Browsing by Author "Zhang, Hao"

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    The Activation and Function of Autophagy in Alcoholic Liver Disease
    (Bentham Science Publishers, 2017) Khambu, Bilon; Wang, Lin; Zhang, Hao; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of Medicine
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    Automated assessment of steatosis in murine fatty liver
    (PLOS, 2018-05-10) Sethunath, Deepak; Morusu, Siripriya; Tuceryan, Mihran; Cummings, Oscar W.; Zhang, Hao; Yin, Xiao-Ming; Vanderbeck, Scott; Chalasani, Naga; Gawrieh, Samer; Computer and Information Science, School of Science
    Although mice are commonly used to study different aspects of fatty liver disease, currently there are no validated fully automated methods to assess steatosis in mice. Accurate detection of macro- and microsteatosis in murine models of fatty liver disease is important in studying disease pathogenesis and detecting potential hepatotoxic signature during drug development. Further, precise quantification of macrosteatosis is essential for quantifying effects of therapies. Here, we develop and validate the performance of automated classifiers built using image processing and machine learning methods for detection of macro- and microsteatosis in murine fatty liver disease and study the correlation of automated quantification of macrosteatosis with expert pathologist’s semi-quantitative grades. The analysis is performed on digital images of 27 Hematoxylin & Eosin stained murine liver biopsy samples. An expert liver pathologist scored the amount of macrosteatosis and also annotated macro- and microsteatosis lesions on the biopsy images using a web-application. Using these annotations, supervised machine learning and image processing techniques, we created classifiers to detect macro- and microsteatosis. For macrosteatosis prediction, the model’s precision, sensitivity and area under the receiver operator characteristic (AUROC) were 94.2%, 95%, 99.1% respectively. When correlated with pathologist’s semi-quantitative grade of steatosis, the model fits with a coefficient of determination value of 0.905. For microsteatosis prediction, the model has precision, sensitivity and AUROC of 79.2%, 77%, 78.1% respectively. Validation by the expert pathologist of classifier’s predictions made on unseen images of biopsy samples showed 100% and 63% accuracy for macro- and microsteatosis, respectively. This novel work demonstrates that fully automated assessment of steatosis is feasible in murine liver biopsies images. Our classifier has excellent sensitivity and accuracy for detection of macrosteatosis in murine fatty liver disease.
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    Autophagy in Alcoholic Liver Disease, Self-eating Triggered by Drinking
    (Elsevier, 2015-09) Wang, Lin; Khambu, Bilon; Zhang, Hao; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of Medicine
    Macroautophagy (autophagy) is an evolutionarily conserved mechanism. It is important for normal cellular function and also plays critical roles in the etiology and pathogenesis of a number of human diseases. In alcohol-induced liver disease, autophagy is a protective mechanism against the liver injury caused by alcohol. Autophagy is activated in acute ethanol treatment but could be suppressed in chronic and/or high dose treatment of alcohol. The selective removal of lipid droplets and/or damaged mitochondria is likely the major mode of autophagy in reducing liver injury. Understanding the dynamics of the autophagy process and the approach to modulate autophagy could help finding new ways to battle against alcohol-induced liver injury.
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    Autophagy induced by calcium phosphate precipitates targets damaged endosomes
    (ASBMB, 2014-04-18) Chen, Xi; Khambu, Bilon; Zhang, Hao; Gao, Wentao; Li, Min; Chen, Xiaoyun; Yoshimori, Tamotsu; Yin, Xiao-Ming; Department of Pathology & Laboratory Medicine, IU School of Medicine
    Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62.
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    Benzisothiazol‐3‐ones through a Metal‐Free Intramolecular N–S Bond Formation
    (Wiley, 2018-11) Yang, Ke; Zhang, Hao; Niu, Ben; Tang, Tiandi; Ge, Haibo; Chemistry and Chemical Biology, School of Science
    The highly efficient synthesis of benzoisothiazol‐3‐ones from thiobenzamides has been described with good functional group compatibility and excellent yields. This work represents the first example of selectfluor‐promoted N–S bond formation processes. This method provides a facile approach to access various important bioactive benzoisothiazol‐3‐ones.
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    Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation
    (Elsevier, 2023) Hadisurya, Marco; Lee, Zheng-Chi; Luo, Zhuojun; Zhang, Guiyuan; Ding, Yajie; Zhang, Hao; Iliuk, Anton B.; Pili, Roberto; Boris, Ronald S.; Tao, W. Andy; Urology, School of Medicine
    Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease, and healthy control individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.
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    Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply
    (Taylor & Francis, 2018) Zhang, Hao; Yan, Shengmin; Khambu, Bilon; Ma, Fengguang; Li, Yong; Chen, Xiaoyun; Puertollano, Rosa; Li, Yu; Chalasani, Naga; Yin, Xiao-Ming; Martina, Jose A.; Pathology & Laboratory Medicine, IU School of Medicine
    Normal metabolism requires a controlled balance between anabolism and catabolism. It is not completely known how this balance can be retained when the level of nutrient supply changes in the long term. We found that in murine liver anabolism, as represented by the phosphorylation of RPS6KB (ribosomal protein S6 kinase), was soon elevated while catabolism, as represented by TFEB (transcription factor EB)-directed gene transcription and lysosomal activities, was downregulated after the administration of a high-fat diet (HFD). Surprisingly, neither the alteration in RPS6KB phosphorylation nor that in TFEB functions was static over the long course of HFD feeding. Instead, the 2 signals exhibited dynamic alterations in opposite directions, which could be explained by the dependence of MTORC1 (MTOR complex 1) activation on TFEB-supported lysosome function and the feedback suppression of TFEB by MTORC1. Disruption of the dynamics by enforced expression of TFEB in HFD-fed mice at the peaks of MTORC1 activation restored lysosome function. Consistently, interference of MTORC1 activation with rapamycin or with a constitutively activated RRAGA mutant at the peak or nadir of MTORC1 oscillation enhanced or reduced the lysosome function, respectively. These treatments also improved or exacerbated hepatic steatosis and liver injury, respectively. Finally, there was a significant inverse correlation between TFEB activation and steatosis severity in the livers of patients with non-alcohol fatty liver diseases, supporting the clinical relevance of TFEB-regulated events. Thus, maintaining catabolic function through feedback mechanisms during enhanced anabolism, which is caused by nutrient oversupply, is important for reducing liver pathology.
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    The impact of mTOR, TFEB and Bid on non-alcoholic fatty liver disease and metabolic syndrome
    (2015-05-18) Zhang, Hao; Yin, Xiao-Ming; Chalasani, Naga P.; Konger, Raymond Lloyd; Murrell, Jill R.
    Non-alcoholic fatty liver disease and metabolic syndrome induced by high nutrient status have increasingly become a global health concern as it cause multiple complications. The mTOR complex is central in regulating anabolic reactions within cells under growth factors or under high nutrients stimulation. Constitutive and persistent activation of mTOR can impair cellular functions. In the first part of this study, we demonstrate a damping oscillation of mTOR activity during a long-term treatment of high fat diet. TFEB translocation and lysosomal enzyme activity also oscillate, but in an opposite direction. TFEB controls the lysosomal activity, autophagic degradation and lipid metabolism. Overexpression of wild type and mutant TFEB could inhibit NAFLD development in mice. In addition, TFEB location in nucleus inversely correlates with NAFLD severity in patients. mTOR activation under hypernutrition status suppresses TFEB translocation, inhibits lysosomal functions and autophagic degradation of lipid droplets. Inhibition of mTOR activity by rapamycin reverse the above phenotypes. Because mTOR activation also requires normal lysosomal function, the inhibition of TFEB by mTOR leads to decreased lysosomal function and mTOR downregulation. This negative feedback may explain the oscillation pattern of mTOR activation in long term high fat diet regimen and is a novel mechanism for inhibition of mTOR. In the second part of study, we report that Bid protein, previously known for its pro-apoptosis function in promoting mitochondrial permeability, plays an unexpected role in regulating fatty acid beta oxidation. Deletion of Bid in mice reprograms the body's response to hyper-nutrition caused by high fat diet, leading to the resistance to the development of obesity, liver steatosis and metabolic syndrome. These mice present a higher oxygen consumption, a lower respiratory quotient, and an increased beta-oxidation rate. Mechanistically, the high fat diet regimen triggers translocation of the full length Bid molecule to mitochondrial membrane. Genetic deletion of Bid also affects the stability of its binding protein, MTCH2 in the mitochondrial membrane. In summary, we describe in this study a mTOR-TFEB-lysosome feedback loop, which can regulate NAFLD development, and a novel Bid-mediated regulatory mechanism in beta-oxidation, which limits energy expenditure and promotes obesity development.
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    Mechanisms of Cytoskeletal Dysregulation in the Kidney Proximal Tubule During ATP Depletion and Ischemia
    (2009-08) Zhang, Hao; Atkinson, Simon J.; Harrington, Maureen A.; Marrs, James A.; Quilliam, Lawrence A.
    Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.
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    Promotion of diet-induced obesity and metabolic syndromes by BID is associated with gut microbiota
    (Wolters Kluwer, 2022) Yan, Shengmin; Zhou, Jun; Zhang, Hao; Lin, Zhen; Khambu, Bilon; Liu, Gang; Ma, Michelle; Chen, Xiaoyun; Chalasani, Naga; Yin, Xiao-Ming; Pathology and Laboratory Medicine, School of Medicine
    A growing body of evidence has indicated an expanding functional network of B-cell lymphoma 2 (BCL-2) family proteins beyond regulation of cell death and survival. Here, we examined the role and mechanisms of BH3 interacting-domain death agonist (BID), a pro-death BCL-2 family member, in the development of diet-induced metabolic dysfunction. Mice deficient in bid (bid-/- ) were resistant to high-fat diet (HFD)-induced obesity, hepatic steatosis, and dyslipidemia with an increased insulin sensitivity. Indirect calorimetry analysis indicated that bid deficiency increased metabolic rate and decreased respiratory exchange ratio, suggesting a larger contribution of lipids to overall energy expenditure. While expression of several genes related to lipid accumulation was only increased in wild-type livers, metabolomics analysis revealed a consistent reduction in fatty acids but an increase in certain sugars and Krebs cycle intermediates in bid-/- livers. Gut microbiota (GM) analysis indicated that HFD induced gut dysbiosis with differential patterns in wild-type and in bid-/- mice. Notably, abrogation of GM by antibiotics during HFD feeding eliminated the beneficial effects against obesity and hepatic steatosis conferred by the bid deficiency. Conclusion: These results indicate that the protective role of bid-deficiency against diet-induced metabolic dysfunction interacts with the function of GM.