nanoplatform (Dox/HCVBP) to overcome this dilemma. Dox/HCVBP contains a tumor extracellular acidity-sensitive detachable PEG shell achieved by a benzoic imine linkage. The and investigations further demonstrated that Dox/HCVBP could be in a "stealth" state at blood stream for a long circulation time due to the buried HA ligands and the minimized nonspecific interaction by PEG shell. However, it could transform into a "recognition" state under the tumor acidic microenvironment for efficient tumor cellular uptake due to the direct exposure of active targeting ligand HA following PEG shell detachment. Such a transformative concept provides a promising strategy to resolve the dilemma of natural ligand-based DDS with conflicting two processes of tumor cellular uptake and nonspecific biodistribution.

Objective To investigate the effect of miR-132-3p on the proliferation of endothelial progenitor cells and its regulatory mechanism in order to provide a new theoretical basis for the treatment of deep venous thrombosis. Methods Real-time quantitative PCR (qPCR) was used to detect the expression of miR-132-3p in the plasma and endothelial progenitor cells of 27 healthy volunteers and 22 thrombus patients, and in endothelial progenitor cells under normoxic and hypoxic conditions. The miR-132-3p analogue and the specific siRNA were transferred into endothelial progenitor cells by the electroporation method. The effect of miR-132-3p on the proliferation of endothelial progenitor cells was detected using MMT and Cell Counting Kit-8 (CCK-8) methods. The effects of miR-132-3p on the expression of FOXO1 in endothelial cells were analyzed using the luciferase assay and western blots. Results The expression of miR-132-3p in clinical patients with thrombosis was significantly decreased to 0.45 ± 0.05 times of that of the healthy volunteers (P＜0.05). The expression of miR-132-3p in endothelial progenitor cells under hypoxia was down-regulated to (0.23 ± 0.13) times of that of under normoxia (P＜0.05). The expression of miR-132-3p of experiment group under hypoxia was up-regulated to (15.72 ± 2.06) times of that of control group (P＜0.05). MMT assay showed that the proliferation of cells in the experimental group under hypoxic condition was up-regulated to (7.79 ± 1.37) times of that in the control group (P＜0.01). CCK-8 assay showed that cell proliferation in experimental group was up-regulated to (6.46 ± 0.38) times of that in the control group (P＜0.01). Software analysis showed that FOXO1 was a direct target of miR-132-3p. Luciferase activity of miR-132-3p mimics transfected endothelial progenitor cells under hypoxic conditions were 0.47 times of that in siRNA treatment group. Western blot showed that the expression of FOXO1 protein in endothelial progenitor cells transfected with miR-132-3p mimics in hypoxia was 0.18 times of that in siRNA treatment group. Conclusions Compared with healthy volunteers, miR-132-3p expression in the blood of patients with thrombosis was significantly reduced that can promote transcription of the FOXO1 gene (and protein expression) and inhibit the proliferation of endothelial progenitor cells. It could be closely related to the formation of venous thrombosis.

Objective To investigate the bacterial spectrum and drug resistance of bone infection after multiple hospitalizations.Methods A retrospective case series study was conducted on 95 patients with bone infection due to injuries admitted in the General Hospital of Shenyang Military Area from January 2009 to December 2016.There were 76 males and 34 females,with an average age of 47 years (range,17-94 years).Bacterial culture and drug sensitivity tests were performed in 246 specimens of the infection secretions and infected tissues.The bacterial species and drug resistance data of all the specimens were statistically analyzed.The numbers and ratios of Gram-positive bacteria and Gram negative bacteria were counted according to the changes of hospitalization frequency,and the changes of drug resistance of Staphylococcus aureus after repeated hospitalizations were also recorded.Results A total of 110 pathogenic bacteria were isolated,and mixed infection was found in 19％ of the bacteria.There were 61 Gram-positive bacteria (55.5％),including 35 Staphylococcus aureus [seven methicillin-resistant staphylococcus (MRSA) strains],accounting for 57％ of Gram-positive strains.Other Gram positive bacteria were mainly Enterococcus faecalis and Staphylococcus epidermidis.There were 48 Gram-negative bacteria (43.6％),including 12 Pseudomonas aeruginosa strains,accounting for 25％ of Gram-negative strains,nine Klebsiella pneumoniae strains,accounting for 19％ of the Gramnegative strains.Staphylococcus aureus had a resistance rate to penicillin of 82％,and the major Gram positive bacteria (Staphylococcus aureus,Enterococcus faecalis,Staphylococcus epidermidis) were all highly sensitive to vancomycin and linezolid.The major Gram-negative bacteria (Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Acinetobacter baumannii) were highly resistant to the second generation of cephalosporins and were sensitive to carbapenem antibiotics.In 95 bone infection patients,the ratio of Gram positive bacteria to Gram negative bacteria increased from 0.98 at the first admission to 3 after repeated hospitalizations,and the ratio change was statistically significant (P ＜0.05).After multiple hospitalizations,the drug resistance of Staphylococcus aureus to gentamicin,ciprofloxacin,levofloxacin,and tetracycline increased gradually.The resistance rate to penicillin was even up to 100％.Conclusions The mixed infection of bone infection is common,among which Staphylococcus aureus and Staphylococcus epidermidis are the main Gram-positive pathogenic bacteria,Pseudomonas aeruginosa and Klebsiella pneumoniae are the main Gram negative pathogenic bacteria.The proportion of Gram-positive bacteria infection increased after multiple hospitalizations and became the major pathogenic bacteria.Penicillin should be avoided in the treatment of Staphylococcus aureus infection in multiple hospitalizations,and gentamicin and ciprofloxacin should be used with caution.Vancomycin or linezolid which is more sensitive is a better option.