Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria

BACKGROUND: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. METHODOLOGY: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. SIGNIFICANCE: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria

Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion

Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS

Case conferences in palliative care: A substudy of a cluster randomised controlled trial

Background In palliative care, case conferences have demonstrated improved maintenance of function and a significant reduction in hospitalisations. This study aimed to define the content and themes of palliative care case conferences. Methods This was a substudy of a cluster randomised controlled trial. Case conferences meeting the requirements for Medicare Benefits Schedule reimbursement were organised by the research officer in conjunction with the general practitioner and the participating palliative care service. All were audiotaped, coded and analysed for content and themes, using qualitative methods and interaction analysis. Results Seventeen case conferences were transcribed and coded. Physical issues were the dominant topic. Management of psychosocial concerns were rarely discussed. Lack of information was a common theme and time was spent during each conference ensuring all people were familiar with the issues and patient history. Healthcare professionals tended to respond to the content of patient concerns, but not the emotion. Discussion The discussions were complex and health professional participants rarely summarised information or checked that patients and carers had understood the information provided

Assessment of the psychometric properties of an english version of the cancer dyspnea scale in people with advanced lung cancer

Context: Dyspnea is a poorly understood subjective sensation. Existing dyspnea measures fail to adequately address its multidimensionality. A Japanese group developed and validated the Cancer Dyspnea Scale (CDS) for assessing dyspnea in patients with advanced lung cancer. Objectives: We evaluated the validity and reliability of the English version of the CDS (CDS-E) that has 12 items and takes, on average, 140 seconds for individuals to complete. Methods: Eligible patients had advanced lung cancer, consented, and were fluent in English. Participants completed a 100 mm visual analogue scale (VAS), the modified Borg scale, the CDS-E, the Hospital Anxiety and Depression Scale, and the Functional Assessment of Cancer Therapy - Lung quality-of-life scale. Demographic, radiographic, and treatment information were obtained from patients' medical records. Results: One hundred twelve participants were enrolled at three sites in the U.S., Australia, and the U.K. Mean age was 64.5 years (SD 11.5); 90% were Caucasian, 68% had Eastern Cooperative Oncology Group performance status 0-1, and 50% had non-small cell carcinoma. All completed the CDS-E independently, without difficulty. The CDS-E had reasonable internal consistency overall (Cronbach's α = 0.71) and for each of the three factors (effort, anxiety, discomfort Cronbach's α = 0.80-0.84). CDS-E scores were significantly correlated with the 100 mm VAS (r = 0.82; P < 0.001) and the modified Borg (r = 0.87; P < 0.001). After factor analysis, the CDS-E was revised by removing three items (r-CDS-E). Conclusion: The CDS-E and r-CDS-E are reliable and valid measures of the sensation and the psychological components of dyspnea, with the shorter version having similar psychometric properties. © 2012 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved

Off-label prescribing in palliative care - A cross-sectional national survey of Palliative Medicine doctors

Background: Regulatory bodies including the European Medicines Agency register medications (formulation, route of administration) for specific clinical indications. Once registered, prescription is at clinicians' discretion. Off-label use is beyond the registered use. While off-label prescribing may, at times, be appropriate, efficacy and toxicity data are often lacking. Aim: The aim of this study was to document off-label use policies (including disclosure and consent) in Australian palliative care units and current practices by palliative care clinicians. Design: A national, cross-sectional survey was conducted online following an invitation letter. The survey asked clinicians their most frequent off-label medication/indication dyads and unit policies. Dyads were classified into unregistered, off-label and on-label, and for the latter, whether medications were nationally subsidised. Setting/participants: All Australian palliative medicine Fellows and advanced trainees. Results: Overall, 105 clinicians responded (53% response rate). The majority did not have policies on off-label medications, and documented consent rarely. In all, 236 medication/indication dyads for 36 medications were noted: 45 dyads (19%) were for two unregistered medications, 118 dyads (50%) were for 26 off-label medications and 73 dyads (31%) were for 12 on-label medications. Conclusions: Off-label prescribing with its clinical, legal and ethical implications is common yet poorly recognised by clinicians. A distinction needs to be made between where quality evidence exists but registration has not been updated by the pharmaceutical sponsor and the evidence has not been generated. Further research is required to quantify any iatrogenic harm from off-label prescribing in palliative care. © The Author(s) 2012

Planning phase III multi-site clinical trials in palliative care: The role of consecutive cohort audits to identify potential participant populations

Multiple sites enable more successful completion of adequately powered phase III studies in palliative care. Audits of the frequency and distribution of the symptoms of interest can better inform research planning by determining realistic recruitment goals for each site. The proposed studies are to improve the evidence-base for registration and subsidy applications for frequently encountered symptoms where current pharmacological interventions are being used 'off-licence'