TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and ‘skimming plane’ imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca The advent of total internal reflection fluorescence (TIRF) microscopy has greatly facilitated the imaging of cellular structures and processes in or very close to the plasma membrane . In esse2+ indicator dyes for fast dynamic imaging of local cytosolic Ca2+ transients generated by ion channels in the plasma membrane [TIRF works by directing excitation light through a glass substrate toward an aqueous specimen at a sufficiently shallow angle so that total internal reflection occurs due to the refractive index decrease at the glass–water interface. However, a very thin electromagnetic field (evanescent wave) is created in the liquid with the same wavelength as the incident light, which decays exponentially with distance from the interface  ,3. This membrane ,5, and bmembrane .2+ signals, this is typically done by taking a ratio of stimulus-evoked Ca2+-dependent changes in fluorescence at a pixel relative to the mean resting fluorescence at that pixel. Nevertheless, this normalization is imprecise, and cannot correct for temporal fluctuations resulting from factors including organelle movement, mechanical drift and vibrations, and ongoing, constitutively active changes in fluorescence. Moreover, uneven illumination also introduces disparities in noise levels between neighboring cellular regions, which cannot be corrected by normalization.TIRF microscopy is typically implemented by introducing a laser beam focused to a single spot at the back focal plane of a specialized, high numerical aperture (NA) objective lens . The ang2+ signals in cultured cells and two-color imaging of the relocalization of STIM and Orai proteins during activation of store-operated Ca2+ entry.Several designs have been published which mitigate these problems and produce a more even illumination field by either introducing light through a continuous annular ring at the back focal plane of the TIRF objective lens ,10, or bThe optical scheme  is desigx and y directions. Moreover the ratio between amplitudes of the sine and cosine waves can be further adjusted to alter the ellipticity of the scan (ratio of width to height), and their respective phase  so as to compensate for any astigmatism in the optics and differences in sensitivity and phase lag between the galvanometer systems. Once a setting is adjusted, a lookup table for a sine and cosine wave with user defined frequencies and 1MHz sample rates is generated and stored on the DAC. In addition to the analog outputs controlling the galvanometer scanners, the software also provides TTL (on/off) and analog (variable power) outputs to control two different lasers (488 nm and 532 nm DPSS lasers in our system) to enable two-color imaging with fluorophores of differing excitation spectra. With the exception of the STIM/Orai relocalization experiment, all data presented here were obtained using 488 nm excitation. All of these settings can be saved as presets, and can be recalled by clicking one of three individual buttons that can be configured to achieve different fluorescence excitation modes: for example, shadowless TIRF ; regular TIRF (with the laser spot at any stationary position within this annulus); ‘skimming-plane’ illumination ; or pseudo-widefield illumination . Moreover, the settings from up to three presets can be interleaved, frame by frame, to accomplish near-simultaneous imaging by different modalities (e.g. TIRF and pseudo-widefield), and/or excitation of different fluorophores by different laser lines.The galvanometers are driven by analog signals generated by a National Instruments DAC PCI-6733 card in a PC running Windows 7. A custom program developed in the open source programming language Python enables control via a graphical user interface . A maste2+ signals, and a Cascade 650 to provide greater pixel resolution for slower (500 ms exposure time) imaging of STIM/Orai dynamics. By utilizing a TTL pulse generated by the scanner software to initiate acquisition by the camera, each frame represents an average across all 360° azimuthal incidence angles, producing the effect of a spatially and temporally uniform field of illumination. High resolution and color images  cameras  were used for acquisition of dynamic fluorescence signals; a Cascade 128 for fast (5 ms exposure time) imaging of Cages Figs  were obtwww.micro-manager.org) would be equally applicable. Processing and analysis was facilitated by a custom software package written in Python (0) image stacks where the value of each pixel is normalized relative to the average value calculated from a specified range of resting frames acquired before stimulation. Duplicate regions of interest can then be defined on multiple image stacks, which are linked so that movement of one region is mirrored by its paired region on the other stack. A window displays superimposed traces over time of average pixel intensities within the paired regions, and these traces can be saved as ASCII files for subsequent analysis and plotting.Images from the Cascade emCCD cameras were acquired using MetaMorph , but other software, including open-source packages such as Micro˗Manager  of the laser spot.To assist in alignment, we use a test specimen made from an aqueous solution of fluorescein (2 μM) and rhodamine (1 μM) in a culture dish with a cover glass as its base (Mattek). The fluorescein remains in free solution, while the rhodamine quickly adheres as a thin film to the coverglass. Thus, when illuminated by wide-field epifluorescence a diffuse green glow is seen through the eyepiece as the laser light excites fluorescein throughout the bulk (a few mm deep) solution. In contrast, when adjusted for TIRF illumination, the film of rhodamine is selectively excited, producing a distinct orange fluorescence outlining the image of the scan mirror. When correctly aligned, displacement of a stationary (non-scanning) laser spot toward the edges of the objective aperture in both Correct alignment of the spinning-spot is most easily accomplished by use of a Bertrand lens (‘centering telescope’) that allows an image of the back focal plane of the objective to be viewed through the microscope. The microscope should be focused on the fluorescein/rhodamine test dish described above. With the scan radius set well below that giving TIRF illumination, a green circle will be apparent when viewed through the Bertrand lens , left. ANote that the images in 2+ puffs were as described [2+ indicator Cal-520, caged iIP3 and the slow Ca2+ buffer EGTA by incubation with membrane permeant esters of these compounds. Cells were imaged in a Ca2+-free medium at room temperature, and Ca2+ puffs were evoked by photoreleasing iIP3 by flashes of UV light focused uniformly throughout the imaging field. Images of tubulin in 2 and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum (Omega scientific) and 2 mM L-glutamine (Sigma-Aldrich). HEK cells were transfected with Lipofectamine 2000 (Invitrogen) as directed by the manufacturer using 2.6 μg mCherry-STIM1 and 1.3 μg EGFP-Orai1 per well of a 6-well petri dish. Cells were split the following day into 35 mm MatTek dishes with integrated coverslip bottoms coated with poly L-lysine  and were imaged at room temperature in Ringer’s solution on the second day after transfection. Calcium store depletion was induced by addition of 2 μM thapsigargin . The plasmid mCherry-STIM1 was constructed using an Infusion kit (Clontech) based cloning approach. Starting with the plasmid YFP-STIM1 [SH-SY5Y, COS-7 and HEK-293 cells were obtained from ATCC. Procedures for imaging Caescribed . In brieFP-STIM1 , the yel2+ signals, it is important that fluctuations in fluorescence excitation are minimal so that they contribute little noise above the irreducible limit imposed by photon shot noise. For dynamic measurements, such as Ca2+ puffs are localized subcellular Ca2+ signals evoked by Ca2+ liberation from the ER through inositol trisphosphate (IP3) receptors. They have durations of several tens to a few hundred ms, so that imaging frame rates of hundreds of Hz are required to resolve kinetic details during individual puffs. We had previously described that TIRF imaging provides enhanced resolution of puffs arising at sites near the plasma membrane [Camembrane , but the2+ indicator Cal-520 together with caged iIP3 and the slow Ca2+ buffer EGTA [3 by a UV light flash delivered within the imaging field then evoked Ca2+ release from the ER through IP3 receptors, which were apparent as transient localized events (puffs) arising discrete sites because the EGTA suppressed Ca2+-induced Ca2+ release between adjacent clusters (0) from three puff sites in We imaged puffs in SH-SY5Y neuroblastoma cells loaded with the fluorescent Cafer EGTA . Cells iclusters . A maximclusters . RepreseA particular advantage of utilizing computer-controlled galvanometer mirrors to scan the laser spot is that effectively simultaneous image sequences can be captured in different excitation modes by alternating exposures with different scan settings. One example of this is illustrated in 2+ puffs. Images were acquired as in left) and in spinning spot TIRF mode (right). The resting, background fluorescence of cells imaged by spinning spot TIRF was spatially quite uniform, but was highly irregular with stationary spot excitation , maximum value projections of ΔF/F0 ratio images remained less clearly defined and failed to reveal some events captured in ΔF/F0 projections from spinning spot illumination  modes.We had previously described the benefit of TIRF imaging for high fidelity recording of Caan cells ,13. Howeo) in TIRF mode, whereas the lower trace illustrates a site where greater amplitudes were obtained in WF mode. We interpret the ratio of puff amplitudes obtained for each event in TIRF/WF modes as reflecting the axial location where that event arose. For example, TIRF imaging would show a small signal from a deep site because fluorescence excited in the evanescent field would be limited to the small fraction of released Ca2+ that diffused near the plasma membrane. vs. WF imaging from 189 events. This shows a broad distribution, with roughly equal numbers of puffs showing amplitudes greater in TIRF than WF (ratio >1) and vice versa (ratios <1). The variability in ratios arises primarily from differences between puff sites, rather than variability among successive puffs recorded at a given site  were acquired in TIRF mode with 488 nm excitation , in TIRF mode with 532 nm excitation , and in skimming-plane mode with 532 nm excitation . Representative images, taken from wo cells , but redwo cells . At restwo cells ). After wo cells . The TIRwo cells .TIRF microscopy has become the technique of choice for cellular imaging of near-membrane structures and dynamic processes ,16. In c2+ signaling. Moreover, it can be readily constructed using commercially available, off-the-shelf components, and we make freely available custom software based on open source platforms for both controlling the scan mirrors, laser excitation and image acquisition and analysis software for de-interlacing and processing image data acquired with interleaved mode switching.Filling an annular ring around the objective lens’ back focal plane to achieve substantially uniform illumination can mitigate this deleterious effect. Various approaches have been described to achieve ‘shadowless’ TIRF in this way, including an annular mask to block laser light from all but a circumferential ring ; by usin2+ puff sites within neuroblastoma cells. We find that sites are distributed over a range of distances from the plasma membrane, with roughly one half of sites generating fluorescence signals that are larger in TIRF than in WF mode. This complicates analysis of puff signals recorded exclusively by TIRF. We had previously assumed a fixed ‘yardstick’ measure of the fluorescence corresponding to the opening of a single IP3R channel by which to estimate how many channels opened during any given puff [A particular feature is that the ability to rapidly alternate the scanning mode enables depth information to be obtained by altering the scan radius to achieve different penetration depths of the evanescent field; or to alternate between TIRF excitation and ‘skimming plane’ or wide-field conical epifluorescence excitation to achieve effectively simultaneous imaging in each mode. Here, we applied this capability to investigate the axial distribution of Caven puff , whereas2+ imaging, for which a fast acquisition rate and corresponding scan speed are essential, it would be equally applicable for imaging many other near-membrane cellular processes such as exocytosis, lipid raft dynamics, and protein diffusion and trafficking.In summary, we describe a simple and inexpensive TIRF illumination system that provides much more even illumination than conventional stationary-spot TIRF systems, and moreover enables fast, near simultaneous imaging in both TIRF and wide-field modes. Although our system was designed primarily for dynamic CaS1 FigThe circular scan of the laser spot is achieved by two galvanometer mirrors driven by sine and cosine voltage waveforms generated by a National Instruments DAC card. The DAC signals are controlled using the GUI, written in Python. The frequency slider controls the frequency of the sine and cosine waves (i.e. the period of a complete circular scan). The radius slider controls the amplitude of both sine (x-deflection) and cosine (y deflection) waves. The ellipticity and phase sliders alter the relative amplitudes and phases of the two signals, respectively. The x-shift and y-shift sliders control the zero-offset of each signal (i.e. the center of the circular scan). The software further enables control of the on/off state and intensity of the lasers. Up to three different settings can be saved and recalled with the buttons in the lower panel. The GUI can alternate between up to three of these different settings during sequential cycles of the circular scan, enabling near simultaneous imaging in multiplexed modes.(TIF)Click here for additional data file.S2 Fig0) images with frames acquired alternately in TIRF and WF modes. Both image stacks can be scrolled in synchrony. Custom regions of interest (ROIs) can be drawn on one stack and mirrored in another, such that moving and resizing one produces the same changes in the other. (D) The average fluorescence intensity over time within mirrored regions of interest in each image stack can be overlaid and plotted in different colors to facilitate comparison. These traces can be exported into ASCII files.The screengrabs show the user interface for custom software used to process and analyze images. The software (FLIKA) was written in Python and can be run in the Linux, OSX, or Windows operating systems. (A) Image stacks saved in MetaMorph.stk file format or multi-plane TIFF format can be loaded and displayed. The user interface enables black level subtraction and pixel-by-pixel normalization relative to average baseline fluorescence, and de-interleaving. (B) The de-interleave window enables separation image stacks interleaving 2 or 3 imaging modalities into separate stacks. (C) Illustration of two de-interleaved image stacks, showing ratio (ΔF/F(TIF)Click here for additional data file.S1 Movieleft; TIRF, right) after averaging 2 sequential frames. Three stationary microspheres had adhered to the coverglass, and others can be seen undergoing Brownian motion in the water. Image is 51 x 51 μm.Aqueous suspension of fluorescent microspheres (0.5 μm diameter) imaged by alternating shadowless TIRF and widefield excitation. Images were acquired by alternating 5 ms exposures in each mode. The montage shows de-interlaced images Click here for additional data file.S2 Movie2+ and induce STIM and Orai relocalization. Each panel is 81 x 61 μm.Images in each panel of the video show EGFP-tagged Orai visualized by shadowless TIRF excitation at 488 nm (top left); mCherry-tagged STIM visualized by TIRF excitation at 532 nm (top right); mCherry-tagged STIM visualized by skimming plane (SP) excitation at 532 nm (bottom left); and a pseudocolored overlay  of these three images. The imaging modes were alternated with 100 ms exposures in each mode. Thapsigargin was applied shortly after the beginning of the record to deplete ER Ca(MP4)Click here for additional data file.S1 Supporting Information(DOCX)Click here for additional data file.


Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes  within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA , and Western blot or immunostaining  showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis.Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of  Despite several advances in ocular surgeries, including sutureless and minimally invasive procedures, infectious endophthalmitis remains one of the most concerning postsurgical complications due to its ability to cause blindness12468Staphylococcus epidermidis exhibit mild acute inflammatory response and the infection is resolved quickly, with restoration of visual acuity close to normal1213Bacillus cereus151617S. aureus319202122The visual outcome following endophthalmitis largely depends upon the virulence potential of the infecting pathogen and the dynamic interplay between the host and the pathogen10One of the cardinal steps in initiating an innate immune response is pathogen recognition by specific receptors present on innate immune cells19202829302193132Principal component analysis (PCA) demonstrated that 12 h and 24 h post-infection samples are separated from controls and 3 h post-infection samples along Principal Component 1 (PC1), which accounts for 65.7% of the variance in transcriptome data. This demonstrates that transcriptional differences were greater at later time points  of SA infection, as compared to an early time point, 3 h post-infection, and compared to uninfected controls. This also suggests that SA infection initially results in a potent minimal response, manifesting as an early qualitative and quantitative changes in gene transcription, which grows and spreads exponentially over time. The analysis also revealed distinctive clusters of samples consistent with the biological groups on the basis of the transcriptional profiles .After preprocessing, we conducted group-wise comparisons using t-tests and fold change to identify differentially expressed genes at each time point after SA infection compared to control/vehicle samples. The group comparison identified the sets of genes that were significantly differentially expressed at each time point compared to vehicle and non-infection samples . All timTo identify gene expression patterns, we performed SOM analysis on 1,234 genes that were differentially expressed at multiple time points after SA infection, as identified by the group comparison analysis aboveTo gain insight into the functional consequences of altered gene expression due to SA infection, we employed the Database for Annotation, Visualization and Integrated Discovery (DAVID). The constitutively regulated genes that show significant enrichment in clusters of the gene ontology (GO) categories related to metabolism , RNA splicing, and protein ubiquitination are shown in As a complementary approach, pathway enrichment analysis was performed on genes with temporal transcriptional changes following SA infection. Pathways enrichment was assessed using Ingenuity Pathway Analysis (IPA) tools. This IPA demonstrated significant enrichment of temporally altered genes involved in numerous inflammatory response and immune system-related pathways . In keepTo further understand the molecular mechanisms of SA infection, we performed systems biology analysis on constitutively or temporally altered transcripts. The systems biology analysis was performed using a master regulator approach that assists in identifying the key transcriptional regulators that might be responsible for pathophysiology. The regulatory analysis depicted significant activation of multiple major immune and inflammation regulators, including STAT1, STAT3, IL-6, NFKB2, JUN, SPP1, IL-1β, CSF1, CXCL2, IGF1, CEBPB, and PTPN1 . The sysWe previously reported that TLR2 activation induces protective responses in SA endophthalmitis19The PAM3-mediated development of a microenvironment suitable for combatting SA infection was studied by PAM3 counter regulation analysis. SOM counter analysis was performed on 1,234 differentially expressed genes induced by SA infection to identify the effects of PAM3 treatment on these genes. Counter-regulation means that PAM3 treatment down-regulates genes that are up-regulated in SA infection vs. controls and vice versa. SOM clustering of differentially expressed transcripts post-infection to 10 bins was performed to identify transcripts that are counter regulated by PAM3 treatment. Bins of transcripts manifesting different degrees of counter regulation were identified and further filtered on the basis of magnitude of counter regulation. The final list of counter regulated genes was generated by considering genes that are counter regulated by a magnitude of fold change (FC) > 1.2–1.5 on treatment, as compared to their expression 24 h post-SA infection. The analysis identified genes that were temporally regulated by SA infection and counter regulated by PAM3 treatment . Figure The SA infection signature genes that were counter regulated by PAM3 treatment were further investigated for functional and pathway enrichment using Ingenuity Pathway Analysis (IPA) tools. The functional groups most significantly enriched related to two major categories, namely, inflammatory processes and basic cellular functions. The top inflammatory functional categories included cellular movement, inflammatory response, immune cell trafficking, immunological disease and infectious disease, and the top functions associated with general cellular functioning included cellular development, cell growth and proliferation, hematological system development and function, cell death and survival, and cellular morphology . FurtherSystems biology analysis of genes associated with the PAM3 counter regulation signature further identified the major networks formed by these genes and predicted the master regulators and there up- or down-regulation based on the expression patterns of the network genes. It is very evident from the analysis that the magnitude of differential expression and its directionality are reversed for these PAM3 counter regulation signature genes before and after PAM3 treatment . The hubTo validate the microarray data, the expression profile of all 13 master regulator genes  was confirmed by qRT PCR in both WT and TLR2 KO mouse retina. Our data revealed that TLR2 deficiency resulted in the downregulation of the hub genes . The oveEndophthalmitis is an inflammatory disease caused by intraocular microbial infections, which result in blindness in some patients. Severe vision loss in endophthalmitis is the result of retinal damage caused by pathogen derived virulence factors  and excessive host inflammatory responses352193226Salmonella, Shigella, and Pseudomonas spp., where the type III and type IV secretion systems (T3SSs and T4SSs) are particularly involved in targeting host ubiquitin systemsS. aureus produces a variety of virulence factors, including toxins, to our knowledge their role in disrupting ubiquitin pathways has not been reported. Our study, showing a constitutive dysregulation of ubiquitination pathways (e.g. UBE3A) in SA-infected retinal tissue, indicates that SA may utilize these mechanisms for survival in the eye and that the activation of specific ubiquitin mechanisms may promote bacterial clearance. Similarly, SA endophthalmitis was found to alter the expression of genes/pathways involved in cellular metabolism, including increased fatty acid synthesis and reduced energy/ATP generating signaling pathways, such as AMPK signaling  pathway, which evokes a wide range of host inflammatory responses to inhibit bacterial growthignaling . Indeed,The genes with temporal transcriptome changes demonstrated that SA infection targeted multiple pathways and biological processes that were central to immune and inflammatory responses and cellular growth and proliferation, as expected, but also revealed novel pathways impacted by SA infection, such as the glucocorticoid receptor signaling, RANK/RANKL signaling, protein ubiquitination, and Notch signaling. Pathway analysis revealed an overall activation of multiple immune and inflammation pathways, including JAK/STAT signaling, interleukin pathways , interferon signaling, and Toll-like receptor (TLR) signaling. The induced expression of genes/molecules involved in TLR signaling was expected, as extensive studies from our21920263228304246Systems biology analysis of the SA infection signature assisted in generating a global picture of the crosstalk between the pathways/genes involved in pathophysiology and to highlight the master regulators and key processes that are most critical for SA endophthalmitis. The systems biology analysis identified multiple immune system and inflammation molecules  as master regulators that are activated upon infection. On the other hand, genes linked to cell proliferation and apoptosis  emerged as the top inhibited master molecules due to infection .Similar to NF-KB signaling, JAK/STAT signaling has been studied extensively as is critical for in that it converges diverse signals from the immune system to perform multiple functions, including resisting infection, maintaining immune tolerance, and enforcing innate barrier functions505354555751Many immune system specific transcription modulators are also temporally dysregulated post-infection. The CCAAT/enhancer-binding protein beta (C/EBPβ) has been shown to bind the IL-1 response element of the IL-6 gene and several other acute-phase response genes. C/EBPβ is a member of the C/EBP subfamily of leucine zipper containing transcription factors61656769Another key master regulator identified in our study is IL-1β, a potent activator of immune responses directed against bacterial infectionsIn summary, we present a comprehensive transcriptomic analysis of retinal gene expression at the early stages of staphylococcal endophthalmitis. Our systems biology analysis revealed that 13 hub genes, including the classical inflammatory genes  which regulate retinal innate responses during infection. Moreover, Pam3 preconditioning dramatically altered the expression of these master regulators, suggesting that Pam3 is a potential candidate for the development of therapeutic modalities to prevent excessive inflammation and tissue damage in endophthalmitis. A thorough mining of our data on the basis of master regulators that are perturbed in response to infection demonstrated that key focus molecules are mostly involved in the innate immune response and that pretreatment of the host with innate immune activators increases resiliency and blunts immune system activation. Moreover, the identified key pathways can be targeted therapeutically to prevent or treat endophthalmitis.C57BL/6 and TLR2 KO mice were purchased from the Jackson Laboratory . The animals were housed in a restricted access DLAR facility at the Kresge Eye Institute, were maintained in a 12 h light to 12 h dark cycle, and fed LabDiet rodent chow  and water ad libitum. Female mice of 8–12 weeks of age were used throughout all experiments. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Wayne State University under protocol # A 08-02-13. All experimental procedures/methods were carried out in accordance with the approved guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.® solution  for downstream processing.The mice received intravitreal injections of 1.0 μl of PBS containing ~5,000 CFU of SA strain RN6390 (left eye) to induce endophthalmitis, as described previously20The mouse tissue gene expression profile was assessed at multiple time points  post-infection and PAM3 treatment using HT 430 2.0 PM Array plate  containing >45,000 probes, allowing for the analysis of >30,000 well characterized mouse genes. Expression data were preprocessed using the Robust Multichip Average (RMA) method in R using the Bioconductor and associated packagesHierarchical clustering to identify outliers and correlation among samples. Before PCA and Clustering, normalized data were preprocessed to remove transcripts with low expression for reducing false-positive results. Only transcripts with absolute expression ≥40 in at least 5% of the samples were included.The unsupervised analysis was performed using Principal Component Analysis and p values (student t-test) between control or vehicle treated samples and infected samples as the cut-off criteria to identify differentially expressed genes at each time point. The genes with an absolute fold change >1.5 and a p value <0.05 are considered to be differentially expressed. The fold change was calculated over multiple biological replicates to reduce false positive rates. It is well accepted in several studies that temporal analysis is the most effective strategy to identify the least false positive genes that are altered in a time-dependent manner to external stimulant using a low cutoff P value < 0.057576We used absolute fold changes (FC) and p value based significance of counter regulation. The final list of counter regulated genes was generated by considering genes that are counter regulated by a magnitude of fold change (FC) >1.2 compared to the degree of gene expression at 24 h after SA infection.To identify genes that are involved in the treatment of infection, upon treatment or pre-treatment with PAM3, we performed counter regulation analysis on the transcripts from the SA infection signature that are differentially expressed due to SA infection. Counter regulation means that PAM3 treatment down-regulates genes that are up-regulated in SA infection and vice versa. SOM clustering of differentially expressed transcripts to 20 bins was performed to identify transcripts that are counter regulated by treatments. Bins of transcripts manifesting different degrees of counter regulation were identified and further filtered on the basis of magnitude and p-value is assigned to each category on the basis of enrichments using jackknife Fisher exact probabilities.To identify over-represented GO categories in differentially expressed genes from different comparisons, we used the Biological Processes and Molecular Functions Enrichment Analysis available from the Database for Annotation, Visualization and Integrated Discovery (DAVID)p values using Fisher’s exact test for each pathway and functions according to the fit of the user’s data to IPA databases. The pathways with p-values < 0.05 were considered significantly affected.Ingenuity Pathway Analysis (IPA) (Qiagen) was applied to early, late, or temporal expression changes due to SA infection and/or PAM3 treatments to identify biological pathways significantly over represented at the time of infection and counter regulated by PAM3 treatment. It calculates p value) is calculated using Fisher’s exact test and indicates the likelihood that a gene will be found in a network due to random chance.In addition to individual gene analysis, interactive network analysis was performed as the genes from different significantly affected pathways and functions crosstalk to each other, often resulting in some degree of pathophysiology. In the case of interactive networks, all of the identified genes were mapped to genetic networks available in the IPA database and were ranked by the score. The score  and understand the underlying biological mechanisms. The analysis will help in identifying first which transcription regulators are significantly affected by the treatment, as well as determine whether they are activated or inhibited. The significance of overlap was determined using one-tailed Fisher’s exact test.® Mini Kit, as per the manufacturer’s instruction . Some RNA was used for the microarray analysis, and the remaining RNA was used for gene validation. cDNA was synthesized using 1.0 μg of total RNA using a Maxima first strand cDNA synthesis kit, as per the manufacturer’s instructions . Primers were purchased from Integrated DNA Technologies . PCR was performed using a StepOnePlus™ instrument . The quantification of gene expression was determined via the comparative ΔΔCT method. The expressions in the test samples were normalized to endogenous reference GAPDH levels. All assays were performed in triplicate and repeated at least twice. The data are presented as the mean ± the SD and statistical analysis were performed using one-way analysis of variance (ANOVA).Total RNA was extracted from retinal tissue using an RNeasyThe detailed methods for validation of target molecules using ELISA, Western Blotting, and Histopathology and Immunostaining are provided in our previous studies3193132How to cite this article: Rajamani, D. et al. Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis. Sci. Rep.6, 21502; doi: 10.1038/srep21502 (2016).


Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles  to 7.0±4.1 dB for the most extreme treatment condition . In parallel,  The most common method of examining treatment response is a biopsy which assesses the cellular characteristics of tumor Apoptosis, or programmed cell death, is a process characterized by distinct structural changes at various stages in vivo tumours are compared to histological sections of excised tumor samples. Spectral changes determined within a −6 dB acoustic window about the center frequency were identified to be closely associated with cell death histological changes for various treatment conditions. The results of this study demonstrated significant correlations between these ultrasound parameters and cell death resulting from vascular disruption caused by the microbubble-stimulated ultrasound and radiation treatments. Furthermore, they suggest that quantitative ultrasound imaging can be used as a sensitive tool for characterizing cell death, and is able to detect cellular changes caused by low doses of radiation enhancing agents.The study here is an addition to previous studies on quantitative ultrasound of cell death, and examines the ability to acoustically detect cell death caused by tumor exposure to ultrasound-stimulated microbubble treatments combined with radiation, and examines the extent of the sensitivity of this method. This study supports previous findings which have demonstrated that ultrasound-stimulated microbubbles trigger endothelial cell apoptosis within tumor microvasculature and facilitate radiation enhancement All animal experiments presented in this manuscript were conducted in compliance with Canadian Council on Animal Care guidelines. Experimental protocols were approved by the Committee on the Ethics of Animal Experiments of Sunnybrook Health Sciences Centre. The title of the Animal Use Protocol (AUP #: 13-395 AUP Expiry: 14 April 2014) was as follows: “Novel Ultrasound Microbubble Potentiated Radiosensitization of Tumours.”Tumor-bearing animals were prepared and handled as previously described Ultrasound-stimulated microbubble treatments were conducted as previously described in Radiation treatments were performed as previously described in vivo. Image acquisition was performed using a Vevo770,  fitted with a RMV  scanhead, 25 MHz single-element transducer (62 µm resolution) . Tumors were imaged in the hip-to-toe direction with the transducer positioned approximately 2 mm above skin surface. An average of 60 frames was acquired, per scan, with 250 lines of radiofrequency per frame and a 0.2 mm scan plane separation. For analyses ROI sizes were rectangular shaped and equivalent from sample to sample. These occupied 2/3 of the tumour diameter in width and height.Conventional high frequency ultrasound imaging was performed prior to and 24 hours after treatment. For each scan, radiofrequency data and B-mode images were obtained volumetrically for tumor specimens Spectral parametric maps were generated using in-house software. Mid-band fit values were mapped within a selected region of interest that outlines the tumor. Regions of interest were selected in a consistent manner, which covered the whole tumor, excluding skin and muscle. The analysis bandwidth ranged from 18 to 30 MHz (−6 dB bandwidth). A sliding window analysis rooted in a Hamming function was used in situ end labelling (ISEL), or cyclophilin A. Unfixed tissue samples were embedded in OCT medium and immersed in liquid nitrogen, and were then stored at −80°C and frozen sections were prepared for ceramide labelling. The immuno-staining procedure for ceramide and cyclophilin A was performed according to the immunohistochemical staining protocol of frozen sections from Relia Tech . For staining a 10× diluted mouse monoclonal primary antibody for ceramide  and 100× diluted polyclonal primary antibody  for cyclophilin A  were used with Histostain-Plus Kit  .Tumor tissue was prepared as described in Radiofrequency data were analyzed using normalized power spectra as previously described Cell death was assessed using low-magnification images of ISEL stained tumors captured under a light microscope attached to a CCD camera as previously described A 2-way ANOVA  was performed in order to test for statistical significance in each spectral parameter. The same was applied for changes in relative fraction of structurally altered nuclei representing an index of histologically detected cell death. GraphPad Prism Software Version 4  was used.In general, quantification of ultrasound data enabled the detection of changes in tumors generated by a range of ultrasound-stimulated microbubble treatment exposures with varying ultrasound intensities and radiation doses. This resulted in closely corresponding ranges of cell death. Statistical tests of significance referenced below are presented in Results demonstrated that higher radiation doses caused increased cell death, resulting in higher backscatter intensity. in vivo with elevated backscatter intensity. The increases ranged from 1.4±3.3 dB to 3.4±5.0 dB when comparing 250 kPa to 570 kPa and 750 kPa, respectively (Tumors treated with higher dose of ultrasound-stimulated microbubbles similarly demonstrated greater backscatter intensity . Mid-banectively . A “platp<0.0001). Overall, the average change in mid-band fit increased as radiation dose, microbubble concentration, or microbubble-stimulating ultrasound responses were increased . At the highest microbubble concentration and ultrasound pressure, cell death increased from 0.2 to 0.4 from 0 Gy to 8 Gy radiation, respectively. Responses appeared sparsely heterogeneous throughout tumor tissue with central core of tumors exhibiting the most response.Low magnification images of ISEL labelling demonstrated that increases in microbubble concentration and radiation dose results in increased cell death in tumors . At the High magnification images of H&E stained sections from treated and control tumors were acquired in order to examine areas of cell death . The purPrevious studies have demonstrated that ceramide primarily mediates this ultrasound-stimulated apoptotic cell death in endothelial cells Previous literature has indicated a proportional relation between the permeabilization of cells and ultrasound treatment. The higher the pressure or the lower the centre frequency of ultrasound, the more cell permeabilization can be achieved This investigation supports the finding that ultrasound-based spectral parameters, such as the MBF parameter and 0-MHz intercept, can detect changes associated with cell death consistent with previously described research in vitro and in vivo, and assessed for changes in intracellular structures In other studies, prostate cancer cells treated with chemotherapeutic agents have been subjected to ultrasound imaging, both Early permeabilization of plasma membrane was identified here using cyclophilin A immuno-stain  [31]. ThIn the study here, treatment with ultrasound-stimulated microbubbles combined with radiation was used as the primary treatment and has been identified to cause rapid cell death, in terms of structural changes within cells (24 hours). Here, destruction of tumor vasculature leading to secondary tumor cell death was the mechanism for cellular changes. In these treatments, low doses (<2 Gy) of radiation combined with ultrasound-stimulated microbubbles have exhibited comparable efficiency as high doses of radiation only (>5 Gy), linked to ceramide-mediated apoptosis in endothelial cells within tumor vasculature The study here does not address the causation of the spectral changes observed and only establishes a co-incidence of spectral changes and the development of cell death. Other research has investigated causal factors in the past and is summarized elsewhere In highly cellular xenograft tumours backscatter signals and spectra are identical to backscatter signals and spectra of centrifuged cell models that mimic the histological packing of xenografts. Such packed cell models have no extracellular matrix and collagen present yet exhibit nearly identical ultrasound scattering profiles.Different cell types may be differentiated in part on the basis of their nuclear signals. This further suggests an important role of nuclear structure in backscattered ultrasound.In cell experiments where nuclear structure is specifically modified, there are changes in backscattered ultrasound. It has been previously demonstrated that such changes specifically cause the types of backscatter changes observed. When cells are treated with colchicine to arrest them in G2/M of mitosis with condensed nuclear material there are significant increases in backscattered ultrasound. It has been demonstrated that such increases can be reversed by digesting cellular DNA through enzymatically treating such colchicine treated cells with DNAse Additionally, if one similarly subjects viable cells or mouse liver tissue to enzymatic digestions with DNAse, backscatter signals drop to at least half their pretreatment values Similarly, other experiments in which cells are treated with sodium butyrate to cause chromatin unfolding exhibit significant decreases in ultrasound backscatter.Calculated scatterer sizes from ultrasound backscatter do not work out to be the same as cell sizes but coincide with smaller sizes suggestive again of an important role of the nucleus.Isolated nuclei from apoptotic cells in comparison to viable cells exhibit greater backscatter see .It is speculated that with the treatments here which induce endothelial cell apoptosis, followed by vascular collapse, and then secondary ischemic cell death, after 12 hour necrosis starts to occur alongside apoptosis leading to swelling of organelles including the nuclei and chromatin In order to demonstrate a biological mechanism behind cell death ceramide production was assessed. Ceramide is a lipid-mediator that is often triggered by a variety of stress stimuli such as heat, reactive oxygen species, and ionizing radiation In situ end-labelling (ISEL), ceramide and cyclophilin A labelling was performed to identify regions of cell death due to DNA fragmentation, ceramide mediated apoptosis, and permeabilization of cells, respectively.In this study SCID mice with human prostate cancer xenografts were treated with different concentrations of microbubbles , with bubbles stimulated with various ultrasound pressures  and tumor tissue exposed to radiation . Each tumor was imaged with ultrasound prior to treatment and 24 h after treatment. Spectral analysis results were in agreement with immunohistochemical analysis which demonstrated that there was an increase in backscatter intensity and quantitative ultrasound parameters co-incident with cell death increases with treatment. The increase in backscatter intensity when compared to control samples ranged from 1.9±1.6 dB for the clinically-recommended dose  to 7.0±4.1 dB for the extreme treatment condition . This type of ultrasound-stimulated radiation-enhancing treatment would offer a great advantage for patients by saving time since less treatment is needed while using quantitative ultrasound to monitor tumors. The former uses ultrasound to stimulate microbubbles to destruct tumor microvasculature and enhance effects of radiation treatment while the latter uses ultrasound to detect such cell death. This preliminary investigation provides significant groundwork for developing a clinical application that benefits patients by offering both ultrasound-mediated microbubble radiosensitization with non-invasive tissue characterization.


MYOIC gene in mammalian cells encodes three isoforms . Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the  Prostate cancer is the most common cancer diagnosed in men worldwide and the second most common cause of cancer death in the US MYOIC gene in mammalian cells encodes three isoforms that are called myosin IC isoforms A, B, and C Myosin IC is a member of the myosin superfamily Our previous analysis of myosin IC isoform expression in murine tissues revealed a tissue-specific expression pattern of specifically myosin IC isoform A with high expression levels in kidney, adrenal gland, pancreas, and in epididymal and retroperitoneal adipose depots This tissue specific expression of myosin IC isoform A led us to explore the expression profiles of the myosin IC isoforms further. We report here the evaluation of myosin IC isoforms A and B expression in a variety of mammalian tissue culture cell lines. We demonstrate that expression of myosin IC isoform A is significantly increased in prostate cancer cell lines when compared to other (non-prostate) cancer cell lines or to a non-cancer prostate cell line. Subsequent analysis of prostate cancer tumor tissues from the murine TRAMP model that closely mirrors the pathogenesis of human prostate cancer Antibodies that recognize specific isoforms of myosin IC: 1. the anti-NMI antibody is a rabbit polyclonal antibody that was raised against the 16 amino acid long N-terminal peptide of NMI, here called isoform B 2.COS-7, A-431, MCF-7, TRAMP-C1, DU 145, 22Rv1, RWPE-1, and PC-3 cells were obtained from American Type Culture Collection . OVCAR-3 cells, originally obtained from ATCC, were generously provided by Dr. Arthur M. Edelman . As4.1 cells Frozen tissue samples from 22 weeks of age TRAMP mice as well as age matched wild type mice were purchased from the Mouse Tumor Model Resource at RPCI . The tissues were collected in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health under RPCI’s 890M Tumor banking protocol for the Mouse Tumor Models Resource. The protocol was approved by Roswell Park Cancer Institute’s IACUC . Animals were sacrificed by anesthesia overdose followed by cervical dislocation. All prostate and tumor tissues had been microdissected at necropsy, flash-frozen in liquid nitrogen, and stored at −80°C until use. Five-micron-thick paraffin embedded matched tissue sections were purchased from the same source.2O before undergoing standard antigen retrieval . Endogenous peroxidase activity was blocked by using 3% H2O2 prior to incubation with either myosin IC isoform A or SV40-TAg antibodies for immunohistochemistry according to standard methods. Images were obtained with a Zeiss Axio Imager Z1  and processed using Adobe Photoshop software.Paraffin embedded tissue sections were de-paraffinized with xylene, rehydrated with alcohol, and placed in dHFor preparation of crude cellular extract, an equal number of cells was lysed in SDS sample-buffer. For each cell line based analysis, three independent experiments were performed. Tissue extract was prepared as described previously homo sapiens: NM_033375.4; mus musculus: XM_006532429.1) and mouse and human myosin IC isoform B . For quantification, triplicates were normalized to the average of the GAPDH housekeeping gene. The primer used for qRT-PCR are described in Total RNA was isolated from cells or tissue samples using Trizol reagent  following the manufacturer’s instructions. 1 µg total RNA from each cell line was used to reverse transcribe into cDNA using Superscript III reverse transcriptase and oligo dT primer according to manufacturer’s instructions . qRT-PCR was performed using the iCycler iQ Real-Time PCR Detection System  and primer that recognize specifically mouse and human myosin IC isoform A  with isoform-specific primer . As showA striking observation of our analysis of myosin IC isoform expression in these cell lines are the high expression levels of myosin IC isoform A in the mouse and human prostate cancer cell lines when compared to the normal (non-cancer) prostate cell line RWPE-1 and to other (non-prostate) cancer cell lines as summarized in To explore this possibility, we analyzed myosin IC isoform A expression in various tissues from the TRAMP mouse. TRAMP is a transgenic line of C57BL/6 mice that express a construct containing the minimal −426/+28 regulatory element of the rat probasin promoter to target expression of the SV40 large T antigen to prostatic epithelium To confirm the presence of myosin IC isoform A in tissues associated with prostate cancer, we analyzed various TRAMP prostate tumor tissues that were comprised either of lateral and ventral (LV) or of dorsal, later, and ventral prostate (DLV). As shown in An analysis of myosin IC isoform A expression in prostate tissue and distant site metastases in TRAMP mice or age matched wild type mice showed that myosin IC isoform A is significantly higher expressed in prostate tumors relative to normal murine prostate, lung, or liver tissues. This is, to our knowledge, the first report documenting a change of a myosin IC isoform expression in connection to cancer. The possible biological and physiological consequences of the overexpression of myosin IC isoform A in tumor tissues are currently under investigation in our laboratory.In addition to establishing a link between myosin IC isoform A expression and cancer, our data suggest that aberrant myosin IC isoform A expression changes can be specifically linked to transcriptional changes associated with the occurrence of prostate cancer. This hypothesis is supported by several observations. Our previous analysis of myosin IC isoform A expression in mouse tissue samples The TRAMP prostate cancer model is based on the prostate-specific expression of the SV40-TAg oncoprotein While the expression pattern of myosin IA isoform A in human and mouse prostate cancer needs to be analyzed further, our results here, that show an overexpression in human and mouse prostate cancer cells lines and in prostate cancer associated murine tissues, strongly suggest that specifically myosin IC isoform A could be a possible diagnostic marker for detection of prostate cancer.


There were no significant differences in age, height, weight, and resting blood pressure between cancer survivors and control participants. Brachial artery FMD was similar between groups (P = 0.98). During exercise at the VT, TOI was similar between groups, but [Hb]total and [HHb] were significantly decreased in cancer survivors compared to controls (P < 0.01) The rate of change for TOI 2 extraction, 2 delivery during exercise.The cardiotoxic effects of adjuvant cancer treatments  have been well documented, but the effects on peripheral cardiovascular function are still unclear. We hypothesized that cancer survivors i) would have decreased resting endothelial function; and ii) altered muscle deoxygenation response during moderate intensity cycling exercise compared to cancer-free controls. A total of 8 cancer survivors (~70 months post-treatment) and 9 healthy controls completed a brachial artery FMD test, an index of endothelial-dependent dilation, followed by an incremental exercise test up to the ventilatory threshold (VT) on a cycle ergometer during which pulmonary  Numerous types of cancer are frequently treated with chemotherapy or a combination of chemotherapy and radiation. While these treatment regimens have contributed in part to increased cancer survival rates , their u2 transport pathway . The stimulus eliciting brachial artery dilation following cuff deflation was calculated as the area under the shear rate curve (AUCSR) determined using the trapezoidal rule . FMD was calculated as the highest absolute (mmΔ) and relative (%Δ) mean average 3-s diameter following cuff release in peak brachial artery diameter from the preceding baseline diameter. The baseline and post-occlusion time-averaged mean velocity (in centimeters per second) values over each 3 s contraction cycle were calculated on the ultrasound system using the manufacturer’s on-screen software. The binned diameter and velocity data were time aligned and used to calculate shear rate ) and deoxygenated ([HHb]) concentrations. The NIRS probe was placed longitudinally on the belly of the right m. vastus lateralis and secured using a cohesive bandage. Location of the m. vastus lateralis was confirmed manually with palpation during active knee extension and visually via 2D ultrasound. The depth of the muscle was measured from the 2D ultrasound image and used to correct for adipose tissue thickness n detail . Brieflyn detail . Due to n detail . In addi nm with n detail .total  throughout the incremental test were taken as an index of changes in total microvascular hemoglobin concentration which can occur due to changes in regional blood volume and/or capillary hematocrit. It is important to note that Δ[Hb]total does not provide a measurement of systemic hemoglobin concentration. Changes in [HHb] (Δ[HHb]) were taken as an estimate of skeletal muscle fractional O2 extraction [2]/ ([HbO2] + [HHb]) x 100. The Δ[Hb]total, Δ[HHb], and ΔTOI were calculated as the difference between the value obtained during the initial 30 seconds of the incremental test and the mean average at VT. These differences were then used to calculate the individual rates of concentration change relative to the change in -1 ramp, the mean work rate during this time was 7.5 W, which is similar to the unloaded cycling baseline used in previous studies  was significantly increased above baseline at 50W 36.1 ± 0.4 vs. 39.6 ± 0.8 μM; P < 0.001) and VT . However, [HHb] was only significantly increased above baseline at the VT in the cancer survivors  suggesting a delayed increases in fractional O2 extraction in the cancer survivor group compared to controls. Muscle [Hb]total significantly increased at 50W  and VT  relative to baseline in controls, whereas [Hb]total only increased at VT in the cancer survivors . The [Hb]total at VT was significantly decreased in the cancer survivors compared to controls . The rate at which TOI  and [HHb]  increased for a given increase in Mean values of NIRS-obtained skeletal muscle microvascular TOI, [HHb], and [Hb]controls .total and [HHb] were significantly decreased during dynamic cycle ergometry in the cancer survivors compared to healthy controls. These decreases suggest that muscle microvascular hemoglobin concentration and muscle O2 extraction were lower in the cancer survivor group. Lastly, these differences resulted in decreased ΔTOI and Δ[HHb] relative to 2 delivery, O2 extraction, and O2 utilization during exercise in cancer survivors, which to our knowledge, represents the first evidence of potential muscle microvascular toxicity in cancer survivors treated with adjuvant treatments.The purpose of this study was to evaluate endothelial function and the skeletal muscle deoxygenation responses to moderate intensity ramp exercise in a group of cancer survivors and healthy controls. This study has three major findings. First, brachial artery FMD, a non-invasive measurement of endothelial function, was not different between cancer survivors and healthy controls. This finding does not support the first hypothesis. The second key finding was that skeletal muscle microvascular [Hb]Treatment with certain chemotherapies can elicit endothelial damage and vascular dysfunction. Ito et al.  and GibsSimilar to the findings that chemotherapy can acutely impact vascular function, Chow et al.  observedThe VT expressed as both a work rate and  V˙O2max . In addi V˙O2max . The sim2 extraction  response during exercise in the cancer survivors could be due to additional factors including heterogeneity in microvascular mean transit time, blood flow, blood flow distribution, capillary density, and decreased mitochondrial activity  were compared to healthy controls. In addition, cancer survivors demonstrated decreased rates of change for TOI and [HHb] relative to metabolic rate, probably due alterations in microvascular function, fiber type distribution, intracellular oxidative capacity, or muscle blood flow. Since skeletal muscle [HHb] is an estimate of O2 extraction and since 2 extraction are related, our data suggest that adjuvant cancer therapies has an effect on the integrated relationships required for the maintenance of dynamic exercise. The mechanistic reasons for these findings and the functional consequences can only be speculated at this point and future investigations should focus providing further mechanistic insight into the long-term effects of adjuvant cancer therapies.From the present study in can be concluded that the pattern of microvascular redox status, evaluated via NIRS, during a moderate intensity ramp exercise test is influenced by prior exposure to adjuvant cancer therapy. In cancer survivors skeletal muscle microvascular [Hb]S1 TREND Checklist(PDF)Click here for additional data file.S1 TableData are presented as means ± SE and ± SD; n, no. of subjects; BMI, body mass index; VT, ventilatory threshold; D, diameter; FMD, flow-mediated dilation; AUCSR, area under the shear rate curve; [THC], total hemoglobin+myoglobin; [HHb], deoxygenated hemoglobin+myoglobin; TOI, tissue oxygenation index.(XLSX)Click here for additional data file.


Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon.Coho salmon  associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish.Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10  contains supplementary material, which is available to authorized users. Oncorhynchus kisutch) and other teleosts is normally tightly coupled to energy intake and is modulated by a large number of environmental factors  to amplify products between 100-200 bp from gene sequences retrieved from a de novo coho salmon skeletal muscle transcriptome . Normalization of gene expression was performed using the geometric average of rpl13 and ef1a. All expression values are expressed as arbitrary units.Primers were designed using Primer 3 .A phylogenetic analysis was carried to confirm the salmonid-specific WGD origin of potential 4R paralogues in the transcriptome. Potential 4R-paralogues were conceptually translated to their amino acid sequence. BLASTp against the non-redundant NCBI database was used to confirm the identities of the translated paralogues. Teleost orthologues for the genes of interest were retrieved from Ensembl including human and mouse orthologues to be used as outgroups. Potential coho salmon 4R paralogues were used as a query against the rainbow trout . The original DESEQ list of genes was further curated, maintaining those genes that showed consistent expression between “sister contigs”, special care was taken to detect 3R and 4R-paralogues annotated with the same ID by investigating the alignment of sister contigs.For global DGE between TR and WT individuals the DESEQ program was used following programmer recommendations and a false discovery rate (FDR) cut-off of FDR < 0.05 was applied for significant differences. The original list of genes differently expressed generated by DESEQ was manually curated and only those contigs with >15 DESEQ-counts and over 2log studies , 46, cohn = 6 for each experimental group). Significant differences were detected by a two-tailed test between WT and TR individuals followed by a Benjamin-Hochberg (FDR) correction for multiple comparisons. Significant differences were accepted when FDR < 0.05.Differences in expression for specific groups of genes were estimated using DESEQ-counts values per individual animal for each gene  prior to correlation analysis. Pearson correlation was estimated using PASW Statistic software v21.Differences in gene expression analysed by qPCR were analysed by t-test and a significant threshold of Gh-transgenic coho salmon show increased appetite and growth relative to wild type fish. We restricted the food intake of transgenic fish (TR) to that of wild-type (WT) fish fed to satiation, resulting in higher levels of muscle Gh expression, but a similar final body size. The two groups had markedly different gene expression signatures, with TR fish showing increased transcript abundance for pathways associated with protein translation and protein folding and reduced expression of genes involved with myotube fusion. The down-regulation of genes with known function in myoblast fusion, particularly cadherin signaling, was correlated with a reduction in average muscle fibre diameter in TR relative to WT fish which is expected to reduce the costs of maintaining ionic homeostasis. This may explain why TR fish are more active than WT fish yet grow at a similar rate.All supporting data for the present manuscript is included as additional files.


Chitotriosidase enzyme (EC: 3.2.1.14) is the major active chitinase in the human body. It is produced mainly by activated macrophages, in which its expression is regulated by multiple intrinsic and extrinsic signals. Chitotriosidase was confirmed as essential element in the innate immunity against chitin containing organisms such as fungi and protozoa; however, its immunomodulatory effects extend far beyond innate immunity. In the current review, we will try to explore the expanding spectrum of immunological roles played by chitotriosidase enzyme in human health and disease and will discuss its up-to-date clinical value.  Chitotriosidase enzyme , belonging to the family of 18 glycosyl hydrolases, was the first active chitinase to be discovered in human plasma [ Plasmodium falciparum [ Wuchereria bancrofti [ Candida albicans [ Madurella mycetomatis [ Cryptococcus neoformans [In man, chitotriosidase is mainly expressed by different lineages of activated blood and tissue macrophages –10 and tlciparum , Wuchereancrofti , Candidaalbicans , Madureletomatis , and Cryoformans .The role of chitotriosidase enzyme inside macrophages is not limited to its chitinolytic activity against the engulfed chitin containing organisms, or even to innate immunity. It has been implicated in the activation and polarization cascades of macrophages, as well as the indirect activation of other immune cells such as T helper cells and eosinophils –19. ReceIn the current review, we will provide a summary of basic information about the enzyme and we will discuss its immunomodulatory effects in humans over both innate and acquired immunity together with its current and possible future clinical applications.All the genes encoding active chitinases and chitinase-like proteins are clustered in two loci on the human chromosome 1 (1p13 and 1q32). This genetic clustering displays a high degree of conservation among different mammals indicating an evolutionary relationship through common ancestral gene duplication events . (CHIT1), extending over 20 kilobases at locus 1q32, consists of 12 exons translating a 466-amino acid protein [ CHIT1 gene , both having equal chitinolytic activities. The 50-kilodalton protein (466 amino acids) is initially produced by macrophages containing the 39-kilodalton N-terminus having the catalytic domain and the C-terminus having the chitin-binding domain connected together by a short hinge region. The 39-kilodalton protein (387 amino acids) is either cleaved post-translationally in the lysosome of macrophages or less commonly formed through differential RNA processing .The enzyme was initially thought to be an exochitinase because it can hydrolyze chitotriose residues and was termed chitotriosidase based on this observation. However, recent structural and binding modes studies revealed the enzyme to be more of an endochitinase rather than an exochitinase , 30. FurAlthough chitotriosidase enzyme is relatively recently linked to human pathology, many aspects of its intracellular and extracellular mechanistic actions, effector and affector molecules, and diseases influenced by the increase or decrease of its expression have been investigated. After its initial association with innate immunity against chitin coated pathogens it was rapidly identified as one of the major protein products of activated macrophages and hence an important nonspecific marker of macrophage activation . ChitotrSeveral intracellular pathways have been proposed to explain the stimulatory molecules and the activation cascades of chitotriosidase enzyme inside human macrophages. α and IFN-γ [κB signaling pathway [α; however, IFN-γ most probably produces its effect through stimulating the Jak-Stat signaling pathway [Chitin naturally is a potent activator of chitotriosidase expression, either phagocytized by the macrophage in the cell walls of different fungi and protozoa or directly introduced to macrophages through an unidentified receptor , 36. Chind IFN-γ  which cand IFN-γ . Lipopol pathway , 39, whi pathway . Another pathway , 42.Prolactin hormone which is structurally related to many human cytokines and is involved in the regulation of monocyte/macrophage functions was also shown to increase macrophage chitotriosidase production . Throughβ, which either can stimulate the NF-κB signaling pathway directly or indirectly induces the production of TNF-α [The inflammasome system is also expected to play some role in the activation of chitotriosidase expression as both ingested bacterial MDP and cystine crystals can stimulate macrophages through the NLRP3 inflammasome system , 46, leaof TNF-α , thus stγ and TNF-α [Another efficient way to activate macrophages and induce chitotriosidase expression is through the paracrine effect of natural killer cells (NK cells), which when exposed to cells infected with viral, bacterial, or fungal pathogens or even neoplastic cells can produce large amounts of INF-nd TNF-α , 48 in tChitotriosidase enzyme expression increases exponentially during the normal monocyte to macrophage maturation process showing a peak of expression between the 5th day and the 7th day of culture  and is rDendritic cells are the most important antigen presenting cells in the human body and one of the members of the monocyte/macrophage lineage. Although AMCase is widely expressed in different tissues, especially in the epithelial cells of the gastrointestinal tract and lungs , chitotr in vitro when incubated with human monocyte derived macrophages were able to activate macrophages in a concentration dependent manner evidenced by the increased concentrations of TNF-α and the concomitant activities of chitotriosidase enzyme in culture supernatant and in cell homogenate. Furthermore, plasma chitotriosidase activities in cystinotic patients correlated positively with leucocytes cystine concentrations, making it a potential target for the disease therapeutic monitoring [Recently, activated macrophages and chitotriosidase elevations were implicated in the pathogenesis of nephropathic cystinosis, another lysosomal storage disorder characterized by cystine crystal accumulation inside macrophages in different body organs. Cystine crystalsnitoring . Cystinoβ and TNF-α in the bronchoalveolar lavage (BAL) of COPD patients supporting the hypothesis of a mutual regulation cascade in the production of these inflammatory mediators [Chit1−/− mice and significantly enhanced in lungs from Chit1 overexpressing transgenic mice. This effect is explained by the activation of fibroblasts through enhancing TGF-β and increasing the expression of TGF-β receptors 1 and 2 leading to the activation of the Smad and MAPK/ERK signaling pathways [Chitotriosidase also mediates many inflammatory processes through the direct stimulation of different inflammatory mediators such as IL-8, MMP9 (collagenase type IV), MCP-1 (CCL2), RANTES (CCL5), and eotaxin (CCL11), thus increasing the migratory capacity of many immunological cells including T lymphocytes, macrophages, and eosinophils , 52. Levpathways . Similarpathways , 22, 54.pathways ; however2O2; however, there was no much evidence to support this hypothesis [Chitinases and chi-lectins could play a detrimental role in human cancer development, especially CHI3L1 (YKL-40) which has been associated with increased tumor angiogenesis and bad prognosis in many human neoplasms such as breast, lung, and cervical cancers –58. On tpothesis . Recentlpothesis . This mupothesis . It is tChitotriosidase is currently an established or a candidate screening marker, severity marker, and/or therapeutic monitor for over 40 different diseases, inherited and acquired. Being a sensitive biomarker of macrophage activation, chitotriosidase activity is currently more and more commonly used in clinical practice to evaluate the status and response to treatment of inflammatory based diseases in which macrophages play a significant role. The interesting mixture of harmful and beneficial immunological effects made human chitinases and chitotriosidase enzyme an intriguing point of research. If proven safe in humans, the development of targeted therapies either to suppress chitotriosidase activity in autoimmune and inflammatory disorders, or to specifically enhance its targeted activity to kill cancer cells or to potentiate immunity against certain infections will not be far in the future.


Standard management of Juvenile Idiopathic Arthritis (JIA) is heavily dependent on the use of methotrexate (MTX), which induces clinical benefit in about 65% of patients. We have previously demonstrated that children with high serum levels of MRP8/14 protein prior to starting MTX have a high chance of good response to MTX. However, among children whose MRP8/14 serum levels are moderate or low, prior to MTX, some respond well whilst others respond poorly to MTX. We have taken a proteomics approach to identify novel biomarkers present in serum prior to treatment that correlate with response or non response to MTX.The aim of this study was to identify novel protein biomarkers in serum from JIA patients to help and inform the clinicians at time of diagnosis whether the patient is likely to respond well to MTX.Blood serum samples and clinical data were obtained from 83 JIA patients from the Childhood Arthritis Response to Medication Study (CHARMS) before starting MTX treatment. The study has full ethical approval and consent. To assess clinical response to medication, core set variables and Definition of Improvement (DOI) for JIA was used, comparing data at 0 and 6 (range 4-8) months. MRP8/14 was measured in these patients and used to classify cases into three groups: non-responders, responders with low MRP 8/14 levels pre MTX, and responders with high MRP 8/14 levels pre MTX. To screen for proteins which distinguish future MTX non-responders from responders, 5 serum samples from age-matched patients from each group were pooled; pooled samples were enriched for low abundant proteins, and fractionated on a 1D PAGE. Proteins were identified and quantified by Mse- label free quantitation and analysed using Nonlinear Dynamics Progenesis LC-MS software for differential expression.A total of 648 unique proteins were identified in serum pools (just prior to commencing MTX treatment). Patients were defined as responders, R (those reaching ACR50 or above) or non-responders, NR (reaching only ACR30 or below). Forty proteins were found to be differentially represented in the serum pools, using a level of >2 fold difference between MTX responders and non-responders. This group included 26 proteins with high abundance in the responder group and 14 proteins with high abundance levels in the non-responder group. Using a more stringent cut off of fold difference > 4 identified 12 proteins that showed a significant difference between R and NR groups. In contrast to our previous data with MRP8/14, these 12 novel biomarker proteins were highly differentially expressed in serum of both the groups of responders (i.e. MRP high and MRP low responder groups), compared to NR cases, prior to MTX use. This indicates that these protein biomarkers may function to distinguish R from NR patients more efficiently than the MRP8/14 biomarker.This study has identified a number of potential novel protein biomarkers which may be able to discriminate between patients who will respond or those who will fail to respond to MTX, when measured prior to starting the treatment. Forty protein biomarkers were able to significantly differentiate between response and non response. Using a higher cut off of >4 fold difference produces 12 proteins that we envisage to be more reliable than MRP 8/14 for predicting response. Further testing and analysis of these proteins is required to validate those which can most reliably recognise patients who will respond to MTX therapy, or those who will fail to respond.None declared


Allogenic osteochondral tissue (OCT) is used for the treatment of large cartilage defects. Typically, OCTs collected during the disease-screening period are preserved at 4°C; however, the gradual reduction in cell viability during cold preservation adversely affects transplantation outcomes. Therefore, improved storage methods that maintain the cell viability of OCTs are needed to increase the availability of high-quality OCTs and improve treatment outcomes. Here, we evaluated whether long-term hydrogen delivery to preservation solution improved the viability of rat OCTs during cold preservation. Hydrogen-supplemented Dulbecco's Modified Eagles Medium (DMEM) and University of Wisconsin (UW) solution both significantly improved the cell viability of OCTs during preservation at 4°C for 21 days compared to nonsupplemented media. However, the long-term cold preservation of OCTs in DMEM containing hydrogen was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology. Our findings demonstrate that OCTs preserved in DMEM supplemented with hydrogen are a promising material for the repair of large cartilage defects in the clinical setting. Allogenic osteochondral tissue (OCT) is one of the materials used for the treatment of large cartilage defects associated with osteonecrosis, osteochondritis dissecans, and traumatic injury due its low rates of donor-site morbidity. The transplantation of fresh OCT leads to high functional outcomes; for example, the treatment of femoral condyle defects with fresh OCTs had a greater than 75% clinical success rate . HoweverNumerous studies have demonstrated that the addition of fetal bovine serum (FBS) to preservation solution significantly improves chondrocyte viability in OCTs during cold storage –6. There in vitro [One promising approach for maintaining the viability of OCTs is the supplementation of preservation solution with molecular hydrogen. The potential efficacy of hydrogen supplementation is supported by the potent antioxidant, anti-inflammatory, and antiapoptotic properties of this gas , 10, whiin vitro , 12. In in vitro , 14. HowHydrogen-rich solutions for the cold storage of organs are typically prepared using high-pressure hydrogen or bubbling with hydrogen , 15, 16.n = 10).All procedures, including the handling of animals, were performed in accordance with the guidelines of the Animal Ethics Committee of Kitasato University. A total of 48 male 14-week-old Sprague-Dawley rats were obtained from Charles River Japan, Inc. . For anesthetization, the rats were first administered diethyl ether for anesthesia induction and were then fully anesthetized with a mixture of medetomidine, midazolam, and butorphanol tartrate by intramuscular injection. To excise distal femora, a skin incision was first made just above the knee. The patellar, cruciate, and collateral ligaments were cut to expose the distal femoral condyles, and the distal parts of the knee extensor muscles then were removed from the bone. Distal femora were cut at the metaphyseal region using a bone saw. The weight of OCT samples was adjusted to approximately 300 mg wet weight by trimming soft tissue and bone tissue with a scalpel or rongeur. Two OCT samples were harvested from the bilateral knees of each Sprague-Dawley rat. The 40 OCT samples obtained from 20 rats were randomly separated into 4 groups  in a 4°C cold room as schematically outlined in We performed cell viability analysis after 21 days of preservation at 4°C because the rat OCT previously exhibited markedly reduced viability after this time point . The celμm thick sagittal sections of the patellar groove of femoral condyles were prepared and then stained with hematoxylin and eosin. The thickest site of articular cartilage in the noncalcified zones was readily observed at 200x magnification using a light microscope. Each set of ten tissue specimens prepared from OCT samples preserved in preservation solution with or without hydrogen was assessed using a computer-assisted image filing system . To determine the proportion of degenerative chondrocytes, the total number of chondrocytes in noncalcified zones was counted, and the number of chondrocytes with normal morphologic features , mild pyknotic and irregular nuclei (mild grade), and severe pyknotic nuclei and eosinophilic shrunken cytoplasm (severe grade) [The assessment of morphologic features of cells in the articular cartilage of OCT samples was performed after the measurement of cell viability. OCT samples were fixed in a 4% paraformaldehyde phosphate buffer solution for 48 h at 4°C and were then decalcified in a 3 mol/L EDTA solution for 2 weeks at 4°C. After embedding the samples in paraffin, 3 e grade)  was deteμL cDNA, the specific primer set , and 12.5 μL SYBR Premix Ex Taq  in a final volume of 25 μL. The sequences of the PCR primer pairs are listed in Total RNA was extracted from harvested OCT samples using TRIzol  according to the manufacturer's instructions and was used as template for first-strand cDNA synthesis using SuperScript III RT (Invitrogen). The PCR reaction mixture consisted of 2 The hydrogen concentrations of DMEM and UW preservation solutions containing OCT samples in polystyrene tubes submersed in the water bath of the hydrogen electrolyzer system were measured periodically over a 48 h period . The hydP < 0.05; P < 0.05). The mean percentages of cell viability of OCT samples maintained in DMEM and UW solution without hydrogen were 10.9% and 20.0%, respectively, whereas those for samples preserved in DMEM and UW solution supplemented with hydrogen were 51.4% and 29.1%, respectively.As determined using a WST assay, OCT samples preserved in DMEM and UW solution supplemented with hydrogen had significantly higher cell viability after 21 days at 4°C than those stored in solution alone  cold preservation. Storage of OCTs in DMEM containing hydrogen at 4°C was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology in comparison with UW solution and nonsupplemented conditions. Our findings show that OCTs cold-preserved in DMEM supplemented with hydrogen are a promising material for the efficient repair of large cartilage defects in the clinical setting.Chondrocyte viability of allografts, particularly at the tissue surface, during cold storage appears to be a strong determinant of tissue properties and long-term repair efficacy after transplantation . Therefoα contributes to chondrocyte apoptosis and cartilage degeneration [α is increased during the cold preservation of OCT and the addition of TNF-α inhibitor to preservation solution was shown to improve the viability of OCT [α expression during organ preservation [α expression of OCT. Although further studies are needed to determine the underlying mechanisms, our findings suggest that hydrogen improves OCT viability through the inhibition of TNF-α production.The inflammatory cytokine TNF-neration , 23. TNFervation , 17. HerTo prevent marked decline in the cell viability of graft material and organs, several antioxidant and anti-inflammatory agents, such as epigallocatechin-3-O-gallate, tumor necrosis factor-alpha inhibitor, and glutathione, have been added to preservation solutions , 24. HowThe continuous delivery of hydrogen to culture medium improved OCT viability during cold preservation. The present findings suggest that OCTs preserved in hydrogen-rich culture media are a promising material for cartilage repair in the clinical setting.


Deforestation in the Brazilian Amazon increased between 2001 and 2004 and declined substantially afterwards, whereas deforestation in the Bolivian Amazon, the Colombian Amazon, and the Peruvian Amazon increased over the study period. The average carbon density of lost forests after 2005 was 130 Mg C•ha−1, ~11% lower than the average carbon density of remaining forests in year 2010 (144 Mg C•ha−1). Moreover, the average carbon density of cleared forests increased at a rate of 7 Mg C•ha−1•yr−1 from 2005 to 2010, suggesting that deforestation has been progressively encroaching into high-biomass lands in the Amazon basin. Spatially explicit, annual deforestation and emission estimates like the ones derived in this study are useful for setting baselines for REDD+ and other emission mitigation programs, and for evaluating the performance of such efforts.Reducing emissions from deforestation and forest degradation (REDD+) is considered one of the most cost-effective strategies for mitigating climate change. However, historical deforestation and emission rates―critical inputs for setting reference emission levels for REDD+―are poorly understood. Here we use multi-source, time-series satellite data to quantify carbon emissions from deforestation in the Amazon basin on a year-to-year basis between 2000 and 2010. We first derive annual deforestation indicators by using the Moderate Resolution Imaging Spectroradiometer Vegetation Continuous Fields (MODIS VCF) product. MODIS indicators are calibrated by using a large sample of Landsat data to generate accurate deforestation rates, which are subsequently combined with a spatially explicit biomass dataset to calculate committed annual carbon emissions. Across the study area, the average deforestation and associated carbon emissions were estimated to be 1.59 ± 0.25 M ha•yr If we were to estimate net carbon emissions, potential uncertainties would also include those associated with other forest dynamics such as degradation and regrowth, those associated with other significant carbon pools  as well as those associated with the land cover dynamics on deforested land. Beside these factors, to reach a conceptually comprehensive estimate of carbon emissions from land cover and land use change as well as associated uncertainty, Houghton oachment .-1 . Methodologically, this suggests that using a biome-level average biomass value in non-spatial carbon accounting models e.g. the bookkeeping model [Closed-canopy forests in the Amazon have high carbon stocks peaked around 150 Mg C∙ha-1 , but def1  [Forest change products over the study area were derived from the yearly MODIS VCF tree cover data. MODIS VCF characterizes the Earth’s land surface as percent tree cover at a spatial resolution of 250 m. The yearly VCF products for the years between 2000 and 2010 were used in this study . These pr (RMSE) . It shouf is tree cover in year x; parameter a defines the magnitude of change where negative values indicate deforestation; b controls change rate where large values indicate abrupt change and small values indicate gradual change; c denotes the inflection time when the change occurs, and d represents the pre-change value (a+d).We designed a novel method to characterize deforestation from time series of tree cover layers. A detailed description of this method and validation of the resulting forest disturbance product was reported in . We provge value . The posa values and passed the goodness-of-fit test (p < 0.01) were labelled as deforestation indicator. If multiple disturbances occurred in the 11-year period, a single logistic fit could not capture these change events. Hence we adjusted the procedure using a 5-year moving window to fit segments of the temporal VCF vector. The window size of 5-year was chosen because (1) this ensured the minimum number of observations required to estimate four parameters and (2) multiple deforestation events were highly unlikely to occur within five years. Goodness-of-fit test was also applied to each fitted segment to identify candidate change pixels. We implemented this curve fitting procedure for every MODIS pixel (n ~ = 111 million) across the study region and labelled every candidate deforestation pixel.The four parameters were initialized and iteratively updated to reach a least-squared fit and the fitted curve was evaluated using a standard F-test based on the chi-square value of the fit. Pixels that had negative Land cover change products derived using MODIS or coarser resolution data are typically considered indicator products and need to be calibrated using Landsat-based products to produce more accurate change estimates , 18, 80.The Landsat data used in calibration were selected from the Global Land Survey (GLS) collections circa 2000 and 2005 . The GLSc indicates the timing of deforestation, we use the fitted parameter c value to select MODIS pixels where deforestation likely occurred within the two Landsat dates.The acquisition dates of the GLS images used in this study range from 2000-01-26 to 2002-11-13 for the 2000 epoch and from 2004-01-02 to 2007-10-29 for the 2005 epoch, which also vary from tile to tile. To resolve the time difference between Landsat and MODIS samples, we linearly normalized the Landsat-based deforestation rates to a common date of 30 June for the particular Landsat acquisition year. Samples contaminated by remaining cloud and cloud shadow (< 10%) were also linearly adjusted, assuming change rates in the cloudy area were the same as cloud-free area of the sample site , 85, 86.a, such that the MODIS-derived deforestation rate matched with the Landsat-derived deforestation rate  ,71.Uncertainties in quantifying carbon fluxes from deforestation arise from two major sources: errors in both deforestation and biomass estimates , 72. HerIndependent deforestation maps produced by the PRODES project were used to evaluate the overall accuracy of the MODIS deforestation. We downloaded a total of 50 Landsat tiles completely covering the Amazon portion of Mato Grosso and Rondonia as reference data. PRODES maps in these areas were chosen because (1) they were generated by local experts using Landsat images and were found highly reliable , 88, 89;The 30 m PRODES maps with pixels labelled as deforestation between 2000 and 2010 were aggregated to 5 km resolution to derive an 11-year deforestation rates per grid. The MODIS deforestation map was also aggregated to 5 km resolution to derive an overall deforestation rate between 2000 and 2010 . We thenεcarbon emission refers to errors in emission estimates; εdeforestation represents errors in deforestation estimates and εcarbon density represents errors in carbon density estimates. Errors in carbon density estimates were quantified from four components including (1) measurement error associated with tree height estimation from Lidar data, (2) allometric error associated with biomass estimation from tree height, (3) sampling error associated with the representativeness of sample plots and the spatial variation of biomass within a 1 km pixel and (4) prediction error of the machine learning model [We generated error bound for emission estimates using the error propagation model defined in the following equation. Both deforestation and carbon density error terms are expressed in terms of percentage of relative error and assuming they are independent , 87, theng model . The err