Measurement of mRNA by quantitative PCR with a nonradioactive label

This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity

“Satan’s Minions” and “True Believers”: How Criminal Defense Attorneys Employ Quasi-Religious Rhetoric and What It Suggests about Lawyering Culture

The notion of law as sacred, and lawyers as righteous saviors, may seem anachronistic in the current context of heavy caseloads and expedited processing in the criminal justice system. Nevertheless, language reflecting these ideals still permeates defense attorneys’ descriptions of their roles, their legal practice, and their relationships to their colleagues and adversaries. We examine this language – specifically, attorneys’ quasi-religious rhetoric – to better understand courtroom dynamics: how attorneys see themselves, their work, their colleagues, and their legal adversaries. In this analysis of semi-structured interviews with 30 defense attorneys, we find that attorneys use of quasi-religious rhetoric manifests as a cultural practice that helps to establish and maintain professional identities, boundaries, and relationships. Our findings also suggest that young and novice public defenders are likely to express especially zealous views, which may compromise their efforts to collaborate within the adversarial system, as well as contribute to burnout

Corrigendum: Causes and Consequences of Innate Immune Dysfunction in Cirrhosis

In the original article, we neglected to include the funder "Equity Trustees." The funding statement has therefore been revised as follows: "KI, IR, and DH are grateful for research funding support from the Mater Research Foundation and Equity Trustees, Australia." The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

US GLOBEC: Program Goals, Approaches, and Advances

This special issue summarizes the major achievements of the US Global Ocean Ecosystem Dynamics (GLOBEC) program and celebrates its accomplishments. The articles grew out of a final symposium held in October 2009 under the auspices of the National Academy of Sciences Ocean Studies Board (http://usglobec.org/Symposium). This special issue updates the US GLOBEC "mid-life" Oceanography issue (Vol. 15, No. 2, 2002, http://tos.org/oceanography/archive/15-2.html), which put forward many of the goals and activities of the program, but was published while field work was still being conducted and results had yet to be synthesized across regional programs. The present special issue highlights the advances in understanding achieved through the synthesis of regional studies and pan-regional comparisons

Altered peripheral blood monocyte phenotype and function in chronic liver disease:Implications for hepatic recruitment and systemic inflammation

Background and Aim

Improvement in Chronic Hepatocerebral Degeneration Following Liver Transplantation

Chronic progressive hepatocerebral degeneration with spastic paraparesis, dementia, dysarthria, ataxia, tremor, and neuropsychiatric symptoms follows long-standing portal-systemic shunting, is associated with structural changes in the central nervous system, and does not respond to conventional therapy for hepatic encephalopathy. A case of advanced chronic liver disease with severe, progressive hepatocerebral degeneration after 23 yr of portal-systemic shunting is reported in whom there was significant objective improvement in intellectual function and in the chronic neurological signs 3 mo after orthotopic liver transplantation and further improvement 12 mo after transplantation

Establishing a basis for ecosystem management in the western Indian Ocean

An ambitious multinational programme, with generous funding for an initial five years, aims to provide understanding of marine resources for the benefit of impoverished island and coastal populations in a much-neglected ocean region

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

Background: Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. Methods: and results We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNACys was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Conclusions: Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy

The toll-like receptor 3 pathway in homeostasis, responses to injury and wound repair

In addition to their established roles in host defence, Toll-like Receptors (TLRs) have emerging roles in control of homeostasis, injury and wound repair. The dsRNA-sensing receptor, TLR3, has been particularly implicated in such processes in several different tissues including the skin, intestine and liver, as well as in the control of reparative mechanisms in the brain, heart and kidneys, following ischemia reperfusion injury. In this review, we provide an overview of TLR3 signalling and functions in inflammation, tissue damage and repair processes, as well as therapeutic opportunities that may arise in the future from knowledge of such pathways

CRIg-expressing peritoneal macrophages are associated with disease severity in patients with cirrhosis and ascites

Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIg(hi) macrophages differed between patients and in the same patient over time, and a high proportion of CRIg(hi) macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIg(lo) macrophages, CRIg(hi) macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIg(hi) cells, human macrophages, and mouse F4/80(hi) resident peritoneal macrophages and among CRIg(lo) macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIg(hi) and CRIg(lo) macrophages may represent a tissue-resident population and a monocytederived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients