Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition characterized by degeneration of upper motor neurons (UMN) arising from the motor cortex in the brain and lower motor neurons (LMN) in the brainstem and spinal cord. Cerebral changes create differences in brain activity captured by functional magnetic resonance imaging (fMRI), including the spontaneous and simultaneous activity occurring between regions known as the resting state networks (RSNs). Progressive neurodegeneration as observed in ALS may lead to a disruption of RSNs which could provide insights into the disease process. Previous studies have reported conflicting findings of increased, decreased, or unaltered RSN functional connectivity in ALS and do not report the contribution of UMN changes to RSN connectivity. We aimed to bridge this gap by exploring two networks, the default mode network (DMN) and the sensorimotor network (SMN), in 21 ALS patients and 40 age-matched healthy volunteers. An UMN score dichotomized patients into UMN+ and UMN- groups. Subjects underwent resting state fMRI scan on a high field MRI operating at 4.7 tesla. The DMN and SMN changes between subject groups were compared. Correlations between connectivity and clinical measures such as the ALS Functional Rating Scale—Revised (ALSFRS-R), disease progression rate, symptom duration, UMN score and finger tapping were assessed. Significant group differences in resting state networks between patients and controls were absent, as was the dependence on degree of UMN burden. However, DMN connectivity was increased in patients with greater disability and faster progression rate, and SMN connectivity was reduced in those with greater motor impairment. These patterns of association are in line with literature supporting loss of inhibitory interneurons. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease with death occurring within 2 to 5 years . It has These aforementioned motor and cognitive changes in ALS may be reflected as changes in baseline neuronal activity in the brain. In the past decade, there has been a surge of studies investigating the neurodegenerative changes in ALS using magnetic resonance imaging [MRI]. Resting state functional MRI (rs-fMRI) techniques have enabled non-invasive investigation of regional brain interactions. It captures low frequency (0.01–0.1 Hz) blood oxygen level dependent (BOLD) signals that reflect spontaneous neuronal activity in cortical and sub-cortical regions at rest, when the brain is not particularly occupied with a goal-oriented task. These spontaneous activities in anatomically separated regions form resting state networks (RSNs) that reflect a level of on-going functional activity between brain regions, and serve as the foundation for higher order motor and cognitive functions , 5.Studies report that RSNs are consistent across healthy participants and are highly reproducible –9. In coThe aforementioned networks were initially reported in healthy subjects in 1995 . The defThe SMN includes somatosensory  and motor  regions and extends to the supplementary motor areas. Studies have indicated that this network is activated during motor tasks such as finger tapping  indicatiWhile lower connectivity has been associated with lower grey matter volumes , 17, repAdditionally, there is limited literature on the role of UMN disease burden and functional changes in ALS. Studies report that structural measures for white matter (WM) integrity such as fractional anisotropy (FA) and mean diffusivity (MD) may predict UMN disease burden in ALS patients . To our We hypothesized that (1) there will be impaired connectivity in both the DMN and SMN in ALS patients as compared to controls; (2) patients with higher UMN disease burden will have more impairments in connectivity as compared to patients with lower UMN disease burden; and (3) impaired RSN connectivity in patients will be associated with higher disease severity, longer symptom duration, increased disease progression rates and higher UMN disease burden.We recruited 21 ALS patients  from the multidisciplinary ALS clinic at the University of Alberta. All patients met research criteria for possible, probable, or definite ALS  and thusDisease severity was inferred from the ALSFRS-R , a quest2 was obtained. Thirty-six axial slices aligned to the AC-PC axis with slice thickness = 3 mm and voxel size = 3 × 3 × 3 mm were obtained. A total of 192 time points were acquired for each subject. Subjects were instructed to remain awake with their eyes closed without thinking about anything in particular during the scan. Anatomical scans were acquired with a high-resolution 3D magnetization prepared rapid gradient echo (MPRAGE) sequence with TR/TE/TI = 8.5/4.5ms/300ms, voxel size = 1 × 1 × 1 mm. matrix size = 256 x 200 points. An MPRAGE sequence with TR/TE/TI = 10.35/4 ms/300ms, voxel size = 1 x 1 x 1mm and matrix size = 256 × 200 points was acquired instead of the former sequence on a proportion of the participants. Both were interpolated to 0.5 × 0.5 × 1mm, matrix size 512 × 400 points.Resting states fMRI (rs-fMRI) was acquired on a Varian Inova 4.7T scanner at the Peter S. Allen MR Research Centre. A single shot T2* weighted echo planar imaging sequence with TR/TE = 2500/19 ms, flip angle = 75°, matrix size = 67 × 80 points, field of view (FOV) = 240 mmIndependent samples t-test was used to compute statistically significant differences in age, education and finger tapping scores between patients and controls. Pearson chi-square was computed to check for differences in distribution of gender between the two groups.www.fil.ion.ucl.ac.uk/spm) on MATLAB R2014a .The images were preprocessed and analyzed using Statistical Parametric Mapping version 8.0 . The posterior cingulate cortex (PCC) was selected as a seed for the DMN [Seeds for the DMN and the SMN were built using MarsBar on a standard MNI template   and hypothesis 2 (UMN+ vs UMN-). Dichotomization of patients into UMN+ and UMN- groups was determined by their median scores on the adapted UMN Scale .Multiple regression models were used to address hypothesis 3 .Age was used as a covariate in all models for group analysis and clinical correlations to control for the effects of age-related brain changes. A primary threshold of p<0.001 was set for all models. Monte Carlo simulations were used to compute cluster size threshold (90 voxels) to correct for multiple comparisons at p<0.05.df = 57, p = 0.6). Patients had significantly lower years of education as compared to controls  and the median UMN Scale score of patients was 9.5. Pearson chi-square indicated a trend towards significance  in the distribution of gender between the two groups; controls had more females as compared to patients. Patients scored significantly lower than controls in the finger tapping test for both the left  and the right  sides.There were no significant differences in age between patients and controls , anterior region of the left superior frontal gyrus (T = 5.30) and posterior region of the right middle temporal gyrus (T = 4.59). Higher SMN connectivity was associated with higher left finger tapping scores in the anterior regions of the left superior frontal gyrus  was associated with longer disease duration . ReHo isWe also report that connectivity between the left superior frontal gyrus and the SMN seed increases with higher left finger tapping. Previous studies using task-based fMRI to study finger tapping, report an increase in ipsilateral activation of premotor areas in ALS patients as compared to controls . AdditioTo our best knowledge, this is the first report exploring UMN burden and functional connectivity in ALS. The novel finding is that functional connectivity is not dependent on UMN burden as analyzed by sub-grouping patients into UMN+ and UMN- groups. The UMN pathway is classically described as motor neurons originating in the motor regions of the cerebral cortex that project to the brain stem and the lower motor neurons. The RSN activity may not be affected with the degeneration of these UMNs which do not have intracerebral connections as would association and commissural fibres. On the other hand, other MRI measures such DTI may be more sensitive to this loss as they primarily reflect the structural integrity of white matter tracts irrespective of their connections.The presence of associations between clinical parameters and RSN connectivity supports aspects of pathology reported in previous literature as well as highlights the possibility of individual differences in disease course. While it is promising to see the association of RSN connectivity with clinical parameters, whether this association implies a functional reorganization that predicts disease-specific parameters would require further investigation. It would be interesting to longitudinally explore whether such changes are indeed re-organization and if the changes can further predict prognosis or survival in ALS patients.Our study employed seed based correlation as opposed to the ICA approach reported by previous studies in ALS. This provided an advantage of studying voxel-wise correlations of the brain and the seed region for each network of interest . We recrNevertheless, our study is not without limitations. We do not report structural changes that may be associated with functional connectivity. Additional multi-modal analysis would provide further insight into the results obtained and may prove to be more comprehensive in understanding functional changes in ALS. Participants in our study were trending towards significance in the distribution of gender between the two groups. While gender differences in RSN connectivity has been reported to be weak by previous studies  includinWe report significant associations between RSN connectivity and clinical variables for the DMN and the SMN. DMN connectivity was increased in those with greater disability and those with a faster progression rate, and SMN connectivity was reduced with greater clinical motor impairment. It appears that although RSN alterations in ALS may not reach statistical significance in comparison with controls, the association of increased or decreased connectivity with clinical parameters suggests underlying functional changes in the cortex that may reflect loss of inhibitory neurons. It would be of interest to employ multi-modal imaging analysis on a larger sample of participants and include additional RSNs such as the executive control network in the future for a comprehensive understanding of functional changes in ALS.S1 FigImages display the seeds used for the (A) Default mode network (DMN) and (B) Sensorimotor network (SMN). Seeds are represented on the Montreal Neurological Institute averaged brain.(PDF)Click here for additional data file.


This paper corroborates three different hybrid modulation strategies suitable for single-phase voltage source inverter. The proposed method is formulated using fundamental switching and carrier based pulse width modulation methods. The main tale of this proposed method is to optimize a specific performance criterion, such as minimization of the total harmonic distortion (THD), lower order harmonics, switching losses, and heat losses. The proposed method is articulated using fundamental switching and carrier based pulse width modulation methods. Thus, the harmonic pollution in the power system will be reduced and the power quality will be augmented with better harmonic profile for a target fundamental output voltage. The proposed modulation strategies are simulated in MATLAB r2010a and implemented in a Xilinx spartan 3E-500 FG 320 FPGA processor. The feasibility of these modulation strategies is authenticated through simulation and experimental results. Pulse width modulation (PWM) strategies are dedicated for power converters to shape the output profile in a way beneficial to the industrial requirements. There have been several control strategies developed to enhance the fundamental component and minimizing the total harmonics distortion (THD) through elimination of lower order harmonics and to create a wider dead band for a given switching frequency –3. SeverThis paper corroborates three different hybrid modulation methods suitable for single-phase voltage source inverter derived using concatenation of fundamental switching and carrier based pulse width modulation (PWM) methods like unipolar PWM, inverted sine carrier PWM, and sixty-degree PWM, respectively. Consequently, with better harmonic profile for a target fundamental output voltage, the harmonic pollution in the power system will be moderated and the power quality will be improved.Vd or from 0 to +Vd. This scheme “effectively” has the effect of doubling the switching frequency as far as the output harmonics are concerned, compared to the bipolar-switching scheme.The PWM schemes developed for single-phase inverters shown in Vd and +Vd; the scheme is called the bipolar PWM. In this scheme the diagonally opposite transistors are turned on or turned off at the same time. For a full-bridge inverter with bipolar PWM scheme the output voltage is between −Vd/2 and Vd/2.Similar to unipolar PWM, the voltage switches between two levels −The inverted sine carrier PWM (ISCPWM) control scheme for single-phase full-bridge inverter eliminates poor output voltage spectral quality and reduced fundamental output voltage. It enhances the fundamental output voltage particularly at lower modulation index ranges while keeping the total harmonic distortion (THD) lower without involving changes in device switching losses.The 60-degree PWM is similar to the modified pwm. The idea behind 60-degree PWM is to “flat top” the waveform from 60 degrees to 120 degrees and 240 degrees to 300 degrees. The power devices are held on for one-third of the cycle (when at full voltage) and have reduced switching losses. The 60-degree PWM creates a large fundamendal (2/3) and utilizes more of available dc voltage than does sinusoidal PWM. The output waveform can be approximated by the fundamental and the first few terms.The main philosophy of the PWM modulation emphasizes providing desired fundamental magnitude and reduced THD by pushing the lower order harmonics to higher carrier band thereby minimizing the filter design. In this direction, a new PWM strategy is developed to offer lesser switching loss with improved harmonic profile while offering desired fundamental output voltage. The proposed hybrid pulse width modulation (HPWM) strategies shown in The hybrid switching controller (HSC) basically consists of logic circuits generating gating signal for each device with two different frequencies, quarter cycle being switched at FFS, while the other quarter cycle is modulated at CBPWM. A random switching scheme is embedded with this hybrid modulation in order to swap both FFS and CBPWM for every quarter cycle of the output waveform. A simple base PWM circulation scheme is also introduced here to get resultant hybrid PWM circulation that inherits the characteristics of CBPWM methods. The hybrid modulation scheme consists of base PWM circulation and hybrid switching controller (HSC) to generate new modulation pulses.X and X′) acquired from a direct comparison of reference sine and carrier waveforms. Based on the selection of carrier, the PWM generation is classified as hybrid unipolar pulse width modulation (HUPWM) obtained from the comparison between sine reference and unipolar carrier, while hybrid inverted sine carrier pulse width modulation (HISCPWM) for inverter module is generated from the inverted sine carrier. The other PWM method reported for fundamental enhancement with improved harmonic profile, namely, H-60° PWM (HSDPWM).The block diagram representation of base PWM circulation design is seen in C and Y) out of three square wave signals having 50% duty ratio and frequencies with half and twice the fundamental frequency. The third signal is FFS (A), a square-wave signal synchronized with the reference sine waveform, (A = 1) during the positive and (A = 0) during negative half cycles of the reference sine.In addition to PWM circulation pulses, the HPWM requires three square wave signals that make every power switch operating at carrier based PWM (CBPWM) and square wave randomly per quarter cycle to equalize the power losses within each device. Two signals are random switching pulse (Y and C), FFS (A), and CBPWM (X and X′) that produces hybrid modulation (HM) pulses. It is designed by using a simple combinational logic and the functions for a single-phase inverter are expressed asA = 1, S1 and S2 are commutated at low frequency in the first and second quarter of first half cycle, while both devices are modulated with PWM in the second and first quarter cycles in the same half cycle of fundamental frequency, respectively. Similarly if A = 0, S3 and S4 are modulated with the same pattern in the second half cycle of the fundamental frequency as those in the devices S1 and S2, respectively. Therefore voltage stress and current stress of four switches are also equalized. The proposed HPWM method allows use of similar power devices with identical heat sinks, thus permitting a more reliable design.HSC combines random signals  = 200 V, switching frequency of PWM signal (fs) = 1 kHz, fundamental frequency (f0) = 50 Hz, R = 150 Ω, and L = 100 mH, respectively.In order to evaluate the quality of output voltage waveform acquired from single-phase inverter using the proposed HPWM methods, the simulation study is carried in MATLAB/Simulink platform. The input parameters for simulation study are taken with dc-link voltage  processor. It incorporates a facility to be either configured or reconfigured by the application engineer. The Spartan 3E-500 FG 320 has on-board high-speed USB port, 500 K logic gates, 16 MB of PSDRAM, 16 MB of Intel strata flash ROM, and 60 FPGA input/output routed to expansion connectors. Xilinx offers CAD tools for the development of ASIC based FPGA and VHDL is used in digital logic design. The algorithm is then complied and simulated using Modelsim software. The bit file is generated in Xilinx software and finally downloaded to the Spartan 3E device through USB cable. The downloaded pulses are applied to single-phase inverter to produce the PWM modulated output voltage waveform. The download pulses, load output voltage waveform along with voltage spectrum, are captured through Tektronix TPS 2024 scope which is shown in Figures The hardware rig is constituted using MOSFETs (IRF 840), laboratory variable dc-link voltage setup and resistive-inductive load of Various HPWM strategies for single-phase inverter operating at low switching frequency with lesser switching commutations are proposed. The proposed HPWM method can be extended to any carrier based PWM methods. In comparison with conventional PWM methods, the proposed methods offer lesser number of switching commutations and reduced switching loss for the same fundamental voltage. The harmonic performance of the HPWM methods are examined in the entire operating range and its performance is better. The proposed methods facilitate balanced conduction loss with reduced switching loss within each device. Also the hybrid PWM strategy reduces conduction losses to a larger extent so that the THD is better when compared to the conventional PWM methods. The experimental results demonstrate that the proposed methods can be extended to any type of dc-ac/ac-ac power conversion systems.


Over the last decade, pharmacogenetics has become increasingly significant to clinical practice. Psychiatric patients, in particular, may benefit from pharmacogenetic testing as many of the psychotropic medications prescribed in practice lead to varied response rates and a wide range of side effects. The use of pharmacogenetic testing can help tailor psychotropic treatment and inform personalized treatment plans with the highest likelihood of success. Recently, many studies have been published demonstrating improved patient outcomes and decreased healthcare costs for psychiatric patients who utilize genetic testing. This review will describe evidence supporting the clinical utility of genetic testing in psychiatry, present several case studies to demonstrate use in everyday practice, and explore current patient and clinician opinions of genetic testing. Mental illnesses are extremely prevalent and debilitating. Depression alone is the leading cause of disability worldwide, leading to a significant patient/economic burden, affecting at least 350 million people . ApproxiThere is significant interindividual variation to psychotropic treatment response, leading psychiatrists to adopt a trial and error approach to treatment . GeneticMuch of the utility of pharmacogenetic testing has been shown in clinical settings other than psychiatry. Many of these tests identify mutations relating to altered expression and functions of genes associated with drug disposition and response and have been useful in clinical practice . Within TPMT). This enzyme is necessary for proper thiopurine treatment in patients suffering from acute lymphoblastic leukemia. Patients who express a defective version of this enzyme can experience life-threatening adverse events in response to treatment [ TPMT testing allows for individualization of therapy and has been shown to be cost effective in patients who are treated with azathioprine [SLC6A4) have been found to help predict how patients will respond to antidepressant treatment. As SLC6A4 is the primary target for selective serotonin reuptake inhibitors (SSRIs), patients with a variation in this protein may show poor response, lower remission rates, and increased side effects leading to medication intolerance with SSRIs [Understanding the utility  of genetic tests applied in one field can facilitate adoption in areas where testing is not presently employed. For example, a genetic test currently used in medical practice analyzes genetic variations in thiopurine methyltransferase (reatment . TPMT tehioprine . Similarth SSRIs .MTHFR) is another gene regularly tested in the field of cancer biology and has growing implications within psychiatry. Patients with variations in MTHFR, particularly the C677T variation, have decreased enzyme activity and variable folate levels [ MTHFR variant depicts a risk for increased side effects in response to methotrexate therapy, a folic acid antagonist [ MTHFR variations has been shown to effectively predict which patients are more likely to suffer from these serious adverse events in response to methotrexate treatment [Methylenetetrahydrofolate reductase , as well as serotonin receptor 2A (5HTR2A) and cytochrome P450 1A2 (CYP1A2) [ CYP2D6, serotonin receptor subtype 2C (5HT2C), SLC6A4, and 5HTR2A [While still evolving, the use of pharmacogenetic testing in psychiatry is expected to become widespread . A recen(CYP1A2) . Adversed 5HTR2A . These ed 5HTR2A .In addition to improved treatment outcomes, genetic testing can reduce high costs related to treatment failures and severe adverse events . The totCYP2D6, CYP2C19, CYP2C9, and CYP1A2), SLC6A4, and 5HTR2A found individuals with genetic variations relating to adverse clinical outcomes had 69% higher total healthcare costs, 67% more general medical visits, 3-fold higher medical absence days, and 4-fold greater number of disability claims than individuals without the associated risk variations [ CYP2D6 and CYP2C19 leading to poor or ultrarapid metabolism was found to reduce overall medical costs by 28% in schizophrenic patients [A retrospective study examining antidepressant response based on variations in cytochrome P450 enzymes , a dopamine receptor subtype (DRD2), catechol-O-methyl transferase (COMT), ankyrin G (ANK3), and MTHFR. A study was conducted analyzing the cost effectiveness and impact on medication adherence in psychiatric patients who received this genetic test compared to a matched set of controls [An example of one commercially available genetic test for psychiatric patients is the Genecept Assay. This assay examines ten genes associated with treatment response, side effects, metabolism, tolerability, and overall efficacy of many psychiatric medications , 33–35. controls . This stcontrols . This ficontrols ; it is pcontrols . These f SLC6A4, DRD2, and 5HT2C, increasing his risk for failure and intolerance with SSRIs and antipsychotic agents. Thus, a treatment strategy which did not target the serotonin transporter or dopamine receptor pathway would likely be better tolerated in this patient [ ANK3 gene [Armed with patient genetic information, clinicians can more quickly identify effective therapies, thus limiting the prolonged suffering and economic burden placed upon many patients with chronic illnesses. Several examples are available which demonstrate the utility and effectiveness of testing in practice for a variety of clinical diagnoses. This is demonstrated in the following published patient cases. One case describes an 18-year-old male diagnosed with intermittent explosive disorder, suffering from uncontrolled anger outbursts and several failed medication trials, who elected to utilize genetic testing . The res patient . In anot patient . In thisNK3 gene . ANK3 isNK3 gene . LamotriNK3 gene , 39. MTHFR which led the clinician to prescribe L-methylfolate, leading to complete remission of symptoms [ MTHFR gene may lead to impaired neurotransmitter synthesis and increased depression risk; therapeutic intervention with L-methylfolate has been shown to be an effective adjuvant therapy for patients suffering from major depressive disorder [Genetic testing not only helps identify conventional treatments which may be most effective, but also can help identify effective alternative therapeutic options. In this last case, a clinician utilized genetic testing for a 69-year-old man suffering from long-term depression symptoms . This pasymptoms . As descdisorder , 42.While pharmacogenetic testing in psychiatry is still emerging, there is a growing body of evidence demonstrating improved outcomes and cost effectiveness to support its utility and validity. Continued education and future research in the field are vital to its widespread acceptance. The knowledge and understanding of pharmacogenetics in psychiatry are continually growing, as is the application and utility into everyday clinical practice.An important factor for the widespread adoption of genetic testing in psychiatry is clinician and patient acceptance. A study which utilized a random sample of US psychiatrists suggested that clinicians would be very open and welcome to pharmacogenetic testing in clinical practice . Eighty-Several studies also examined patient perceptions to genetic testing. One study from UCLA found that cancer patients overwhelmingly (98.98%) would elect to receive predictive genetic testing at time of treatment even if no further treatment was available . The stuPharmacogenetic testing in psychiatry is a newly evolving field and is rapidly gaining wide acceptance. Numerous articles and reviews, as well as a growing number of case studies, have been published showing the clinical utility and cost effectiveness of genetic testing for psychiatric patients. To further substantiate the utility of genetic testing in psychiatry large randomized controlled trials are needed. The prevalence and burden of depression are predicted to continually grow ; the ideIn addition to the growing body of evidence on the utility of genetic testing to aid in the treatment of psychiatric disorders, there is also growing literature and promise regarding the utility of other genomic markers. Some of these other markers include small sections of ribonucleic acid (RNA) called microRNA (miRNA) responsible for playing a role in gene regulation, and gene expression levels analyzed using messenger RNA (mRNA). An additional layer of gene regulation is controlled by epigenetic modification which could also impact how genes are expressed.MicroRNAs are small sections of RNA which can regulate up to several hundred genes and dysregulation of certain miRNAs may play a role in psychiatric and neurological disorders . One exaBDNF). This gene has long been thought to be involved in antidepressant treatment response via genetic variation, epigenetic modification, and varied mRNA expression. BDNF mRNA expression levels have been found in several meta-analyses to be significantly decreased among psychiatric patients, and effective antidepressant treatments increase BDNF serum mRNA levels [ BDNF may also be an indicator of antidepressant treatment response; however, these studies are still preliminary [Epigenetic modifications and mRNA gene expression levels are closely related as epigenetic changes can impact levels of mRNA gene expression. Epigenetic modification is defined as heritable changes in gene activity and expression which occurs without variation to the DNA sequence. These changes include DNA methylation and histone modification. A good example of both epigenetic and mRNA expression variations related to psychiatric treatment response can be found when examining the gene, brain-derived neurotrophic factor (A levels –53. Thesliminary , 54, 55.In the years to come, it is appears that pharmacogenetic testing will become integrated into everyday psychiatric practice. There is also great promise for additional genomic markers to be researched and developed to add information to further improve patient care. Pharmacogenetic/genomic testing will become a valuable tool to help improve patient outcomes, lower healthcare costs, and increase patient medication adherence. Just as pharmacogenetic testing has revolutionized clinical practice in areas such as cancer treatment, it has the potential to do so in psychiatry.


We find marked spatiotemporal heterogeneity in uplift from mid-Holocene to present: some areas accrue more permanent uplift than others. Thus, uplift imposed during the 1 April 2007 Mw 8.1 event may be retained in some locations but removed in others before the next megathrust rupture. This variability suggests significant changes in strain accumulation and the interplate thrust process from one event to the next.Histories of vertical crustal motions at convergent margins offer fundamental insights into the relationship between interplate slip and permanent deformation. Moreover, past abrupt motions are proxies for potential tsunamigenic earthquakes and benefit hazard assessment. Well-dated records are required to understand the relationship between past earthquakes and Holocene vertical deformation. Here we measure elevations and  Information regarding tectonic motion from before instrumental records can be found from palaeoshorelines and the reconstruction of sea level from observations. Here, the authors study corals uplifted by past earthquakes near the Solomon Islands and assess the Holocene deformation that took place there. Mw 8.1 earthquake in the Western Province of the Solomon Islands ruptured across the Australian-Woodlark-Pacific triple junction12324On 1 April 2007, a large Mw 7.2 were instrumentally recorded between 156°E and 158°E from 1900 to 2007  than in any previous study. U-Th chemistry24in situ coral levels (see Methods) with longer-term net upliftIn this work, using the elevation and ages of uneroded, in situ, dated coral uplift levels described in the Methods section  at the site. Thus, we infer that much of the uplift imposed by prehistoric earthquakes and that of the 2007 uplift at Lale is balanced by subsidence in the years preceding each megathrust earthquake as found at margins where there are large coseismic uplifts but very little net Holocene upliftThe 2007 coseismic uplift profile along the long axis of Ranongga follows a fairly smooth classic elastic strain release curve because of slip on a reverse fault1in situ corals documenting paleo-earthquakes. At locations such as Konggu, Newbare and Kolomali, we find a high mid-Holocene coral terrace . By in situ we refer to corals found attached in growth position .Supplementary Figures 1-2, Supplementary Tables 1-5 and Supplementary References


Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. Immunotherapy with anti–PD-1 monoclonal antibodies such as pembrolizumab shows robust, durable anti-tumor activity in multiple indications including patients with advanced melanoma. A more complete understanding of the mechanism of action and biology associated with both response and resistance to pembrolizumab is critical to better inform on future clinical development and will also provide insight into the development of additional immuno-oncology therapies.To further support the clinical development of pembrolizumab, muDX400 (an anti-PD1 murine surrorgate) was tested in syngeneic mouse models. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Using a multifaceted approach, tumors from these models were extensively characterized at the molecular and cellular level by gene expression profiling and whole exome sequencing to help elucidate mechanisms of action and biology associated with response and resistance to anti-PD1 treatment.These findings are also being compared to data that we are obtaining from ongoing clinical trials with pembrolizumab to help better understand the translatability of these preclinical models to the clinic. For example, both the IFN-γ and the expanded-immune signatures showed statistically significant associations with ORR and PFS in Keynote 001 Phase Ib melanoma trial. Analysis of top-ranked genes on the platform led to the discovery of two new signatures, “TCR-signaling” and “De novo” that were enriched in T cell markers and MHC Class I and II genes. Measuring immune-related biomarkers, including T cell specific, antigen presentation–related, and IFN-γ signaling–related genes, may allow for improved selection of patients likely to respond to anti–PD-1 therapy with pembrolizumab.The translatability of molecular features such as gene expression and mutation burden will be assessed in syngeneic mouse models.These preclinical syngeneic tumor models allow us the opportunity to formulate and test specific hypotheses to more completely understand the biology behind the clinical successes currently observed with the novel cancer immunotherapies including anti-PD-1 antibodies such as pembrolizumab.


Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript  Cone photoreceptors are the light sensitive cells in the retina that mediate colour and high acuity vision. Because these cells are enriched in the macula of the human retina, their degeneration results in a significant impairment of the central visual field. Cone degeneration can occur as a consequence of mutations in cone-specific genes or can be secondary to rod degeneration, with the most prevalent cone degenerative condition being age-related macular degeneration (AMD). AMD is thought to involve an inflammatory component coupled with dysfunction of the choroid and retinal pigment epithelium (RPE), which is essential for structural, trophic and metabolic support of photoreceptors  and medium wavelength (green) light, respectively. Cones are generated between embryonic day 11 (E11) and E18 in the mouse, and like rods, are produced as postmitotic precursors (reviewed in13Coiled-coil domain containing 136 (Ccdc136)/nasopharyngeal carcinoma-associated gene 6 (NAG6) locus. Ccdc136 is predicted to be a single-pass membrane protein with coiled-coil domains. Although the function of Ccdc136 is largely unknown, it is frequently deleted in nasopharyngeal, gastric, breast and ovarian cancers, suggesting a role in tumor suppressionCcdc136 expression has been reported in the outer nuclear layer (ONL) of cone only Nrl−/− and cone/rod hybrid Nr2e3−/− mouse retinas1617Ccdc136GFP mouse, reporter gene expression from the Ccdc136GFP allele is initiated in embryonic cone precursors located at the apical surface of the retina and marks S and S/M cones, as well as rod bipolar cells in the adult. Consistent with expression in cones in the adult retina, the GFP reporter was expressed in the majority of cells in the photoreceptor layer in the cone-dominant Nrl−/− backgroundNrl−/− mice integrated into the photoreceptor layer of adult recipient mice and adopted photoreceptor morphologies. This study describes a reporter strain that can be used to identify cone precursors that are competent to integrate into recipient retinas, demonstrating a powerful resource for monitoring early cone development and as a tracer for cone transplantation.We describe the development of a novel GFP-reporter mouse line with a gene trap insertion into the Ccdc136 gene is located 3′ of Opn1sw (S opsin) on mouse chromosome 6, consists of 15 exons spanning 31.1 kb  . RT-Q-PCR with primers spanning exons 2 and 3 of Ccdc136 amplified a product in the Ccdc136GFP/GFP retina , suggesting the possibility that the trapped allele could produce wildtype transcripts where the gene trap sequences are spliced out  . Ccdc136iced out . The preanscript . We wereCcdc136GFP dosage  /outer segment (OS) boundary and basal processes terminating at the outer plexiform layer    and bothckground , which iwith PKC . GFP+ ce S opsin , which irm layer . All of in (PNA) . While G subsets . FinallyGFP mice , furtherCcdc136GFP/GFP mice. We found no evidence of cone degeneration in Ccdc136GFP/GFP mice at 3 and 8 months , cells expressing Ccdc136 transcripts and GFP were located on the apical side of the retina, however, after P0 we noted a change in the distribution of GFP+ cells and Ccdc136 transcripts in the ONL  and progenitor (Vsx2) markers  However, postnatal cods were not fully mature, based on the absence of cone opsin and cone arrestin expression at P1 (data not shown). At this stage the GFP+ cells were not rod bipolar cells, as IHC analysis of cod-GFP retinas confirmed that these cells develop after P6 . This observation suggests that induction of the Ccdc136GFP reporter occurs after Crx expression is initiated in the cone differentiation cascade.As another approach to investigate the relationship between  markers . However markers , which iafter P6  and lackafter P6 . We alsoCcdc136GFP mice makes this mouse strain an ideal donor line to investigate the feasibility of cone engraftment into recipient retinas. Rod photoreceptor precursors capable of integrating in the adult retina are present in mixed dissociates isolated from P1–P7 donorsCrx−/− mice are non-functional with extensive degeneration of the ONL at 4–6 monthsCrx−/− recipient mice this study. As a second degeneration model, we used mice mutant for the combination of rod transducin (Gnat1−/−), cone cyclic nucleotide-gated channel (Cnga3−/−), and melanopsin (Opn4−/−) , which lack all light perception but maintain an ONL when they were transplanted at 4 months+ cell bodies that lacked apical or basal processes  rather than cod-like  (+ cells in cod-transplanted retinas we cannot entirely rule out the possibility that some of the integrated cells arise from fusion with host cells.Because we could not detect cone marker expression in GFP lack OS . Moreove lack OS . We did romatin) . Howeverromatin) . In summCcdc136GFP reporter line to investigate integration of endogenous cone photoreceptors. Because of the low frequency of cones on a wildtype background, we modified our transplantation protocol by first FACS enriching for GFP+ cells and reducing the number of cells transplanted . We chose E17 donor cells, as we reasoned the cones at this stage would be similar in maturation to P1 cod-GFP cells, with low to undetectable levels of mature cone marker expression+ cells in the ONL, several examples of GFP+ cells with apical and basal processes with the number of integrated cells ranging from 40 to 170 (n = 5 eyes), representing approximately 1% of injected cells. Similar to transplanted cod-GFP cells, transplanted cones were undifferentiated, based on the absence of cone arrestin expression . This difference in nuclear positioning between Crx-GFP and Ccdc136GFP embryonic cones could be explained by an influence of co-transplanted rods in the Crx-GFP model.Here we have investigated the feasibility of using this reporter line to track transplanted cone photoreceptors. We show that GFP-marked cods and cones can integrate in the adult retina and undergo morphological differentiation. Though it has been previously reported that the cell bodies of integrated embryonic Crx-GFP marked cones were located on the apical side of the ONLCcdc136GFP-tagged cods and cones did not mature fully in terms of expression of the phototransduction cascade proteins, it is unlikely they would mediate vision rescue in blind mice. Moreover, the numbers of integrated cods in our study was considerably lower than the number of functional photoreceptors required to measure an ERG responseet al.et al.in situ5Because integrated + cods and cones prompted us to consider whether some of these cells result from fusion with endogenous host photoreceptors. Simple cellular fusion is an unlikely explanation as the integrated cells had single nuclei and fusion events have not been reported in rod and cod transplantation studies where this possibility was tested using dual reporter systems42734+ cods with processes  in degenerated Crx−/− retinas, demonstrating that intact host photoreceptors are not required to detect transplanted cod-GFP cells. We did, however, observe GFP+ cells in the host ONL that had host instead of donor cell nuclear morphology. Whether this nuclear mismatch represents cell fusion or donor cells changing nuclear morphology in their new environment will require further investigation.The lack of mature cone marker expression in the integrated GFPGFP reporter to stratify the cone precursor pool and identify those with the best morphological and functional integration.While post mitotic rod precursors have the best integration potential, the molecular basis for this property is not well understood. In our study the integration rates of P1 and P6 donor cods were comparable to those reported for unsorted P3–P6 rod precursors34274et al.In contrast to integrated cods and cones, expression of mature cone markers was readily observed in un-integrated cells located in the subretinal space and the vitreous, which is consistent with the differentiation of un-integrated rod photoreceptor precursors in other studies26Nrl−/− cods exhibit some differences from wildtype cone photoreceptors1922Nrl−/− retina. Consistent with an environmental contribution to cod dysmorphology, transplanted cods exhibit better morphology compared to cods that differentiate in situ in the Nrl−/− retina  for 60 minutes at 95 °C. Samples were neutralized with 300 μl neutralization reagent (40 mM Tris-HCl) and genotyped by PCR using primer sets indicated in All experiments were approved by the University of Ottawa Animal Care Ethics Committee and adhered to the guidelines of the Canadian Council on Animal Care. Animals were maintained under standard laboratory conditions and all procedures were performed in conformity with the University of Ottawa Animal Care and Veterinary Service, and the University of Calgary Animal Care Committee (protocol #AC11–0053) in compliance with the Guidelines of the Canadian Council of Animal Care. Wildtype C57BL/6 mice were obtained from Charles River. The following transgenic mouse strains used in this study were maintained as homozygous lines: In situ hybridization was carried out as previously reportedEyes were harvested at various stages beginning at embryonic day (E) 13.5 to adult. Mice younger than postnatal day (P) 10 were culled and entire heads with eyes were collected. Mice older than P10 were perfused with 4% paraformaldehyde (PFA) and eyes were marked on their corneas for dorsal orientation with a silver nitrate stick before enucleating. Tissues were fixed in 4% PFA overnight then cryoprotected overnight in 30% sucrose in phosphate buffered saline (PBS) . After sinking in sucrose, tissues were equilibrated in 50:50 30% sucrose: OCT (Tissue-Tek) for 1–2 hours, followed by orienting and embedding in plastic molds in sucrose:OCT and freezing. Samples were stored at −80 °C. Tissue was sectioned at 12 μm onto Superfrost Plus slides (Fisher Scientific) on a Leica cryostat, air-dried for 1 to 2 hours and stored at −20 °C with desiccant. Retinal sections and dissociated cells were permeabilized with 70% ethanol for 5 minutes then washed in PBS for 20 minutes. Following an antigen retrieval step , sections were blocked with 50 mM Tris buffer (pH 7.4), 10 mM lysine, 145 mM NaCl and 1% BSA  for 1 hour at room temperature. Primary antibodies  and 10,000 trypsin (embryonic FACS enriched Ccdc136GFP/+) dissociated cells were transplanted to the subretinal space of adult recipient C57Bl/6, Crx−/− and TKO mice (1–6 months old). Donor cell integration was assessed three weeks after transplantation.Detailed methods are available in the Crx−/− recipients, in or adjacent to the INL. The average number of integrated cells per eye was determined by counting all integrated GFP+ cells in alternate sections of each eye and multiplying that number by two. Only animals with successful transplantations, where GFP+ cells were located in the subretinal space, were included in the quantitative analysis. If there was evidence of intravitreal cell delivery, bleeding into the eye or if no GFP+ cells were detected in the subretinal space, these animals were considered as “failed transplants” and excluded from further analysis. All data are presented as mean ± SEM. Statistical significance was evaluated using a two-tailed paired t-test or 2-way ANOVA.Fluorescent images were captured using an Axioimager M1  or an Axioimager 2 . Confocal images were acquired using a FluoView 1000 confocal microscope (Olympus), a LSM 700 (Carl Zeiss) or a LSM 780 . Wildtype and mutant retinas were imaged with identical exposure times at each magnification. Images were processed using Photoshop CS4 (Adobe) and any adjustments were made to the entire image and equally for each genotype. Quantification of dissociated cells was performed by counting marker+ cells and dividing by the total number of nuclei (5 images taken per stain with a minimum of 200 cells counted). Transplanted eyes were harvested three weeks post surgery. Retina cryosections, prepared as described above, were stained with anti-GFP and cell type specific antibodies . Cells wHow to cite this article: Smiley, S. et al. Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line. Sci. Rep.6, 22867; doi: 10.1038/srep22867 (2016).


The val7 were used to evaluate how the platelet response measures associate with clinical outcomes. This trial was discontinued early owing to concerns that the potential small benefit of bleeding reduction did not outweigh the potential risk for diagnosis of acute myeloid leukemia (AML) as defined by pathology or initiation of AML-type treatment; the final analysis of the 58-week data set showed comparable AML rates in both arms.7 We describe here to what extent the currently clinically available platelet response criteria were applicable in this study and whether they were associated with platelet transfusions, bleeding and overall survival.Data from a 58-week placebo-controlled trial of romiplostim in patients with lower-risk MDS and thrombocytopeniaN=250) were randomized 1:2 to 26 weeks of weekly placebo (N=83) or romiplostim (N=167), with dose adjusted for platelet count, followed by a 4-week washout period and bone marrow biopsy, another 24 weeks as randomized (extended treatment period), and a second 4-week washout period and bone marrow biopsy .7 The 4-week washouts occurred before bone marrow biopsies so that the study drug would not affect results, as romiplostim can be associated with transient blast cell count elevations. Patients then entered long-term follow-up.Patients with International Prognostic Scoring System (IPSS) low/int-1 risk MDS (9/l or (2) ⩽50 × 109/l and a history of bleeding. Patients were stratified by baseline IPSS status  and platelet count . Platelet response was assessed using various platelet response criteria , with platelet counts (1) ⩽20 × 10criteria . Clinicapost hoc. The Fisher's exact test was used to test the association of romiplostim and platelet response. The Mantel–Haenszel method was used to calculate a pooled odds ratio of romiplostim and placebo across the randomization stratification factors of baseline platelet count and IPSS. A Cox regression model including prognostic factors and platelet response as covariates was used to predict overall survival in romiplostim-treated patients. Poisson regression models, including baseline platelet count, IPSS status and platelet response as covariates, were used to predict bleeding events and platelet transfusions in romiplostim-treated patients. As this was an analysis based on response, a landmark sensitivity analysis was performed using platelet counts from the first 26 weeks to determine platelet response, excluding patients who discontinued the trial within 26 weeks, and analyzing outcomes that occurred after 26 weeks; survival curves by response status were plotted per landmark at week 26. CSBEs were too infrequent for the landmark sensitivity analysis to be meaningful. Overall survival was recorded up to the last observation in long-term follow-up, and bleeding events and platelet transfusions were measured in the extended treatment period only.All analyses were performed post hoc analyses. Patients  were mostly male (59.2%) and Caucasian (94.0%). Median  age was 70.0  years and median  baseline platelet count was 19.3  × 109/l. Median  MDS duration was 0.44  years. Most patients were MDS WHO classification refractory cytopenia with multilineage dysplasia (67.6%).The six platelet response measures  were useRomiplostim treatment was significantly associated with platelet response by all criteria studied . For exa10 and durable response were significantly associated with less CSBE. These same measures, and International Working Group (IWG) 2000 criteria, were significantly associated with improved overall survival. Survival curves for HI-P, the platelet response measure most significantly associated with survival, are shown in The association between platelet response criteria and clinical outcomes such as bleeding  were evaluated, as in P-values, although results were generally consistent with the original analyses. Differences included that complete response10 and IWG 2000 major response11 were marginally significantly associated with overall survival . All platelet response measures remained significantly associated with all bleeding and platelet transfusions (data not shown).Landmark sensitivity analyses were performed for all measures described above to determine whether this being an analysis based on response and discontinuation of patients affected trial results. For the overall survival end point, after excluding patients who discontinued in the first 26 weeks, the sample size decreased from 167 to 143. Among the 24 subjects who were excluded, 12 died. Smaller sample size and fewer events contributed to slightly larger per se has been associated with worse prognosis in MDS, including an increased risk of disease progression.3 For the first time, we show that platelet response to a thrombopoietin mimetic is positively associated with overall survival. Possibly, romiplostim has a beneficial effect through reducing potentially life-threatening thrombocytopenia or other as-yet-unrecognized broader effects. Thrombopoietin has previously been shown to stimulate other hematopoietic lineages.12 It is unclear whether the improved outcomes are associated with response to romiplostim or that, inherently, patients that respond have better outcomes. While no difference in survival was seen with romiplostim vs placebo,7 better selection of patients could lead to improved survival outcomes with romiplostim.Data from this large placebo-controlled romiplostim trial indicate that platelet response criteria, developed empirically from clinical experience and trials using disease-modifying agents, are heterogeneous, result in a wide range of response rates for the same patient population and are predictive of clinical outcomes in thrombocytopenic MDS patients treated with a thrombopoietin mimetic. This is in keeping with the finding that thrombocytopenia 13 For studies examining survival and treatment with erythropoiesis-stimulating agents (ESAs) in MDS, results have been mixed. A retrospective multivariate analysis of patients treated with ESAs with or without granulocyte colony-stimulating factors (G-CSFs) reported that survival, but not disease progression, was improved in the ESA-responsive cohort compared with an untreated IPSS/IMRAW  cohort.14 Another multivariate analysis, comparing patients treated with ESAs plus G-CSFs with a control cohort of untreated MDS patients, found better survival with ESA treatment, particularly for those requiring fewer than two red blood cell units transfused per month.15 However, a small randomized phase 3 Eastern Cooperative Oncology Group (ECOG) study of patients receiving supportive care alone or supportive care plus ESAs with or without G-CSFs found no difference in survival, and that survival was increased for those who responded to ESA treatment.16 Whether ESA treatment improves survival in MDS may become clearer as ongoing studies report results.A positive association between treatment and survival is also seen for the disease-modifying therapy azacitidine in higher-risk MDS.In summary, these data indicate that platelet response criteria, specifically those that incorporate durable response, such as IWG 2006, correlate with overall survival and have the potential to be used as interim markers for clinically significant outcomes. However, a limitation of this data set is that the study drug treatment was ended early owing to concerns regarding transient increases in peripheral blast cell counts with romiplostim that put patients at risk for the diagnosis of and treatment for AML. Therefore, the data set was incomplete, and it is possible that different results regarding the association of platelet response measures and clinical outcomes would have been obtained with a fuller data set. Evaluation is needed of these associations in either past MDS clinical trials of interventions to raise platelets or future ones to confirm that these findings occur in the broader context.


Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes  As major producers of energy and oxidative stress, the maintenance of the mitochondrial network is critically important for cells. Mitochondrial quality control is especially crucial for long-lived cells with high energetic demands, such as neurons.Previous methodologies for studying mitochondrial aging have severe limitations including a lack of spatial resolution and live-cell applicability.The fluorescent Timer protein fluoresces green post-synthesis. Within 24 h the emission spectra shifts to red fluorescence. We fused the fluorescent Timer to the mitochondrial targeting sequence of COX8A subunit, to form the MitoTimer construct. To control the timing of transcription of MitoTimer in cells, a doxycycline-inducible vector (pTRE-tight) was used to express MitoTimer. The resulting mitochondrial-targeted Timer localizes to mitochondria as assessed by co-localization with TOMM20 staining in MEF cells .C) in transiently-transfected cells. Expressing MitoTimer in primary hippocampal neurons revealed similar time-dependent changes in green and red MitoTimer fluorescence  will depend on rates of synthesis/import of new (green) protein and degradation of old (red) protein. To validate these associations, we quantified fluorescence in mitochondrial networks with pulsed MitoTimer synthesis, decreased protein import, or inhibited degradation by autophagy.P < 0.05, n = 4).Limiting synthesis of new MitoTimer with a 4 h pulse-exposure to doxycycline altered the age profile relative to continuous 24 h doxycycline treatment. Pulsed expression shifted the age profile of MitoTimer in the mitochondrial network to increase the proportion of red relative to green fluorescence compared with continuous doxycycline . PlottinP < 0.05, n = 3). Importantly this decrease in green MitoTimer occurred only in the presence of doxycycline, which is required to allow transcription of new MitoTimer protein. Without doxycycline for the last 24 h before imaging, there was no significant change in green MitoTimer fluorescence after FCCP treatment  treatment in COS cells stably-expressing MitoTimer . Similar results were seen with chloroquine treatment, which neutralizes lysosomal acidity. In order to address the effects of inhibiting mitochondrial turnover on MitoTimer age profile in steady-state condition, we generated stably-expressing MitoTimer COS cells via lentivirus infection. Treatment with bafilomycin or chloroquine similarly induced an accumulation of MitoTimer protein, with a greater proportion of red (old) protein accumulating . This effect was similar in cells that had not received doxycycline treatment for the last 24 h, which suggests the effect of bafilomycin and chloroquine was via inhibition of autophagy and independent of transcriptional effects  material without significantly changing the amount of newly synthesized (green) protein , confirming that reduced protein turnover by autophagy leads to the accumulation of red (aged) MitoTimer. Collectively these findings are in agreement with the expected influence of mitochondrial turnover on accumulation of aged material within mitochondrial networks.Inhibition of autophagy was also achieved upstream of the lysosome with genetic knockout of We next examined whether mitochondrial dynamics influences the equilibration of protein age among individual mitochondria within the network. While individual mitochondria within the cell may import and degrade proteins at different rates, their involvement in continuous cycles of fusion and fission events may counter subcellular heterogeneity by allowing for the equilibration of matrix protein content. We rationalized that the ability to become engaged in fusion events may influence the level of heterogeneity in protein age among mitochondria in the cell.Mfn1 and Mfn2 (Mfn1/2 KO) show significant subcellular heterogeneity of MitoTimer age profiles compared with wild-type MEFs . We also determined that inhibition of mitochondrial fusion by Opa1 knockdown in INS1 cells resulted in increased subcellular heterogeneity of MitoTimer age profile . Overall these data indicate the importance of mitochondrial fusion in equilibrating the levels of new and old protein within the network.Double-knockout MEFs lacking both ype MEFs .7 This Mfor 24 h , but wasfor 24 h . Assessiild-type . While a KO MEFs . OverallWe next turned to primary hippocampal neurons to examine MitoTimer age profiles in a cellular system with complex morphology and large spatial dimensions. As such, the neuron is a unique model in which one can study the impact of separate cellular compartments with disparate movement, fusion and biogenesis kinetics on the subcellular distribution of young and old protein.MitoTimer expressed in primary hippocampal neurons for 24 h displays an overlap of green and red fluorescence in the soma producing an overall yellow appearance. In contrast, mitochondria in distal regions of neurites appeared more orange or red in fluorescence compared with the soma . This efP = 0.01). Constitutively active RHOT1-KK increased homogeneity of the age profile between neurites and soma (distribution shifted toward zero). Overexpressing RHOT1-WT significantly reversed the normal distribution of aged material between the neurites and soma . These data illustrate the importance of mitochondrial motility in equilibrating protein age between the soma and neurites.Given that transport greatly influences subcellular spatial distribution of mitochondria, we chose to investigate the role of mitochondrial motility in the age equilibration pattern of MitoTimer in neurons. To increase mitochondrial transport, we co-expressed MitoTimer with RHOT1 wild-type (RHOT1-WT) or a hyperactive, calcium-insensitive mutant of RHOT1 (RHOT1-KK). RHOT1 is a motor adaptor protein that selectively facilitates mitochondrial movement along microtubules.To better understand the connection between spatial location and age heterogeneity, we performed time-lapse imaging to assess the dynamics of green and red MitoTimer in mitochondria located in the soma or neurites. Over the 12 h imaging period (imaging started after 24 h of MitoTimer expression), red MitoTimer fluorescence intensity increases in the soma and neurites as MitoTimer in both compartments transitions to red . OverallMechanisms for turnover of cellular constituents are central to the pathophysiology of age-related diseases. MitoTimer represents a novel tool to study mitochondrial turnover at cellular and subcellular levels  and Atg5−/− (Atg5-KO) mouse embryonic fibroblasts (MEFs) transfected with pEF321-T and an SV40 large T antigen expression vector were a generous gift from N Mizushima and were previously described.Mfn1 and Mfn2 double-knockout (Mfn1/2 KO) MEFs were described previously.Matched Hippocampal tissue was harvested from timed pregnant (E14) CD1 mice and maintained in Neurobasal media  with B27 supplement  and Pen/Strep  with half-media changes every other day. Neurons were cultured on poly-d-lysine/laminin  coated MatTek glass bottom dishes for confocal imaging. With the exception of the live-cell time-course studies, cells were fixed with 4% paraformaldehyde  in phosphate buffered saline (PBS) prior to imaging.MEFs were transfected using Lipofectamine LTX . Neurons were transfected with calcium phosphate  on the seventh day of in vitro culture (DIV7). Doxycycline  was added to the media the day after transfection to induce MitoTimer expression.Mfn1/2 KO cell lines were infected with MitoTimer lentivirus and exposed to doxycycline to induce MitoTimer expression. After 72 h of doxycycline exposure, fluorescent cells were sorted into medium- and high-expressing populations with a BD FACSARIA II SORP cell sorter. Cells were then maintained without doxycycline for 2–3 passages before cells were plated and used for experiments. In order to conduct experiments with cells expressing steady-state levels of MitoTimer, stable cell lines were exposed to doxycycline for at least 5 d (doxycycline replaced daily) to obtain steady-state expression of MitoTimer before specific treatments or imaging was performed.COS, INS1, MEF wild-type, Atg5 KO, and Opa1-shRNA lentiviruses targeted against different portions of the rat Opa1 sequence. Both viruses were found effective at knocking down Opa1 levels in INS1 cells resulting in a fragmented mitochondrial architecture. Cells were cultured for 6–7 d after infection with Opa1-shRNA lentiviruses before imaging.INS1 cells stably-expressing MitoTimer were infected with 2 different To assess MitoTimer co-localization with mitochondrial Tomm20, immunofluorescence staining for Tomm20 was performed on MEFs transfected with MitoTimer. Briefly, transfected cells were fixed with 4% paraformaldehyde for 15 min. After washing in PBS, cells were incubated with 3% BSA  in PBS with 0.2% Triton-X-100  for 30 min. Cells were then incubated overnight at 4 °C with a polyclonal rabbit anti-Tomm20 antibody  diluted 1:200 in PBS containing 1% BSA. After PBS washes, cells were treated with anti-rabbit Alexa Fluor 405  secondary antibody diluted 1:200 in PBS containing 1% BSA. Cells were washed with PBS and imaged shortly after to visualize colocalization of MitoTimer with Tomm20 staining.To quantify green and red fluorescence intensity per cell in MEFs and COS cells, cells were imaged using the Celigo Imaging Cell Cytometer (Brooks Life Science Systems). Green  and red  fluorescence channels were imaged for each well.For subcellular imaging of MitoTimer expression, MEF, INS1 cells and primary hippocampal neurons were imaged by confocal microscope  with a 63× oil immersion objective. MitoTimer was excited with 488 nm argon. Green fluorescence emission was collected in the 497–531 nm range while red fluorescence emission was collected in the 583–695 nm range. We observed that the red fluorescence will bleach faster than the green during multiple acquisitions. To control for possible bleaching during multiple acquisitions, cells were imaged multiple times with the laser settings used for experiments. Resulting images were then analyzed for the fluorescence intensity of red, green, and the red/green ratio in order to verify that successive imaging of the cells did not bleach either the green or red fluorescence. To observe individual mitochondria, z-stack images were acquired in series of six slices ranging in thickness from 0.5 to 0.8 µm. Tile imaging was performed to acquire the complete arborization of neurons, typically yielding 1–2 neurites per neuron used for analysis. Data for comparisons in neurites of control, RHOT1 WT, and RHOT1 KK groups were compiled from experiments done in replicate (3 < n < 5).Analysis parameters for images acquired by Celigo Imaging Cell Cytometer were optimized to identify individual MEF and COS cells based on fluorescence, and the average green and red fluorescence intensity per cell was determined at the different time points assessed. Specifically, the “Target 1+2 Merge” application setting in the Celigo software was used to determine the average green and red fluorescence for each cell selected based on the analysis parameters. Analysis settings were determined to identify fluorescent cells distinguishable from background fluorescence of cells not treated with doxycycline (no MitoTimer expression). Exposure and analysis settings were kept constant for each time-course experiment. Average fluorescence intensity per cell values were determined by the average integrated intensity per cell values in order to exclude error from background pixels included in identified cell regions. At least 1,000 cells were analyzed per well, with 3 well replicates per experiment.To characterize MitoTimer fluorescence in individual mitochondria, a ratiometric image analysis approach was performed. An average of 8–12 cells was analyzed per experiment with experiments repeated at least 3 times (n = 3). Image processing and analysis was done with Metamorph software (Molecular Devices). Red and green images were used to generate a ratio (red/green or green/red) image. A maximum intensity projection of original z-stack images was generated to visualize the entire mitochondrial volume. Minimum thresholding for mitochondrial areas was set at 15% of the maximum pixel intensity for red images. Prior to analysis, all images were scanned to verify that all intensity measurements were below saturation; therefore an upper threshold was not necessary. The thresholded images were then binarized and single pixels removed. The identified mitochondrial areas were then applied to the green image, and a ratio image was generated by dividing the red by the green image (or green divided by red for green/red ratio images). Pseudocoloring was used to visualize the variation in red/green ratio among mitochondria. Fluorescence intensity (red/green) values of identified mitochondria were then determined to assess the differences in MitoTimer fluorescence among mitochondria in MEF cells or primary neurons, thus generating an age profile.Additional material


Climate in part determines species’ distributions, and species’ distributions are shifting in response to climate change. Strong correlations between the magnitude of temperature changes and the extent of range shifts point to warming temperatures as the single most influential factor causing shifts in species’ distributions species. However, other abiotic and biotic factors may alter or even reverse these patterns. The importance of temperature relative to these other factors can be evaluated by examining range shifts of the same species in different geographic areas. When the same species experience warming in different geographic areas, the extent to which they show range shifts that are similar in direction and magnitude is a measure of temperature’s importance. We analyzed published studies to identify species that have documented range shifts in separate areas. For 273 species of plants, birds, mammals, and marine invertebrates with range shifts measured in multiple geographic areas, 42-50% show inconsistency in the direction of their range shifts, despite experiencing similar warming trends. Inconsistency of within-species range shifts highlights how biotic interactions and local, non-thermal abiotic conditions may often supersede the direct physiological effects of temperature. Assemblages show consistent responses to climate change, but this predictability does not appear to extend to species considered individually. Climate change has altered ecological phenomena across the globe ,2]. Met. Met2]. 2 = 0.59 and r2 = 0.37 for latitudinal and elevational range shifts, respectively . As . As 5]. We conducted a literature search to identify studies documenting climate-induced range shifts. We based our search on papers cited within two previous meta-analyses ,4] or a or a4] o-1 for elevational shifts and km*year-1 for latitudinal shifts ),2 analyses to test this idea. In three out of four χ2 analyses, the observed within-species consistency was more supportive of the null expectation that species respond independently between areas  group of species that are representative of climate-induced range shifts in general. Similarly, publication bias is unlikely to be influential in this analysis. We excluded studies that published single species accounts, as these are more likely to selectively focus on exceptional responses. Moreover, our analysis could only be done for species whose distributions had been studied in earlier time periods meaning the selection of which species are included was set by ecologists working several decades ago, before climate-induced range shifts were a consideration.In any analysis of published literature, there is concern that bias in the selection of studies can shape the trends observed. However, it is unlikely that selective publication or inclusion of studies is influencing the trends we measured. When range shifts are considered across species, the proportion of uphill or poleward range shifts closely matches previous estimates ,25]. Th. Th25]. 2 test indicates the populations appear to be much less predictable than assemblages. This implies that rising temperatures act as a force pushing most species to track temperatures, although which species do so in an area is strongly influenced by non-thermal factors.We interpret the collective results as evidence that temperature is sufficient to predict mean range shifts across an assemblage of species but insufficient to explain the results at the level of individual species. Each method of analyzing range shifts within species produced similar results. At the broadest level, the direction species moved was frequently inconsistent. The magnitude of a population’s range shift in one area was a poor predictor of the magnitude in another area, and even for the subset of species shifting in the same direction, there was about an order of magnitude difference in the response. The χImportantly, the results we observed do not contradict previous meta-analyses: most populations (61%) in our study shifted in the direction expected from warming, which agrees well with estimates from other analyses , 25]. A. A25]. AIf species shift their distributions one way in one region and shift their distributions in another direction in a separate region, what is the effect on their rangewide distribution? Presently, there is little evidence to suggest changes in geographic distribution in a single area could be used to extrapolate to a species’ entire range. Studies documenting changes in distribution throughout a species’ entire range are rare . C). C38]).st century may be less variable than projections of precipitation trends. Locally-refined estimates of climate change and incorporation of biotic interactions will produce better predictive frameworks but will require more effort and cost to collect the necessary data ..st centuS1 File(DOCX)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S7 Fig(TIFF)Click here for additional data file.S8 Fig(TIFF)Click here for additional data file.S9 Fig(TIFF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.S11 Fig(TIFF)Click here for additional data file.S12 Fig(TIFF)Click here for additional data file.S13 Fig(TIFF)Click here for additional data file.S14 Fig(TIFF)Click here for additional data file.S15 Fig(TIFF)Click here for additional data file.


To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT).Subcutaneous fat biopsies were obtained before, at 6- and 18–24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis.There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18–24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18–24 months (adjusted p<0.05) and focal adhesions and tight junction at 6 months (p<0.5). Genes controlling leukocyte transendothelial migration (p<0.05) and ECM-receptor interactions (p = 0.04) were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18–24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF.After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens. Antiviral therapies for HIV infection have been associated with abnormalities in serum lipids, an increWe have previously reported changes in the expression of genes controlling lipid metabolism and mitochondrial respiratory chain in biopsies taken from patients 6 and 18–24 months after starting antiretroviral regimens containing different nucleoside analogue regimens combined with efavirenz. ,20 Here Details of the patients have been previously reported and the design of the study is summarised in 10 (IRQ) were 5.1 (4.4–5.7), 4.6 (4.2–5.3) and 4.5 (4.4–4.9) respectively for AZT, TDF and ABC. All but two patients (both on AZT) had undetectable viral load when tested at 6 month following treatment  but all had undetectable viral loads at 18–24 m. Both admitted to poor compliance. None developed any clinical evidence of lipodystrophy during the period of study and post treatment weight, BMI, DEXA and abdominal CT results were not significantly different between groups at both time points after treatment. Serum lipids, fasting glucose and other parameters were similar between groups at each time point.[In the second non-randomised part of the study patients who were on their initial antiretroviral regimen containing abacavir (ABC) and 3TC in fixed-drug combination plus EFV for 6 months (n = 8) or 18–24 months (n = 11) were also tested in a similar way. Patients and controls from both parts of the study were matched for age, ethnicity, gender, weight, and BMI. None had ever had an AIDS defining disease. All three HIV groups were also matched with regards to pre-treatment CD4 count, viral load ,20 and fme point.,20Samples for microarray were available from 103 biopsy specimens . There warrayQualityMetrics package.[Agi4x44PreProcess package with options to use the AFE Processed Signal as foreground signal and BG Used signal as background adjusted signal.[quantile method using the limma function normalize Between Arrays.[Agi4x44PreProcess filter method using AFE provided flags to identify features with quantification errors of the signal, reducing the 45,015 features on the Agilent Whole Human Genome Microarray 4×44K array by 34%, or 15,339 features. Residual technical batch effects were corrected using the ComBat method implemented in the SVA R package.[RNA was extracted for microarray and array data generated using a Qiagen RNeasy Lipid Tissue Mini Kit. Total RNA was converted into labeled cDNA using the WT-Ovation Pico RNA Amplification System and samples were hybridized to an Agilent Whole Human Genome Microarray 4×44K . The Agilent arrays were scanned with the Agilent Technologies DNA Microarray Scanner G2505C with default settings. Scanned images were extracted and initial quality control performed in Agilent Feature Extraction (AFE) software version 10.7.3.1. Additional quality assessment was performed using the R  package. As no mad signal. The datan Arrays. Probeset package.Since patients were not randomised to the ABC treatment all samples were treated as unrelated. Gene expression differences between treatment arms were assessed using the linear modeling features implemented in the limma package in R. MultiplThe study was approved by the South Birmingham Research Ethics Committee (Reference 05/Q2707/40) and the Medicines and Healthcare Products Regulatory Agency—MHRA (Eudract number 2005/004021-26). All patients signed an informed consent to participate in the study.Multi-dimensional scaling (MDS) on all 103 samples was performed using profile level gene expression data from individual samples to see if subjects group according to treatment, time points or both. Distances were plotted using the limma plotMDS function with default parameters. Along the first dimension there was a clear separation of samples by samples time whereas in the second dimension the greatest distance was between the ABC treatment groups (6 m and 18–24 m) versus the other treatment groups including the untreated patients and the controls .Groups of differentially expressed genes were next generated using the R/Bioconductor limma library. Statistical criteria for identification of differentially expressed genes were FDR adjusted P* < 0.01 and fold change greater than 2.0. The cross sectional comparisons GOStats . Statistical significance of gene set enrichment was performed using the Benjamini-Hochberg correction method. Selected KEGG pathways showing over-representation in the three treatment groups compared to the controls are shown in the  terms and KEGG terms using the conditional hypergeometric test algorithm implemented in R package n in the .Adherence junction was significantly enriched compared to controls in the ABC group at both time points but not in the other two treatment groups. Six months after treatment 24 genes were up-regulated and 8 genes were down regulated compared to controls in the ABC group (<0.01). At 18–24 months after treatment the corresponding figures were 22 up-regulated and 7 down regulated (p<0.01) Individual adherence junction genes significantly up-regulated or down-regulated in the ABC treatment arm compared to controls are tabulated in Focal adhesions and tight junctions were also significantly enriched compared to the controls in the ABC treated group at 6 months but not in the other treatment groups. GO terms significantly enriched with ABC but not the other treatment groups at 6 months after treatment include cell adhesion , apical junction assembly , cell-cell junction assembly , cell-cell junction organisation , and cell matrix adhesion . At 18–24 months Go terms significantly enriched with ABC treatment alone include cell-cell—junction assembly , cell-cell signaling  and for ABC and TDF signaling .We next performed enrichment analysis using statistical list of DEG from contrast of the three different treatment regimens at 6 and 18–24 months (excluding the HIV negative samples) in a cross sectional analysis. A Venn analysis was performed for each time point and Volcano plots were created filtering by fold change >1.5 and Benjamini-Hochberg corrected p-value <0.05 at 6 months and 18–24 months .Six months after treatment enrichment of gene pathways involved with WNT signalling and leukocyte transendothelial migration were seen in ABC compared to patients receiving TDF and neuroactive ligand receptor interaction, leukocyte transendothelial migration and cytokine-cytokine receptor interactions compared to patients receiving AZT .KEGG pathways significantly over expressed when ABC was compared with the combined other two treatment groups at six months  included ECM receptor interaction, leukocyte transendothelial migration, tight junction, focal adhesion, WNT signalling, and cell adhesions (p<0.02).Go terms over represented in ABC compared to AZT treatments at 6 months include signalling , cell-cell signalling . Most of the above pathways are involved with cell processes and cell communication .The differences in the expression of genes relating to tight junctions and gap junction were particularly apparent between ABC and TDF at 6 m. Compared to TDF, 27 ABC genes controlling tight junctions were significantly up-regulated and 10 down-regulated (p<0.01). For gap junction genes the corresponding numbers were 19 up-regulated and 8 down regulated (p = 0.01). Similarly at 6 months compared to TDF and AZT 12 and 13 ABC genes respectively controlling AJ were up-regulated and 8 and 6 genes down-regulated (p<0.01) while at 18–24 months only one gene in each treatment groups was significantly up-regulated and none down-regulated.At 18–24 months there were insufficient genes for a KEGG pathways or GO term gene enrichment analysis.Enrichment analysis was next performed using statistical list of DEG in the three treatment groups and HIV infected treatment naïve subjects. The AZT and TDF pre-treatment samples were combined (n = 31) to form the HIV treatment naive group. These were then contrasted with the three treatment groups at 6 m and 18–26 months using limma R/Bioconductor limma library. A Venn analysis was performed for each time point and the unique gene sets expressed were tested for over representation analysis of KEGG pathways and GO-terms using the standard Hypergeometric test.Significant number of genes were only available for a comparions between treatment groups six months after treatment, but not at the later time point. There was enrichment of the MAPK signalling pathway for ABC group compared to HIV treatment naïve patients (p<0.05) which was not seen with the other two groups. None of the pathways relating to environmental processing or signal transduction was enriched in the TDF and AZT treatment groups.In view of the findings showing enrichment of pathways relating to endothelial function we examined a number of genes relating to endothelial adhesion molecules some of which that had previously been reported to be affected by HIV and its treatment as well as some genes involved with tight junction, adherence junction, leukocyte transendothelial migration, and cytokine-cytokine receptor interaction. As can be seen  there waMoreover, when we performed enrichment analysis against the GO term ‘biological adhesions’ (GO:22610) compared to ABC six months after treatment, of the 39 genes expressed, 21 genes in the TDF group and 12 genes in the AZT group were either significantly up-or down regulated (p<0.05). There was no significant difference when AZT and TDF were compared at 6 months. However at 18 m the three regimens had converged such that significant enrichment was only seen in two genes for TDF and one gene for AZT compared to ABC, with the two latter treatment groups again showing no difference when compared against one another. A similar difference was observed when the three treatment groups were compared with controls with 29 genes in GO term ‘biological adhesions’ registered as significant at 6 months for ABC against none for the other two groups while the number of genes being significantly enriched at 18–24 months against the treatment naïve patients were 27, 17 and 15 for ABC, TDF and AZT respectively. A selected list of GO terms relating to cell adhesion comparing controls with the three treatment groups as well as between group comparisons are shown in A complete list of Kegg pathways that are enriched for all three treatment groups at different times after initiating treatment compared to controls is shown on In order to validate the microarray study we looked at genes that we had previously shown to be significantly up or down-regulated in the adipose tissue by real time PCR on the same subcutaneous fat samples. ,20 Thus Biological validation also comes from the observation that KEGG pathway enrichment was observed for fatty acid metabolism , glycolysis and gluconeogenesis , and steroid hormone biosynthesis  at six months. At 18–24 months after treatment PPAR signalling pathway  and biosynthesis of fatty acids  were enriched in all three treatment groups, an observation in keeping with return to health reported in many studies, and the observed increase in limb and central fat in the first 24 weeks of antiviral therapy. The consIn this study we generated microarray data from mRNA prepared from subcutaneous fat samples from HIV negative controls, treatment naïve HIV infected individuals, and after 6 months and 18–24 months into their initial antiretroviral treatment. The regimens contained AZT, TDF or ABC in combination tablets with lamivudine or emtricitabine. All three regimens also contained efavirenz. The patients were matched for age, ethnicity, sex and pre-treatment CD4 count and viral loads. None had experienced any AIDS defining conditions.We have shown that at all time points patients receiving ABC displayed a markedly different pattern of gene expression within subcutaneous adipose tissue, with a large percent of the overall variance accounted for by ABC compared to the other two treatment groups. The differences were particularly marked at 6 months after treatment initiation but were still evident at 18–24 months. After 6 months of treatment, 4185 genes showed significant differentially expressed sequences (DET) in patients receiving ABC relative to the controls while the number of DETs in the AZT and the TDF group were 895 and 381 respectively. After 18–24 months of treatment the differences between the three groups had narrowed. We next examined the specific pathways over expressed in the three treatment groups compared to controls and to treatment naïve HIV-infected patients. All groups showed an enrichment of PPAR signalling and biosynthesis of fatty acids in agreement with previous studies of the effect of antiretroviral therapy ,20,29,30With the greatly increase survival observed with the use of combination anti-retroviral therapy long term complications of such therapies have assumed increasing importance. HIV itself  and antiPossible mechanisms for the cardiovascular effects of ABC include an association with inflammatory markers,–43 abnorThe observation that the increased risk of cardiovascular events was only seen with recent or current use of ABC,,14,18,41Pathways involved with environmental information processing  tended to be either enriched earlier in ABC  or were Cell adhesion molecules (CAMs) ,33 and iin vitro and in vivo[De Pablo et al. reported that ABC significantly induces leucocyte accumulation through activation of MAC-1 which reacts with its endothelial ligand ICAM-1 both d in vivo,61. The Our study does have several limitations. Patients receiving AZT and TDF were randomised while those on ABC were only matched as to gender and ethnicity. While there was no statistical difference in weight (pre-treatment and at test), BMI, CD4 count, smoking history, and family history of diabetes and cardiovascular disease between the three groups, an unintended bias cannot be excluded. Moreover the number of subjects in each treatment group was necessarily small, and not all subcutaneous fat samples were available for study, further limiting the conclusions. However we adjusted for multiple comparisons by using the Benjamini-Hochberg False Discovery Rate (FDR) as well as Bonferroni multiple testing corrections. Finally as discussed above any extrapolation for gene expression from samples obtained from one tissue must only be guardedly extended to the whole subject, not least because we were unable to comment on post-transcriptional and regulation or post-translational modifications of gene expression.Our study has highlighted novel mechanisms by which ABC may interfere with endothelial adhesion process and endothelial function in subcutaneous adipose tissue, particularly early in the treatment. In particular abnormalities in tight junction and adherence junction brought about by ABC use, if observed in other tissues, may be a contributing factor to cardiovascular events in persons already with some underlying risk for atherosclerosis, and may