HIV infection, nucleoside analogue therapy and somatic mitochondrial DNA mutation :implications for ageing?

PhD ThesisIt has been hypothesised that patients with long-term treated HIV infection may exhibit features of accelerated physiological ageing. Given previously established links between, a) anti-retroviral therapy and mitochondrial DNA (mtDNA), and b) mtDNA and ageing, I hypothesised that anti-retroviral therapy may lead to the accumulation of mtDNA mutations, in an acceleration of the molecular process seen in normal human ageing. Using a combination of single cell molecular analyses and ultra-deep sequencing (UDS), I demonstrated that HIV-infected patients with prior exposure to polymerase (pol) γ inhibiting NRTI (nucleoside analogue reverse transcriptase inhibitor) therapy show increased accumulation of somatic mtDNA mutations within cells, in a pattern similar to that seen much later in life due to normal ageing. Empirical data and in silico modelling suggested this is likely to be mediated by the accelerated clonal expansion of pre-existing (age-associated) mtDNA mutations, rather than by increased mutagenesis. I went on to further develop the UDS methodology, to explore more fundamental questions about the characteristics of mtDNA mutations in ageing, health and disease. In so doing, I showed that low-level mtDNA heteroplasmic mutation appears to be universal, and that many ostensibly somatic mutations may in fact have been maternally transmitted at very low levels. I explored the utility of serum FGF-21 (fibroblast growth factor 21) and phosphorus magnetic resonance spectroscopy (31P-MRS) as non-invasive measures of muscle mitochondrial dysfunction in anti-retroviral treated patients. Both showed significant abnormalities, although neither proved sensitive or specific in my patient group. Finally I explored the frequency and severity of fatigue in contemporary HIV-infected patients, showing that half of all patients have excessive fatigue despite good immune function and suppressed HIV viraemia. Patients with prior exposure to pol γ inhibiting NRTIs were almost universally fatigued, suggesting that persistent mitochondrial dysfunction due to accumulated mtDNA mutations may be important in driving fatigue in this patient group.Medical Research Council (MRC) UK (Clinical Research Training Fellowship), British Infection Association (BIA) (Clinical Research Fellowship), National Institute for Health Research (NIHR) Biomedical Research Centre for Ageing and Age-Related Disease (awarded to Newcastle-upon-Tyne Hospitals NHS Foundation Trust and Newcastle University), Newcastle Healthcare Charity

Skeletal muscle mitochondrial dysfunction in contemporary antiretroviral therapy: a single cell analysis

OBJECTIVE: To quantify mitochondrial function in skeletal muscle of people treated with contemporary antiretroviral therapy. DESIGN: Cross-sectional observational study. METHODS: Quantitative multiplex immunofluorescence was performed to determine mitochondrial mass and respiratory chain complex abundance in individual myofibres from tibialis anterior biopsies. Individual myofibres were captured by laser microdissection and mitochondrial DNA (mtDNA) content and large-scale deletions were measured by real-time PCR. RESULTS: Forty five antiretroviral therapy (ART) treated people with HIV (PWH, mean age 58 years, mean duration of ART 125 months) were compared with 15 HIV negative age-matched controls. Mitochondrial complex I (CI) deficiency was observed at higher proportional levels in PWH than negative controls (P = 0.008). Myofibre mitochondrial mass did not differ by HIV status.No ART class was significantly associated with mitochondrial deficiency, including prior exposure to historical NRTIs (nucleoside analogue reverse transcriptase inhibitors) associated with systemic mitochondrial toxicity.To exclude an effect of untreated HIV, we also studied skeletal muscle from 13 ART-naïve PWH (mean age 37). These showed negligible CI defects, as well as comparable myofibre mitochondrial mass to ART-treated PWH.Most CI-deficient myofibres contained mtDNA deletions. No mtDNA depletion was detected. CONCLUSION: Here, we show that PWH treated with contemporary ART have mitochondrial dysfunction in skeletal muscle, exceeding that expected due to age alone. Surprisingly, this was not mediated by prior exposure to mitochondrially toxic NRTIs, suggesting novel mechanisms of mitochondrial dysfunction in contemporary ART-treated PWH. These findings are relevant for better understanding successful ageing in PWH

Age-related mitochondrial DNA depletion and the impact on pancreatic beta cell function

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes

Use of stereotypical mutational motifs to define resolution limits for the ultra-deep resequencing of mitochondrial DNA.

Massively parallel resequencing of mitochondrial DNA (mtDNA) has led to significant advances in the study of heteroplasmic mtDNA variants in health and disease, but confident resolution of very low-level variants ( C, from patient with MNGIE, mitochondrial neurogastrointestinal encephalomyopathy) and comparing mutational pattern distribution with healthy mtDNA by ligation-mediated deep resequencing (Applied Biosystems SOLiD). We empirically derived mtDNA-mutant heteroplasmy detection limits, demonstrating that the presence of stereotypical mutational motif could be statistically validated for heteroplasmy thresholds ≥ 0.22% (P = 0.034). We therefore provide empirical evidence from biological samples that very low-level mtDNA mutants can be meaningfully resolved by massively parallel resequencing, confirming the utility of the approach for studying somatic mtDNA mutation in health and disease. Our approach could also usefully be employed in other settings to derive platform-specific deep resequencing resolution limits

Randomized phase II study of stereotactic body radiotherapy and interleukin-2 versus interleukin-2 in patients with metastatic melanoma.

BACKGROUND: A pilot study of stereotactic body radiation therapy (SBRT) followed by high-dose interleukin-2 (IL-2) showed a higher than anticipated objective response rate (ORR) among patients with metastatic melanoma (MM). We performed a prospective randomized study to determine if the ORR of SBRT + IL-2 was greater than IL-2 monotherapy in patients with advanced melanoma. METHODS: Patients with MM who had adequate physiological reserve for IL-2 and at least one site suitable for SBRT were eligible. There was a 1:1 randomization to SBRT + IL-2 or IL-2 monotherapy. Patients received one or two doses of SBRT (20 Gy per fraction) with the last dose administered 3 days before starting the first cycle of IL-2. IL-2 (600,000 IU per kg via intravenous bolus infusion) was given every 8 hours for a maximum of 14 doses with a second cycle after a 2-week rest. Responding patients received up to six IL-2 cycles. Patients assigned to IL-2 monotherapy who exhibited progression of melanoma after cycle 2 were allowed to crossover and receive SBRT and additional IL-2. Response Evaluation Criteria in Solid Tumors 1.1 criteria were applied to non-irradiated lesions for response assessment. RESULTS: 44 patients were included in the analysis. The ORR in the SBRT + IL-2 group was 54%: 21% complete response (CR), 33% partial response (PR), 21% stable disease (SD) and 25% progressive disease (PD). The ORR in patients receiving IL-2 monotherapy was 35%: 15% CR, 20% PR, 25% SD and 40% PD. Seven patients assigned to IL-2 subsequently received SBRT + IL-2. One CR and two PRs were observed in the crossover group. There was no difference in progression-free or overall survival (OS). At 5 years the OS was 26% in the SBRT + IL-2 group and 25% in the IL-2 monotherapy group. The disease control rate (DCR) was higher in the SBRT + IL-2 group (75% vs 60%, p=0.34). CONCLUSIONS: SBRT + IL-2 induced more objective responses with a higher DCR compared to IL-2 monotherapy in MM. IL-2 monotherapy resulted in a significantly higher ORR than anticipated. Some patients in the crossover group also achieved objective responses. TRIAL REGISTRATION NUMBER: NCT01416831

Universal heteroplasmy of human mitochondrial DNA.

Mammalian cells contain thousands of copies of mitochondrial DNA (mtDNA). At birth, these are thought to be identical in most humans. Here, we use long read length ultra-deep resequencing-by-synthesis to interrogate regions of the mtDNA genome from related and unrelated individuals at unprecedented resolution. We show that very low-level heteroplasmic variance is present in all tested healthy individuals, and is likely to be due to both inherited and somatic single base substitutions. Using this approach, we demonstrate an increase in mtDNA mutations in the skeletal muscle of patients with a proofreading-deficient mtDNA polymerase γ due to POLG mutations. In contrast, we show that OPA1 mutations, which indirectly affect mtDNA maintenance, do not increase point mutation load. The demonstration of universal mtDNA heteroplasmy has fundamental implications for our understanding of mtDNA inheritance and evolution. Ostensibly de novo somatic mtDNA mutations, seen in mtDNA maintenance disorders and neurodegenerative disease and aging, will partly be due to the clonal expansion of low-level inherited variants

Retired A Stars and Their Companions VI. A Pair of Interacting Exoplanet Pairs Around the Subgiants 24 Sextanis and HD200964

We report radial velocity measurements of the G-type subgiants 24 Sextanis (=HD90043) and HD200964. Both are massive, evolved stars that exhibit periodic variations due to the presence of a pair of Jovian planets. Photometric monitoring with the T12 0.80m APT at Fairborn Observatory demonstrates both stars to be constant in brightness to <= 0.002 mag, thus strengthening the planetary interpretation of the radial velocity variations. 24 Sex b,c have orbital periods of 453.8 days and 883~days, corresponding to semimajor axes 1.333 AU and 2.08 AU, and minimum masses (Msini) 1.99 Mjup and 0.86 Mjup, assuming a stellar mass 1.54 Msun. HD200964 b,c have orbital periods of 613.8 days and 825 days, corresponding to semimajor axes 1.601 AU and 1.95 AU, and minimum masses 1.85 Mjup and 0.90 Mjup, assuming M* = 1.44 Msun. We also carry out dynamical simulations to properly account for gravitational interactions between the planets. Most, if not all, of the dynamically stable solutions include crossing orbits, suggesting that each system is locked in a mean motion resonance that prevents close encounters and provides long-term stability. The planets in the 24 Sex system likely have a period ratio near 2:1, while the HD200964 system is even more tightly packed with a period ratio close to 4:3. However, we caution that further radial velocity observations and more detailed dynamical modelling will be required to provide definitive and unique orbital solutions for both cases, and to determine whether the two systems are truly resonant.Comment: AJ accepte

Electronic and Structural Properties of a 4d-Perovskite: Cubic Phase of SrZrO3_3

First-principles density functional calculations are performed within the local density approximation to study the electronic properties of SrZrO$_3$, an insulating 4d-perovskite, in its high-temperature cubic phase, above 1400 K, as well as the generic 3d-perovskite SrTiO$_3$, which is also a d^0-insulator and cubic above 105 K, for comparison reasons. The energy bands, density of states and charge density distributions are obtained and a detailed comparison between their band structures is presented. The results are discussed also in terms of the existing data in the literature for both oxides.Comment: 5 pages, 2 figure

Absorption Coefficient (ABSCO) Tables for the Orbiting Carbon Observatories: Version 5.1

The accuracy of atmospheric trace gas retrievals depends directly on the accuracy of the molecular absorption model used within the retrieval algorithm. For remote sensing of well-mixed gases, such as carbon dioxide (CO₂), where the atmospheric variability is small compared to the background, the quality of the molecular absorption model is key. Recent updates to oxygen (O₂) absorption coefficients (ABSCO) for the 0.76 μm A-band and the water vapor (H₂O) continuum model within the 1.6 μm and 2.06 μm CO₂ bands used within the Orbiting Carbon Observatory (OCO-2 and OCO-3) algorithm are described here. Updates in the O₂ A-band involve the inclusion of new laboratory measurements within multispectrum fits to improve relative consistency between O₂ line shapes and collision-induced absorption (CIA). The H₂O continuum model has been updated to MTCKD v3.2, which has benefited from information from a range of laboratory studies relative to the model utilized in the previous ABSCO version. Impacts of these spectroscopy updates have been evaluated against ground-based atmospheric spectra from the Total Carbon Column Observing Network (TCCON) and within the framework of the OCO-2 algorithm, using OCO-2 soundings covering a range of atmospheric and surface conditions. The updated absorption coefficients (ABSCO version 5.1) are found to offer improved fitting residuals and reduced biases in retrieved surface pressure relative to the previous version (ABSCO v5.0) used within B8 and B9 of the OCO-2 retrieval algorithm and have been adopted for the OCO B10 Level 2 algorithm

Absorption Coefficient (ABSCO) Tables for the Orbiting Carbon Observatories: Version 5.1

The accuracy of atmospheric trace gas retrievals depends directly on the accuracy of the molecular absorption model used within the retrieval algorithm. For remote sensing of well-mixed gases, such as carbon dioxide (CO₂), where the atmospheric variability is small compared to the background, the quality of the molecular absorption model is key. Recent updates to oxygen (O₂) absorption coefficients (ABSCO) for the 0.76 μm A-band and the water vapor (H₂O) continuum model within the 1.6 μm and 2.06 μm CO₂ bands used within the Orbiting Carbon Observatory (OCO-2 and OCO-3) algorithm are described here. Updates in the O₂ A-band involve the inclusion of new laboratory measurements within multispectrum fits to improve relative consistency between O₂ line shapes and collision-induced absorption (CIA). The H₂O continuum model has been updated to MTCKD v3.2, which has benefited from information from a range of laboratory studies relative to the model utilized in the previous ABSCO version. Impacts of these spectroscopy updates have been evaluated against ground-based atmospheric spectra from the Total Carbon Column Observing Network (TCCON) and within the framework of the OCO-2 algorithm, using OCO-2 soundings covering a range of atmospheric and surface conditions. The updated absorption coefficients (ABSCO version 5.1) are found to offer improved fitting residuals and reduced biases in retrieved surface pressure relative to the previous version (ABSCO v5.0) used within B8 and B9 of the OCO-2 retrieval algorithm and have been adopted for the OCO B10 Level 2 algorithm