The Role of Parkin and Mitophagy in Acetaminophen and Alcohol-induced Liver Injuries

Acetaminophen (APAP) is the leading cause of acute liver failure in the United States, and alcoholic liver disease (ALD) is a worldwide health problem that claims two million lives per year. Currently, the only cure for either disease is liver transplantation in severe disease states. Therefore, new therapeutic options for treatment of these liver diseases are greatly needed. To develop new therapeutic options, the mechanisms involved in APAP and alcohol-induced liver toxicities must be better understood. We previously demonstrated that autophagy was protective against both APAP and alcohol-induced liver injuries by removing damaged mitochondria by mitophagy, which is a selective form of autophagy specific for mitochondria. However, the mechanisms for induction of mitophagy in the liver are unknown. Parkin is an E3 ubiquitin ligase that is well known to be required for mitophagy induction in mammalian cell models after mitochondrial depolarization. Therefore, we evaluated the role of Parkin in inducing mitophagy as a protective mechanism against APAP and alcohol-induced liver injuries. For alcohol treatment, acute-binge and chronic-plus-binge (Gao-binge) models were used. First, we found that APAP and alcohol produced opposite responses in Parkin KO mice. Parkin KO mice were protected after APAP treatment, but they had more liver injury and steatosis after alcohol treatment compared to WT mice. It is well known that c-Jun N-terminal kinase (JNK) activation exacerbates APAP-induced liver injury, and it has recently been shown that myeloid leukemia cell differentiation protein (Mcl-1) mediates protection against APAP-induced liver injury. In addition, liver regeneration has been shown to be the most important repair mechanism for APAP-induced liver injury. We found that Parkin KO mice had decreased JNK activation, increased Mcl-1 expression, and increased hepatocyte proliferation after APAP treatment in their livers compared to WT mice. In contrast to protection after APAP treatment, Parkin KO mice were more susceptible to alcohol-induced liver injury than WT mice because of Parkin’s role in maintaining a healthy population of mitochondria, likely through activation of mitophagy. Alcohol caused greater mitochondrial damage in Parkin KO livers compared to WT livers. Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae after alcohol treatment, which were not seen in WT mice. In addition, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute-binge alcohol treatment compared to WT mice. Furthermore, Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared to WT mouse liver mitochondria. The fact that Parkin KO mice were protected against APAP-induced liver injury but had increased liver injury and steatosis after alcohol treatment compared to WT mice suggests that Parkin has multiple roles in maintaining cellular homeostasis in addition to its role in initiation of mitophagy, and these various roles my differ in importance depending on the amount and type of liver injury produced. Therefore, Parkin’s roles in regulating proliferation, JNK activation, and Mcl-1 expression were likely more important than mitophagy during APAP-induced liver injury while its role in mitophagy induction to maintain mitochondrial homeostasis was likely more important during alcohol-induced liver injury. Second, we surprisingly found that even though Parkin has been shown to be required for mitophagy induction in in vitro models, Parkin was not essential for mitophagy induction in the liver. We found that mitophagy still occurred in Parkin KO mice based on electron microscopy analysis after APAP and alcohol treatments. However, mitophagy levels were reduced in Parkin KO mice compared to WT mice, and Parkin translocated to mitochondria in WT mouse livers after both APAP and alcohol treatments. These results suggest that Parkin-induced mitophagy is still likely an important protective mechanism in the liver even though compensatory adaptive mechanisms exist for mitophagy induction in the absence of Parkin. In addition, these compensatory mechanisms may not be as efficient as Parkin in inducing mitophagy in the liver because mitophagy levels were reduced in Parkin KO mice compared to WT mice after APAP and alcohol treatments. Parkin-independent mechanisms for mitophagy induction in the liver are currently unknown, but they may involve activation of other proteins known to mediate mitophagy, such as Mul1, which has been shown to act in parallel to the Parkin pathway in Drosophila. Third, we found that whole-body knockout of Parkin and acute knockdown of Parkin had opposite responses to APAP overdose. While Parkin KO mice were protected against APAP-induced liver injury compared to WT mice, acute knockdown of Parkin in mouse livers resulted in increased liver injury compared to WT mice after APAP treatment. Whole-body Parkin KO mice were protected because they had increased Mcl-1 and proliferation levels and decreased JNK activation, and mice with acute knockdown had exacerbated APAP-induced liver injury because they had reduced Mcl-1 and proliferation levels and increased JNK activation. In addition, mitophagy was reduced in both Parkin KO mice and mice with acute knockdown of Parkin compared to WT mice. However, mitophagy levels were only slightly reduced in whole-body knockout mice while they were significantly reduced in mice with acute Parkin knockdown. These opposite responses between Parkin whole-body KO and acute knockdown mice were likely due to a lack of time to develop compensatory and adaptive mechanisms in the acute knockdown mice that were present in the whole-body knockout mice. Overall, our findings indicate that Parkin-induced mitophagy is likely a mechanism of protection in the liver, but compensatory mechanisms exist for induction of mitophagy in the absence of Parkin. However, these compensatory mechanisms may only play a minor role in the liver. In addition, Parkin has multiple roles in maintaining cellular homeostasis in addition to mitophagy, which causes Parkin KO mice to respond differently to various types of liver injury. However, our findings from Parkin KO mice likely better reflect physiological conditions in people that have chronic loss of Parkin function, such as Autosomal-Recessive Parkinson’s disease patients, who have mutations in the Park2 (Parkin) gene. Finally, our results indicate that evaluation of drug-induced liver injury mechanisms using whole-body knockout mice should be interpreted with caution due to adaptive and compensatory mechanisms that may be activated in knockout mice. In addition, modulating Parkin-mediated mitophagy may be a promising therapeutic approach for targeting drug and alcohol-induced liver injuries

Examining public knowledge and preferences for adult preventive services coverage

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Introduction: To examine (1) what individuals know about the existing adult preventive service coverage provisions of the Affordable Care Act (ACA), and (2) which preventive services individuals think should be covered without cost sharing. Methods: An online panel from Survey Monkey was used to obtain a sample of 2,990 adults age 18 and older in March 2015, analyzed 2015–2017. A 17-item survey instrument was designed and used to evaluate respondents’ knowledge of the adult preventive services provision of the ACA. Additionally, we asked whether various preventive services should be covered. The data include age, sex, race/ethnicity, and educational attainment as well as measures of political ideology, previous insurance status, the number of chronic conditions, and usual source of care. Results: Respondents correctly answered 38.6% of the questions about existing coverage under the ACA, while on average respondents thought 12.1 of 15 preventive services should be covered (SD 3.5). Respondents were more knowledgeable about coverage for routine screenings, such as blood pressure (63.4% correct) than potentially stigmatizing screenings, such as for alcohol misuse (28.8% correct). Blood pressure screening received the highest support of coverage (89.8%) while coverage of gym memberships received the lowest support (59.4%). Individuals with conservative ideologies thought fewer services on average should be covered, but the difference was small—around one service less than those with liberal ideologies. Conclusions: Overwhelmingly, individuals think that most preventive services should be covered without cost sharing. Despite several years of coverage for preventive services, there is still confusion and lack of knowledge about which services are covered

Development of a Novel Real-Time Polymerase Chain Reaction Assay for the Sensitive Detection of Schistosoma Japonicum in Human Stool

Background Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasiscausing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. Methodology/Principal findings Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum. Conclusions/Significance The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts

Trained immunity or tolerance : opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors

Article Accepted Date: 29 January 2014. ACKNOWLEDGMENTS D.C.I. received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement HEALTH-2010-260338 (“Fungi in the setting of inflammation, allergy and autoimmune diseases: translating basic science into clinical practices” [ALLFUN]) (awarded to M.G.N.). M.G.N. and J.Q. were supported by a Vici grant of the Netherlands Organization of Scientific Research (awarded to M.G.N.). This work was supported, in part, by National Institutes of Health grant GM53522 to D.L.W. N.A.R.G. was supported by the Wellcome Trust.Peer reviewedPublisher PD

Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformaticsbased analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent

Why Wait?: Early Enteral Feeding After Pediatric Gastrostomy Tube Placement

Purpose Early initiation of feedings after gastrostomy tube (GT) placement may reduce associated hospital costs, but many surgeons fear complications could result from earlier feeds. We hypothesized that, irrespective of placement method, starting feedings within the first 6 h following GT placement would not result in a greater number of post-operative complications. Methods An IRB-approved retrospective review of all GTs placed between January 2012 and December 2014 at three academic institutions was undertaken. Data was stratified by placement method and whether the patient was initiated on feeds at less than 6 h or after. Baseline demographics, operative variables, post-operative management and complications were analyzed. Descriptive statistics were used and P-values < 0.05 were considered significant. Results One thousand and forty-eight patients met inclusion criteria. GTs were inserted endoscopically (48.9%), laparoscopically (44.9%), or via an open approach (6.2%). Demographics were similar in early and late fed groups. When controlling for method of placement, those patients who were fed within the first 6 h after gastrostomy placement had shorter lengths of stay compared to those fed greater than 6 h after placement (P < 0.05). Total post-operative outcomes were equivalent between feeding groups for all methods of placement (laparoscopic (P = 0.87), PEG (P = 0.94), open (P = 0.81)). Conclusions Early initiation of feedings following GT placement was not associated with an increase in complications. Feeds initiated earlier may shorten hospital stays and decrease overall hospital costs

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

The soil transmitted helminths are a group of parasitic worms responsible for extensive mor- bidity in many of the world’s most economically depressed locations. With growing empha- sis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing- based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1\u2013dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1\u2013mediated organelle transport

Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair

SummaryThe Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs