Hepatology Commons^™

A Pilot Genome-Wide Analysis Study Identifies Loci Associated With Response To Obeticholic Acid In Patients With Nash., Samer Gawrieh, Xiuqing Guo, Jingyi Tan, Marie Lauzon, Kent D Taylor, Rohit Loomba, Oscar W Cummings, Sreekumar Pillai, Pallav Bhatnagar, Kris V Kowdley, Katherine Yates, Laura A Wilson, Yii-Der Ida Chen, Jerome I Rotter, Naga Chalasani

Articles, Abstracts, and Reports

A significantly higher proportion of patients with nonalcoholic steatohepatitis (NASH) who received obeticholic acid (OCA) had histological improvement relative to placebo in the FLINT (farnesoid X nuclear receptor ligand obeticholic acid for noncirrhotic, NASH treatment) trial. However, genetic predictors of response to OCA are unknown. We conducted a genome-wide association study (GWAS) in FLINT participants to identify variants associated with NASH resolution and fibrosis improvement. Genotyping was performed using the Omni2.5 content GWAS chip. To avoid false positives introduced by population stratification, we focused our GWAS on white participants. Six regions on chromosomes 1, 4, 6, 7, 15, and 17 …

Acute Diagnosis Of Wilson’S Disease In A Teenage Patient, Sarah Irvin, Ryan Mccarthy

Marshall Journal of Medicine

Wilson’s Disease, a rare autosomal recessive genetic disease, is caused by a mutation in the ATP7B enzyme gene. Without this enzyme, copper builds up in the brain, liver, and cornea causing a multitude of symptoms. It is important to consider Wilson’s disease because the prognosis is dependent on timely diagnosis. This is an interesting case of a 19-year-old male who presented with suicidal thoughts and rapid weight loss. After many months and an extensive work-up, Wilson’s Disease was diagnosed. Due to his rapid decline, he was transferred to a larger university healthcare center where he is currently enrolled in clinical …

Characterization Of Patients With Both Alcoholic And Nonalcoholic Fatty Liver Disease In A Large United States Cohort., George Khoudari, Amandeep Singh, Mazen Noureddin, Danielle Fritze, Rocio Lopez, Imad Asaad, Eric Lawitz, Fred Poordad, Kris V Kowdley, Naim Alkhouri

Articles, Abstracts, and Reports

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome (MetS) and is characterized by steatosis in the absence of significant alcohol consumption. However, MetS and significant alcohol intake coexist in certain individuals which may lead to the development of BAFLD.

AIM: To assess the clinical characteristics of patients with both alcoholic and NAFLD (BAFLD) in a large cohort in the United States.

METHODS: Adults from the National Health and Nutrition Examination Survey between 2003-2014 were included. NAFLD was diagnosed based on elevated alanine aminotransferase (ALT) and being overweight or obese in the absence of other …

Can We Predict Low Quality Colonoscopy? Establishing Open Access Colonoscopy At A Fellow-Run Gastroenterology Clinic, Matthew Sullivan Do, Abdul Aleem Md, Rajesh Essrani Md, Patrick Hickey Do, Robert Andrews Md, Anam Malik Md, Hiral N. Shah Md, Shashin Shah Md

Department of Medicine

No abstract provided.

The Evaluation Of Liver Fibrosis Regression In Chronic Hepatitis C Patients After The Treatment With Direct-Acting Antiviral Agents – A Review Of The Literature, Olga H. Orasan, George Ciulei, Sorina C. Coste, Bianca A. Cibu, Adela V. S Taut, Simina F. Tarmure, Iulia O. Pfingstgraf, Teodora G. Alexescu, Ionela E. Popovici, Flaviu Mureșan, Fabian Ovidiu, Vasile Negrean, Angela Cozma

Journal of Mind and Medical Sciences

The second-generation of direct-acting antiviral agents are the current treatment for chronic viral hepatitis C infection. To evaluate the regression of liver fibrosis in patients receiving this therapy, liver biopsy remains the most accurate method, but the invasiveness of this procedure is its major drawback. Different non-invasive tests have been used to study changes in the stage of liver fibrosis in patients with chronic viral hepatitis treated with the second-generation of direct-acting antiviral agents: liver stiffness measurements (with transient elastography or acoustic radiation force impulse elastography) or different scores that use serum markers to calculate a fibrosis score. We prepared …

Association Of Histologic Disease Activity With Progression Of Nonalcoholic Fatty Liver Disease., David E Kleiner, Elizabeth M Brunt, Laura A Wilson, Cynthia Behling, Cynthia Guy, Melissa Contos, Oscar Cummings, Matthew Yeh, Ryan Gill, Naga Chalasani, Brent A Neuschwander-Tetri, Anna Mae Diehl, Srinivasan Dasarathy, Norah Terrault, Kris V Kowdley, Rohit Loomba, Patricia Belt, James Tonascia, Joel E Lavine, Arun J Sanyal

Articles, Abstracts, and Reports

Importance: The histologic evolution of the full spectrum of nonalcoholic fatty liver disease (NAFLD) and factors associated with progression or regression remain to be definitively established.

Objective: To evaluate the histologic evolution of NAFLD and the factors associated with changes in disease severity over time.

Design, Setting, and Participants: A prospective cohort substudy from the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) NAFLD Database study, a noninterventional registry, was performed at 8 university medical research centers. Masked assessment of liver histologic specimens was performed, using a prespecified protocol to score individual biopsies. Participants included 446 adults with NAFLD enrolled in …

Silymarin In Non-Cirrhotics With Non-Alcoholic Steatohepatitis: A Randomized, Double-Blind, Placebo Controlled Trial., Victor J. Navarro, Steven H. Belle, Massimo D'Amato, Nezam Adfhal, Elizabeth M. Brunt, Michael W. Fried, K. Rajender Reddy, Abdus S. Wahed, Stephen Harrison

Division of Gastroenterology and Hepatology Faculty Papers

The botanical product silymarin, an extract of milk thistle, is commonly used by patients to treat chronic liver disease and may be a treatment for NASH due to its antioxidant properties. We aimed to assess the safety and efficacy of higher than customary doses of silymarin in non-cirrhotic patients with NASH. This exploratory randomized double-blind placebo controlled multicenter Phase II trial tested a proprietary standardized silymarin preparation (Legalon®, Rottapharm|Madaus, Mylan) and was conducted at 5 medical centers in the United States. Eligible adult patients had liver biopsy within 12 months showing NASH without cirrhosis with NAFLD Activity Score (NAS) ≥4 …

A Challenging Case Of Spontaneous Bacterial Empyema In A Cirrhotic Patient, Saad Emhmed Ali, Olalekan Akanbi, Macy Godman, Mohanad Soliman, Wesam M. Frandah, Karim Benrajab

Internal Medicine

Spontaneous Bacterial Empyema (SBEM) is distinguished as a primary infection of pre-existing hepatic hydrothorax in the absence of cardiopulmonary or pleural disease. It is an under-recognized complication of cirrhosis and is associated with high morbidity and mortality. The diagnosis requires chest imaging to rule out underlying pneumonia as an etiology; a diagnostic thoracentesis is crucial to make the diagnosis and should be obtained in any patient with pleural effusion and evidence of infection. Expeditious administration of antibiotics is essential to ameliorate the outcome. We present an interesting case of spontaneous bacterial empyema in a cirrhotic patient with hepatic hydrothorax.

Irisin Functions To Inhibit Malignant Growth Of Human Pancreatic Cancer Cells Via Downregulation Of The Pi3k/Akt Signaling Pathway., Deguo Zhang, Ping Zhang, Luan Li, Nan Tang, Fei Huang, Xianguo Kong, Xueying Tan, Guangjun Shi

Department of Medicine Faculty Papers

Introduction: Irisin is a newly identified cytokine that has gained increasing attention because of its potential therapeutic applications in metabolic diseases and human cancers. Recently, accumulating evidence indicates that irisin plays an important role in the development and metastasis of various tumors. The aim of this study was to evaluate the effects and underlying mechanisms of irisin on malignant growth of pancreatic cancer cells.

Materials and methods: The anti-proliferative effect of irisin was examined using the CCK-8 assay. Irisin-induced apoptosis was determined by the annexin V-FITC/PI staining assay. The effects of irisin on cell migration and invasion were assessed using …

Geriatric Hepatology: The Hepatic Diseases Of The Elderly And Liver Transplant, Shima Ghavimi, Hamed Azimi, Neel Patel, Oleg Shulik

Internal Medicine

Introduction: With the aging US population, chronic liver diseases are becoming more commonly diagnosed in the geriatric population. Advanced age leads to changes in liver blood flow, volume, morphology and normal physiology. This predisposes elderly patients to develop certain chronic liver diseases. Also, the clinical course and management differ in an older patient when compared to a younger patient. Some causes of chronic liver disease in the geriatric population include Hepatitis A, B, C, Non-Alcoholic Fatty liver disease, prolonged alcohol use and inflammation. Many chronic liver diseases are characterized by a slow, indolent course of progression with non-specific symptoms and …

Proton Pump Inhibitors Increase The Severity Of Hepatic Encephalopathy In Cirrhotic Patients, Matthew J. Fasullo, Prashanth Rau, Dong-Qi Liu, Erik Holzwanger, Jomol Mathew, Yurima Guilarte-Walker, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND: Liver cirrhosis is the late stage of hepatic fibrosis and is characterized by portal hypertension that can clinically lead to decompensation in the form of ascites, esophageal/gastric varices or encephalopathy. The most common sequelae associated with liver cirrhosis are neurologic and neuropsychiatric impairments labeled as hepatic encephalopathy (HE). Well established triggers for HE include infection, gastrointestinal bleeding, constipation, and medications. Alterations to the gut microbiome is one of the leading ammonia producers in the body, and therefore may make patients more susceptible to HE.

AIM: To investigate the relationship between the use of proton pump inhibitors (PPIs) and HE …

Current Perspectives Into The Evaluation And Management Of Hepatitis B: A Review., Vignan Manne, Eric Gochanour, Kris V Kowdley

Articles, Abstracts, and Reports

Hepatitis B is a widespread disease which affects millions of people worldwide. Chronic hepatitis B (CHB) can lead to significant morbidity and mortality due to complications such as cirrhosis and hepatocellular carcinoma. The pathophysiology of hepatitis is critical to diagnosing CHB. Deciding which patients with CHB should be treated is an important decision as treatment can often lead to better outcomes in the appropriate patient population. The nucleos(t)ide analog inhibitors entecavir and tenofovir are currently the mainstay of treatment as they are able to successfully suppress the virus and lead to fewer complications. Novel therapies are currently being developed which …

Segmental Distribution Of Hepatocellular Carcinoma Correlates With Microvascular Invasion In Liver Explants Undergoing Transplantation, Yasir Al-Azzawai, Eva Rouanet, Ryan J. Hendrix, Lidia Spaho, Hesham Malik, Deepika Devuni, Gyongyi Szabo, Graham Barnard

Gyongyi Szabo

Introduction: Microvascular invasion (MVI) in hepatocellular carcinoma (HCC) patients is a poor prognostic factor after liver transplantation and/or resection. Any correlation between MVI and segmental location of HCC has yet to be studied. Our aim is to evaluate the segmental location of HCC and any correlation with the presence of MVI, portal vein thrombosis (PVT) in explanted livers, and the recurrence of HCC after transplantation. Another objective of the study is to assess the treatment history (ablation or transarterial chemoembolization (TACE)) and size of the tumor with respect to the risk of MVI.

Methods: A single center, retrospective chart review, …

Acute-On-Chronic Liver Failure: Consensus Recommendations Of The Asian Pacific Association For The Study Of The Liver (Apasl): An Update, Shiv Kumar Sarin, Ashok Choudhury, Manoj K. Sharma, Rakhi Maiwall, Mamun Al Mahtab, Salimur Rahman, Saeed Hamid, Amna S. Butt, Wasim Jafri, Zaigham Abbas

Section of Gastroenterology

The first consensus report of the working party of the Asian Pacific Association for the Study of the Liver (APASL) set up in 2004 on acute-on-chronic liver failure (ACLF) was published in 2009. With international groups volunteering to join, the "APASL ACLF Research Consortium (AARC)" was formed in 2012, which continued to collect prospective ACLF patient data. Based on the prospective data analysis of nearly 1400 patients, the AARC consensus was published in 2014. In the past nearly four-and-a-half years, the AARC database has been enriched to about 5200 cases by major hepatology centers across Asia. The data published during …

Thalidomide For The Treatment Of Metastatic Hepatic Epithelioid Hemangioendothelioma: A Case Report With A Long Term Follow-Up, Felipe Salech, Sebastián Valderrama, Bruno Nervi, Juan Carlos Rodriguez, Danny Oksenberg, Alvaro Koch, Gladys Smok, Ignacio Duarte, Rosa María Pérez-Ayuso, Nicolás Jarufe, Jorge Martínez, Alejandro Soza, Marco Arrese, Arnoldo Riquelme

Internal Medicine Faculty Publications

Hepatic epithelioid hemangioendothelioma (HEH) is an unusual, low-grade malignant vascular tumor of the liver. Here we describe a case of a 40-year-old woman who presented with abdominal pain in the upper right quadrant and giant hepatomegaly, in which imaging studies and a fine-needle liver biopsy confirmed the presence of a large EHE with an isolated lung metastasis. After balancing all possible therapeutic modalities the patient was treated conservatively with thalidomide (300 mg/day). The drug was well tolerated with minimal toxicity and thepatient continues on therapy 109 months after treatment was started with no disease progression. Current therapeutic options for HEH …

Novel Mechanisms And Biomarkers In Alcohol-Induced Organ Injury., Christine E. Dolin

Electronic Theses and Dissertations

Background. Ethanol (EtOH) consumption is known to affect multiple organs; this is unsurprising, as the concentration of EtOH in the blood at relevant doses reaches the millimolar range. The overarching goal of this dissertation was to elucidate mechanisms of alcohol-induced organ injury, specifically the effects of alcohol on the hepatic extracellular matrix (ECM) proteome, the alcoholic hepatitis (AH) plasma peptidome, and the effects of alcohol on the renal cortex proteome and transcriptome. Methods. Mice were pair-fed ethanol-containing liquid diet chronically, and then some mice were administered lipopolysaccharide (LPS). Liver sections from these mice were processed in a series of increasingly …

Rare Sarcomatoid Carcinoma Of The Liver In A Patient With No History Of Hepatocellular Carcinoma: A Case Report, Kevat B. Patel, Rita H. Hawarny, Elizabeth Saunders, Marc Hettlinger, Niru Nahar

Marshall Journal of Medicine

Sarcomatoid carcinoma is a rare malignant tumor of unknown pathogenesis characterized by poorly differentiated carcinoma tissue containing sarcoma-like differentiation of either spindle or giant cell and rarely occurs in the gastrointestinal tract and hepatobiliary-pancreatic system.¹ Primary hepatic sarcomatoid carcinoma accounts for only 0.2 % of primary malignant liver tumors, and 1.8% of all surgically resected hepatocellular carcinomas.² The majority of hepatic sarcomatoid carcinoma cases appear to occur simultaneously with hepatocellular or cholangiocellular carcinoma.³ The preferred treatment for hepatic sarcomatoid carcinoma is surgical resection and the overall prognosis is poor.⁴ This case depicts a 62-year-old female whom …

Transplanted Adult Human Hepatic Stem/Progenitor Cells Prevent Histogenesis Of Advanced Hepatic Fibrosis In Mice Induced By Carbon Tetrachloride, Yanzhen Bi, Xiyu Liu, Chuanping Si, Ye Hong, Yongke Lu, Pengfei Gao, Yonghong Yang, Xiaobei Zhang, Yibo Wang, Huabao Xiong, Zhongping Duan, Yu Chen, Feng Hong

Pharmacology, Physiology and Toxicology

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl₄) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl₄ (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl₄ induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. …

Extracellular Vesicles In Liver Diseases: Meeting Report From The International Liver Congress 2018, Jesus M. Banales, Ariel E. Feldstein, Hanna Sanger, Veronika Lukacs-Kornek, Gyongyi Szabo, Miroslaw Kornek

Gyongyi Szabo

Extracellular vesicles (EVs) are small and heterogeneous membrane-bound structures released by cells and found in all biological fluids. They are effective intercellular communicators, acting on a number of close and/or distant target cells. EV cargo may reflect the cell of origin as well as the specific stress that induces their formation and release. They transport a variety of bioactive molecules, including messenger RNA, noncoding RNAs, proteins, lipids, and metabolites, that can be transferred among cells, regulating various cell responses. Alteration in the concentration and composition of EVs in biological fluids is a typical hallmark of pathologies in different liver diseases. …

Inhibition Of Triggering Receptor Expressed On Myeloid Cells 1 Ameliorates Inflammation And Macrophage And Neutrophil Activation In Alcoholic Liver Disease In Mice, David Tornai, Istvan Furi, Zu T. Shen, Alexander B. Sigalov, Sahin Coban, Gyongyi Szabo

Gyongyi Szabo

Alcoholic liver disease (ALD) is characterized by macrophage and neutrophil leukocyte recruitment and activation in the liver. Damage- and pathogen-associated molecular patterns contribute to a self-perpetuating proinflammatory state in ALD. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a surface receptor that amplifies inflammation induced by toll-like receptors (TLRs) and is expressed on neutrophils and monocytes/macrophages. We hypothesized that TREM-1 signaling contributes to proinflammatory pathway activation in ALD. Using an in vivo ALD model in mice, we tested the effects of ligand-independent TREM-1 inhibitory peptides that were formulated into human high-density lipoprotein (HDL)-mimicking complexes GF9-HDL and GA/E31-HDL. As revealed …

Aortoduodenal Fistula Forms From Primary Aortic Stump Graft In A Two-Time Multi-Visceral Transplant Patient With Presentation Of Gastrointestinal Bleed And Bowel Perforation: A Case Report, Brielle Corrente

Graduate Student Research Symposium

Usually not diagnosed until open laparotomy, aortoduodenalfistulas (ADF) are one of the rarest complications of intestinal transplant surgery. With an incidence rate of only 0.04% at autopsy and only 250 documented cases since the early 1800’s, aortoduodenal fistulas are the most deadly complications of intestinal transplantation with a mortality rate of 100% without surgical intervention. A 39 year old, two-time multi-visceral transplant African American female patient suffered from a primary aortoduodenal fistula formation in a primary modified multi-visceral transplant aortic stump graft site. With emergency open laparotomy repair, revascularization of the secondary multi-visceral transplant was performed, saving the life of …

A Multicenter Evaluation Of A New Eus Core Biopsy Needle: Experience In 200 Patients., Douglas G. Adler, V. Raman Muthusamy, Dean S. Ehrlich, Gulshan Parasher, Nirav C. Thosani, Ann Chen, Jonathan M. Buscaglia, Anoop Appannagari, Eduardo Quintero, Harry Aslanian, Linda Jo Taylor, Ali Siddiqui

Division of Gastroenterology and Hepatology Faculty Papers

Background and Objectives: We present a multicenter study of a new endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) needle (Acquire, Boston Scientific, Natick, MA). The aim of the study was to analyze the needle's clinical performance when sampling solid lesions and to assess the safety of this device.

Methods: We performed a multicenter retrospective study of patients undergoing EUS-FNB during July 1-November 15, 2016.

Results: Two hundred patients (121 males and 79 females) underwent EUS-FNB of solid lesions with the Acquire needle. Lesions included solid pancreatic masses (n = 109), adenopathy (n = 45), submucosal lesions (n = 34), cholangiocarcinoma (n = …

Parallel Transjugular Intrahepatic Portosystemic Shunt With Viatorr® Stents For Primary Tips Insufficiency: Case Series And Review Of Literature, Driss Raissi, Qian Yu, Michael Nisiewicz, Steven Krohmer

Radiology Faculty Publications

BACKGROUND

Transjugular intrahepatic portosystemic shunts (TIPS) can alleviate complications of portal hypertension such as ascites and variceal bleeding by decreasing the portosystemic gradient. In limited clinical situations, parallel TIPS may be only solution to alleviate either variceal bleeding or ascites secondary to portal hypertension when the primary TIPS fails to do so. Data specifically addressing the use of this partially polytetrafluoroethylene covered nitinol stent (Viatorr^®) is largely lacking despite Viatorr^® being the current gold standard for modern TIPS placement.

CASE SUMMARY

All three patients had portal hypertension and already had a primary Viatorr^® TIPS placed previously. …

Cellular Senescence And Their Role In Liver Metabolism In Health And Disease: Overview And Future Directions, Matthew Schade, Jacqueline A. Sanabria, Rodrigo Aguilar, Milad Modarresi, Brad Gillon, Zach Hunter, Jacqueline Fannin, Amrita Mallick, Henri Brunengraber, Juan Sanabria

Juan R. Sanabria

Chronic liver disease has globally risen mainly due to a prevalent Hepatitis C Virus (HCV) infection rate and an epidemic of obesity. It is estimated by the year 2030, 2.2 billion people around the world will be overweight and 1.1 billion people will be obese. Diabetes and obesity are the main risk factors for the development of the metabolic syndrome and in the liver of Non-Alcoholic Fatty Liver Disease (NAFLD) which could progress to NASH related cirrhosis and liver malignancy. At present there is not effective therapy for NASH besides loss of weight and exercise. Furthermore, optimal management of HCC …

Identification Of Novel Regulatory Genes In Apap Induced Hepatocyte Toxicity By A Genome-Wide Crispr-Cas9 Screen., Katherine Shortt, Daniel P. Heruth, Nini Zhang, Weibin Wu, Shipra Singh, Ding-You Li, Li Qin Zhang, Gerald J. Wyckoff, Lei S Qi, Craig A. Friesen, Shui Qing Ye

Manuscripts, Articles, Book Chapters and Other Papers

Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, …

3d Printed Biliary Anatomy For Surgical Planning, Jason Core, Erik Massenzio, Tamar Wasserman, Kevin Xie

Phase 1

Background: Studies demonstrated that 3D-printed livers from CT or MRI data can be accurate models of actual patient anatomy. However, it has yet to be established if 3D printing offers improvements to clinical outcomes in surgery. This project seeks to optimize these applications for use at Jefferson by producing 3D-printed models from patient CT scans to guide liver resection surgeries.

Methods: A liver transplant attending was interviewed about challenges encountered during hepatectomies. A publicly available abdominal computed tomography scan was used to render a liver and its vasculature in 3DSlicer. The liver surface was cut into two halves in MeshMixer …

Progressive Hypoxia-On-A-Chip: An In Vitro Oxygen Gradient Model For Capturing The Effects Of Hypoxia On Primary Hepatocytes In Health And Disease, Young Bok Abraham Kang, Jinsu Eo, Beyza Bulutoglu, Martin L. Yarmush, O. Berk Usta

Faculty Publications - Biomedical, Mechanical, and Civil Engineering

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient …