Potential use of oxygen as a metabolic biosensor in combination with T2*-weighted MRI to define the ischemic penumbra

We describe a novel magnetic resonance imaging technique for detecting metabolism indirectly through changes in oxyhemoglobin:deoxyhemoglobin ratios and T2* signal change during ‘oxygen challenge’ (OC, 5 mins 100% O2). During OC, T2* increase reflects O2 binding to deoxyhemoglobin, which is formed when metabolizing tissues take up oxygen. Here OC has been applied to identify tissue metabolism within the ischemic brain. Permanent middle cerebral artery occlusion was induced in rats. In series 1 scanning (n=5), diffusion-weighted imaging (DWI) was performed, followed by echo-planar T2* acquired during OC and perfusion-weighted imaging (PWI, arterial spin labeling). Oxygen challenge induced a T2* signal increase of 1.8%, 3.7%, and 0.24% in the contralateral cortex, ipsilateral cortex within the PWI/DWI mismatch zone, and ischemic core, respectively. T2* and apparent diffusion coefficient (ADC) map coregistration revealed that the T2* signal increase extended into the ADC lesion (3.4%). In series 2 (n=5), FLASH T2* and ADC maps coregistered with histology revealed a T2* signal increase of 4.9% in the histologically defined border zone (55% normal neuronal morphology, located within the ADC lesion boundary) compared with a 0.7% increase in the cortical ischemic core (92% neuronal ischemic cell change, core ADC lesion). Oxygen challenge has potential clinical utility and, by distinguishing metabolically active and inactive tissues within hypoperfused regions, could provide a more precise assessment of penumbra

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error

Re A (A Child) and the United Kingdom Code of Practice for the Diagnosis and Confirmation of Death: Should a Secular Construct of Death Override Religious Values in a Pluralistic Society?

The determination of death by neurological criteria remains controversial scientifically, culturally, and legally, worldwide. In the United Kingdom, although the determination of death by neurological criteria is not legally codified, the Code of Practice of the Academy of Medical Royal Colleges is customarily used for neurological (brainstem) death determination and treatment withdrawal. Unlike some states in the US, however, there are no provisions under the law requiring accommodation of and respect for residents’ religious rights and commitments when secular conceptions of death based on medical codes and practices conflict with a traditional concept well-grounded in religious and cultural values and practices. In this article, we analyse the medical, ethical, and legal issues that were generated by the recent judgement of the High Court of England and Wales in Re: A (A Child) [2015] EWHC 443 (Fam). Mechanical ventilation was withdrawn in this case despite parental religious objection to a determination of death based on the code of practice. We outline contemporary evidence that has refuted the reliability of tests of brainstem function to ascertain the two conjunctive clinical criteria for the determination of death that are stipulated in the code of practice: irreversible loss of capacity for consciousness and somatic integration of bodily biological functions

Extrinsic primary afferent signalling in the gut

Visceral sensory neurons activate reflex pathways that control gut function and also give rise to important sensations, such as fullness, bloating, nausea, discomfort, urgency and pain. Sensory neurons are organised into three distinct anatomical pathways to the central nervous system (vagal, thoracolumbar and lumbosacral). Although remarkable progress has been made in characterizing the roles of many ion channels, receptors and second messengers in visceral sensory neurons, the basic aim of understanding how many classes there are, and how they differ, has proven difficult to achieve. We suggest that just five structurally distinct types of sensory endings are present in the gut wall that account for essentially all of the primary afferent neurons in the three pathways. Each of these five major structural types of endings seems to show distinctive combinations of physiological responses. These types are: 'intraganglionic laminar' endings in myenteric ganglia; 'mucosal' endings located in the subepithelial layer; 'muscular–mucosal' afferents, with mechanosensitive endings close to the muscularis mucosae; 'intramuscular' endings, with endings within the smooth muscle layers; and 'vascular' afferents, with sensitive endings primarily on blood vessels. 'Silent' afferents might be a subset of inexcitable 'vascular' afferents, which can be switched on by inflammatory mediators. Extrinsic sensory neurons comprise an attractive focus for targeted therapeutic intervention in a range of gastrointestinal disorders.Australian National Health and Medical Research Counci

RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells

Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity

Impact of Ocean Warming and Ocean Acidification on Larval Development and Calcification in the Sea Urchin Tripneustes gratilla

Background: As the oceans simultaneously warm, acidify and increase in P-CO2, prospects for marine biota are of concern. Calcifying species may find it difficult to produce their skeleton because ocean acidification decreases calcium carbonate saturation and accompanying hypercapnia suppresses metabolism. However, this may be buffered by enhanced growth and metabolism due to warming.Methodology/Principal Findings: We examined the interactive effects of near-future ocean warming and increased acidification/P-CO2 on larval development in the tropical sea urchin Tripneustes gratilla. Larvae were reared in multifactorial experiments in flow-through conditions in all combinations of three temperature and three pH/P-CO2 treatments. Experiments were placed in the setting of projected near future conditions for SE Australia, a global change hot spot. Increased acidity/P-CO2 and decreased carbonate mineral saturation significantly reduced larval growth resulting in decreased skeletal length. Increased temperature (+3 degrees C) stimulated growth, producing significantly bigger larvae across all pH/P-CO2 treatments up to a thermal threshold (+6 degrees C). Increased acidity (-0.3-0.5 pH units) and hypercapnia significantly reduced larval calcification. A +3 degrees C warming diminished the negative effects of acidification and hypercapnia on larval growth.Conclusions and Significance: This study of the effects of ocean warming and CO2 driven acidification on development and calcification of marine invertebrate larvae reared in experimental conditions from the outset of development (fertilization) shows the positive and negative effects of these stressors. In simultaneous exposure to stressors the dwarfing effects of acidification were dominant. Reduction in size of sea urchin larvae in a high P-CO2 ocean would likely impair their performance with negative consequent effects for benthic adult populations

Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis.MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression.RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients.INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.</p

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis

Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus

Polymorphous adenocarcinoma of the salivary glands : reappraisal and update

Although relatively rare, polymorphous adenocarcinoma (PAC) is likely the second most common malignancy of the minor salivary glands (MiSG). The diagnosis is mainly based on an incisional biopsy. The optimal treatment comprises wide surgical excision, often with adjuvant radiotherapy. In general, PAC has a good prognosis. Previously, PAC was referred to as polymorphous low-grade adenocarcinoma (PLGA), but the new WHO classification of salivary gland tumours has also included under the PAC subheading, the so-called cribriform adenocarcinoma of minor salivary glands (CAMSG). This approach raised controversy, predominantly because of possible differences in clinical behaviour. For example, PLGA (PAC, classical variant) only rarely metastasizes, whereas CAMSG often shows metastases to the neck lymph nodes. Given the controversy, this review reappraises the definition, epidemiology, clinical presentation, diagnostic work-up, genetics, treatment modalities, and prognosis of PAC of the salivary glands with a particular focus on contrasting differences with CAMSG.Peer reviewe