Alternative methods in which time-frequency dynamics are preserved are considered. Sparse-coding and source separation algorithms  are proposed as a means to access and summarise time-frequency dynamics which may be more ecologically-meaningful.Passive acoustic monitoring is emerging as a promising non-invasive proxy for ecological complexity with potential as a tool for remote assessment and monitoring , 2015. R Operating within the conceptual and methodological framework of the burgeoning field of ecoacoustics,   and identification of ‘ecological-disturbance indicator species’ , 2010. RVarious forms of remote visual sensing technologies have been explored. Global satellite imaging has been investigated to monitor biophysical characteristics of the earth’s surface by assessing species ranges and richness patterns indirectly e.g., , 2010. TAcoustic surveys have most obvious relevance for the identification of vocal animals. In terrestrial habitats, bird species in particular are of interest as their importance as indicator species of environmental health has been demonstrated in temperate , 2010 anSoundscape Ecology   apply in the shared sonic environment; the soundscape is seen as a finite resource in which organisms (including humans) compete for spectro-temporal space. These ideas were first explicitly captured in Krause’s es (ANH) . Referries (ANH) , 1957 thMorphological Adaptation Hypothesis; (2) through direct adaptation to physical features of the signalling environment, the Acoustic Adaptation Hypothesis; and (3) to facilitate species recognition, the Species Recognition Hypothesis. Field studies of the Neotropical suboscine antbird (Thamnophilidae) provide direct evidence that species recognition and ecological adaptation operate in tandem, and that the interplay between these factors drives the evolution of mating signals in suboscine birds  as a by-product of morphological adaptation, the ne birds , 2005. Ane birds , 2014, ine birds , 2015.or magnitude differences between frequency bands of a spectrogram.This emerging framework, coupled with the technical feasibility of remote acoustic sensing and pressure to meet strategic biodiversity targets, fuels a growing research interest in ecological applications of acoustic indices; several dozen have been proposed over the last 6 years . In Normalized Difference Soundscape Index (NDSI)  animals vocalize outside the 2–8 kHz range (Under the assumption that anthropogenic noise contribution is band-limited to a frequency range (anthropophony: 0.2–2 kHz) below that of the rest of the biological world (biophony: 2–8 kHz), the x (NDSI) , 2012 sex (NDSI) , 2012. HHz range , 2014, api is the proportion of individuals belonging to the ith species in the data set of interest; it quantifies the uncertainty in predicting the species identity of an individual that is taken at random from the dataset. A range of entropy indices are based on the assumption that the acoustic output of a community will increase in complexity with the number of vocalising individuals and species. A summary of the complexity of the sound is assumed to give a proxy of animal acoustic activity. Complexity here is used as a synonym of heterogeneity and many indices derive from classical ecological biodiversity indices. Shannon Entropy , 1949 EqAcoustic Entropy Index, H (sh) and temporal (th) entropies which are calculated on the mean spectrum and Hilbert amplitude envelope of a time wave respectively. H ranges from 0 for pure tones to 1 for high-energy, evenly distributed sound. The index was first tested against simulated choruses, generated by mixing together samples of avian vocalisations and systematically varying the number of species in each track. H values increased with species richness S following a logarithmic model. Field trials were carried out in pristine and degraded African coastal forests and H was shown to reflect assumed variation in species richness  is similarly derived by calculating the Gini index  was developed specifically to capture the dynamic changes in the soundscape: “many biotic sounds, such as bird songs, are characterised by an intrinsic variability of intensities, while some types of human generated noise (such as car passing or airplane transit) present very constant intensity values” , this is a significant constraint. Although existing indices are very cheap in computational terms, the fear is that what is gained in computational efficiency is lost of ecological efficacy. Rather than collapsing the signal into one or other domain, we propose that the theoretical and practical strands of ecoacoustic research could be advanced by developing tools in which time-frequency structure is preserved.By virtue of being based on either time-averaged spectrograms Time-frequency tradeoffs are an important issue in all signal processing tasks. Sparse coding is gaining popularity in brain imaging, image analysis and audio classification tasks as an alternative to vector-based feature representations in part because it is seen to have more time-frequency flexibility than standard Fourier transform representations. Sparse coding aims to construction efficient representations of data as a combination of a few typical patterns (atoms) learned from the data itself. For a given set of input signals, a number of atoms are sought, such that each input signal can be approximated sparsely by a linear combination of a relatively small number of this set of atoms (the dictionary). The dictionary size is higher than the dimensionality of the signal such that a subset of atoms can span the whole signal space—an overcomplete dictionary , 2011. SSparse decomposition using dictionaries of atoms based on biologically informed time-frequency atoms such as Gabor and Gammatone functions–which are seen to resemble characteristics of cochlea filters–are intuitively attractive as they can provide a feature set which is oriented in a two dimensional time-frequency space with which to approximate the original signal. This has been shown to be more efficient than Fourier or wavelet representations , 2005 anV into the product of two multinomial probability distributions, W and H, and a mixing weight, Z. In the auditory domain, V would be a matrix representing the time-frequency content of an audio signal: W can be thought of as a recurrent frequency template and each row of H as the excitations in time of the corresponding basis. z and K is the number of bases in W  is one of a family of techniques used for source separation, which similarly provides a tool for extracting components according to common frequency-amplitude statistics. PLCA is a probabilistic variant of non-negative matrix factorization (NMF) , 2001. Ises in W , 2010. EPLCA can be compared to more familiar component analysis tools such as Principle or Independent Component Analyses  and can be used to perform dimensionality reduction, feature extraction or to explore structure in a data set. The non-negativity constraint is a valuable property for audio and image decompositions, where non-negative representations are prevalent, as the non-negative elements are often perceptually meaningful decompositions which can be easily interpreted. By comparison, methods using non-negativity are bound to return bases that contain negative elements and then employ cross-cancellation between them in order to approximate output , 2008. SSparse and shift-invariant PLCA (SI-PLCA) extends PLCA to enable the extraction of multiple shift-invariant features from analysis of non-negative data of arbitrary dimensionality and was first demonstrated as an effective unsupervised tool for extracting shift-invariant features in images, audio and video , 2008. TA common strategy used throughout the NMF literature is to favour sparse settings in order to learn parsimonious, parts-based decompositions of the data. Sparse solutions can be encouraged when estimating the parameters of the convolution matrix by imposing constraints using an appropriate prior distribution , 2008. Uqualitative patterns of interaction between them. By way of musical analogy, we don’t care what the specific instruments of the orchestra are, rather we wish to assess characteristics of the arrangement and how the voices interact as an ensemble toward a coherent global composition through time, timbre and frequency space. Frequency-based indices may succeed in tracking species richness in simulated communities by measuring gross changes in frequency band occupancy. Perhaps their failure to track variation in species richness in the wild is because the defining feature of acoustic communities are global patterns of interaction across a more complex spectro-temporal domain, rather than frequency band occupancy or amplitude variation alone. As outlined above, current indices based on frequency or amplitude statistics inherently throw away information crucial to the analysis of spectro-temporal patterns: SI-PLCA provides a tool for extracting dynamic sound components grouped by common frequency-amplitude statistics, even when pitch or time shifted. That it has been demonstrated to be effective in extracting the perceptually-meaningful but nebulous concept of ‘groove’  and composition tasks, SI-PLCA provides a tool for accessing perceptually meaningful decompositions—time-frequency shifted patterns in a dynamic signal. From the perspective of community level ecoacoustics we are not necessarily concerned with the identification of specific species, so much as achieving a numerical description of the ‘groove’ , 2013 suIn this paper we take a first look at how these methods might provide a complementary approach to current acoustic indices for investigation of soundscape dynamics and ultimately for biodiversity assessment. Taking a small sample of field recordings across different terrestrial habitats in an Ecuadorian cloud forest reserve we compare existing spectral and temporal indices with sample analyses of a number of approaches to sparse approximation, including dictionaries built using mini-batch gradient descent, Gabor functions and SI-PLCA2D. The potential value of this approach is illustrated with example reconstructions from a new variant of SI-PLCA using dual dictionaries.1Field work data collection was authorised by the Ministerio del Ambiente, Ecuador (permit No 008-15 -IC-FAU-DNB/MA)The SLR  is situated on the western (Pacific) slopes of the Andes in northwestern Ecuador and spans an elevational range of 1,400–2,560 m. The forest is lower montane rain forest (cloud forest). The area has a humid subtropical climate and is composed of fragmented forest reserves surrounded by a matrix of cultivation and pasture lands. It lies within the Tropical Andes biodiversity hotspot and exhibits high plant species endemism and diversity. Topography is defined by steep-sloping valley systems of varying aspect.The data reported here is a subset of that collected during an 8 week field survey (June–August 2014) in the Ecuadorian Andean cloud forest at the Santa Lucia Cloud Forest Reserve (SLR)The SLR was awarded reserve status 20 years ago, prior to which areas of Primary Forest had cleared for fruit farming. The SLR therefore consists of a complex mosaic of habitat types: Ancient Primary Forest (FP) punctuated by small areas of secondary regrowth of around 20 years (FS) and silvopasture (S), typically elephant grass pastures used as grazing paddocks for the mules, which provide local transport. These areas are less than 5 ha. In contrast to other studies where dramatically different sites have been used to validate indices, this complex patchy habitat provides subtle habitat gradients.Acoustic data was collected using nine digital audio field recorders Song Meter SM2+‘ (Wildlife Acoustics), giving three replicates of each of the three habitat types. Minimum distance between recorders was 300 m to avoid pseudo sampling (the sound of most species being attenuated over this distance in cloud forest conditions). Altitudinal range was minimised. Recording schedules captured the full dawn (150 min), dusk choruses (90 min) plus midday (60 min) activity; throughout the rest of the period 3 min recordings were taken every 15 min and ran for a minimum of 14 days at each study site.The SM2+ is a schedulable, off-line, weatherproof recorder, with two channels of omni-directional microphone (flat frequency response between 20 Hz and 20 kHz). Gains were set experimentally at 36 dB and recordings made at 16 bit with a sampling rate of 44.1 kHz. All recordings were pre-processed with a high pass filter at 500 Hz (12 dB) to attenuate the impact of the occasional aircraft and local generator noise.A local expert ornithologist carried out point count surveys , 1995, nhttp://cran.r-project.org/web/packages/seewave/index.html) and soundecology (Available at: http://cran.r-project.org/web/packages/soundecology/index.html) packages in R.For the purposes of this illustrative exercise, analyses were carried out on dawn chorus recordings from just one day at one recording station for each of the three habitat types sampled. A range of indices frequency and time domain indices were calculated: NDSI, H, ADI, AEI, ACI and BI. Indices were calculated for the same 10 min periods during which point counts were made at each site. Calculations were made using the seewave (https://github.com/bregmanstudio/audiospectrumpatchapproximation): dictionary learning using mini-batch gradient learning, a Gabor field dictionary and, shift invariant 2D Principle Latent Component Analysis (SI-PLCA2D). Each uses Orthogonal Matching Pursuit (OMP) to build the component reconstructions. Samples were extracted from analyses of 1 min sections of the field recordings. These examples are aimed at illustrating the potential of a two-dimensional (2D) atomic rather than 1D vector approach in general, rather than experimental validation of any particular algorithm. Default parameters were used in all cases.Three approaches to audio decomposition are illustrated using the Bregman Media Labs Audio Patch Approximation Python package  using 2D dual dictionaries , 2013 bak from a K maximum components  Are the component reconstructions meaningful soundscape objects in ecological terms? Are vocalising species separated in any meaningful ecological way either by soundscape component  or acoustic community ? (2) Might the statistics generated be meaningful? Does the number of components (k) returned reflect ‘complexity’ or ‘decomposability’ in a way which may reflect the status of the acoustic community? Could the entropy summaries of each component be used as a measure of diversity within or between communities?The entropies of each distribution are given in the subfigure captions of acoustic niche in order to develop the understanding necessary to create more ecologically meaningful monitoring tools.The ability of PLCA to separate streams of distinct sonic components is well recognised. Within the conceptual framework of ecoacoustics, such techniques provide a means to investigate the composition of the acoustic community as a whole in terms of dynamic interactions between spectro-temporal patterns of vocalising component species, providing a new tool to begin to experimentally interrogate the concept of or frequency domain may be insensitive to the dynamic patterns of interaction in the soundscape which characterise specific acoustic communities and propose SI-PLCA2D as a promising new tool for research. This was illustrated with example analyses of tropical dawn chorus recordings along a gradient of habitat degradation. It seems likely that if acoustic niches exist that they do not lie neatly along 1D vectors in the frequency or time domain but dance dynamically across pitch-timbre-time space. SI-PLCA2D and related sparse-coding methods are computationally expensive and do not offer an instant ready-to-use proxy for biodiversity monitoring. What they do provide is a tool for extracting shift-invariant spectro-temporal patterns in a dynamic soundscape, structures which are impervious to analysis with current tools. In future work we are testing the approach on more extensive data sets to establish the ecological meaning of the extracted components.Monitoring subtle changes in complex ecosystems is crucial for ecological research and conservation but far from straight forward. Acoustic indices hold promise as a rapid assessment tool, but are subject to the same trade-offs as traditional ecological research of quality versus quantity: any metric necessarily throws away some information. In this paper we have provided an overview of the motivational premises of community-level ecoacoustics, including the concept that acoustic communities may be structured according to competition across acoustic niches through spectro-temporal partitioning. We suggest that existing indices operating in time 10.7717/peerj.2108/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2108/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.2108/supp-3Supplemental Information 3Click here for additional data file.


Following myocardial infarction (MI), patients are at risk of sudden cardiac death (SCD) due to ventricular arrhythmia, which can be prevented with an implantable cardioverter-defibrillator (ICD). Recommendations for ICD implantation is currently based on left ventricular ejection fraction (LVEF), however less than a quarter of patients who receive ICDs based on LVEF have appropriate therapy. Heterogeneity of scar has been implicated in the development of re-entrant arrhythmias and SCD. Texture analysis (TA) is a method of quantifying heterogeneity of tissues in imaging, usually using statistical-based methods to evaluate distribution of grey-level pixels. This study aimed to determine whether TA could be used to quantify heterogeneity of scar as a method of accurately risk stratifying patients.This was a retrospective blinded analysis of late-gadolinium enhanced cardiac magnetic resonance (LGE-CMR) images. Twenty post-MI patients who received ICDs were followed up for up to 686 days after implantation. Ten of these patients went on to have events and were categorised as high risk, while the remaining ten had no events on follow up and were categorised as low risk. TA was performed on regions of interest delineating scar on short axis LGE images of all patients, which involved an initial filtration step, highlighting objects of 2-6mm radii using a laplacian of Gaussian filter, denoted by the term spatial scaled factor (SSF) 2-6, followed by histogram analysis of pixel intensity from which a set of statistical parameters could be derived. Further statistical analysis was performed to determine group-wise differences in TA statistical parameters as well as survival analysis to predict time to event.Significantly different (p<0.05) statistical parameters in group-wise comparison included higher kurtosis of the histogram in the high risk group when SSF 5 and 6 were applied, and more negative skewness of the histogram in the high risk group when no filter was applied. Receiver operating characteristic (ROC) curves were drawn for these three parameters and coordinates with the highest combined sensitivity and specificity were extracted for use as threshold values in Kaplan-Meier graphs to compare time to event, which were statistically different in all three cases (p<0.05).This preliminary study showed that TA of scar identified by LGE in CMR images revealed significant differences in specific statistical parameters between high and low risk groups, as well as significant differences in time to event. There is therefore basis for expanding the analysis and comparing with other clinical and CMR parameters to explore potential for a more accurate method of risk stratifying post-MI patients for ICD implantation.N/A.


CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the  CTNS gene encoding cystinosin, a lysosomal cystine transporter [th cytosolic loop [Nephropathic cystinosis MIM 219800) is an autosomal recessive disorder caused by mutations in the 9800 is alic loop . A seconlic loop , 5. Cystlic loop , 7, thusCTNS gene [The most severe infantile nephropathic clinical form of cystinosis is typically associated with mutations resulting in a complete loss of function of cystinosin . Among tTNS gene . Cells dTNS gene .The initial clinical manifestation of cystinosis developing during infancy is renal Fanconi syndrome, a generalized renal proximal tubular dysfunction, characterized by polyuria and abnormal urinary loss of amino acids, glucose, low-molecular-weight (LMW) and intermediate weight proteins and other solutes , 11, 12.-/- mice) have shown a decreased expression of the multi-ligand receptors megalin and cubilin at the proximal tubule apical surfaces associated with cell dedifferentiation [The mechanism of renal Fanconi syndrome in cystinosis is still not completely understood . Impairentiation , 21. Thein vivo [The major challenge in studying the pathogenesis of renal dysfunction in cystinosis during many years was the absence of a suitable model. The first mouse model developed on a FVB/N genetic background showed no signs of kidney disease despite pronounced cystine accumulation in the kidney . Subsequin vivo .CTNS gene with specific siRNA and confirmed the results in proximal tubular cells derived from patients with cystinosis. We found that the absence of cystinosin resulted in an acute disorganisation of endo-lysosomal compartments, with clustering of endocytotic vesicles in the perinuclear region, decreased expression of multiligand receptors on the cell surface and delayed processing of the ligands. These data, together with our finding of abnormal co-localization of late endosomal and lysosomal marker LAMP-1 with the motor protein kinesin-1 in cystinosin-deficient cells, suggest that cystinosin functions as an important regulator of endo-lysosomal dynamics. Thus, the absence of cystinosin might lead to a defective movement of endosomes to the cell surface. Importantly, pre-treatment with cysteamine resulted in partial improvement of the surface expression of receptors and rescued ligand processing, but could not restore the abnormal morphology of the endo-lysosomal compartments. Our work makes a contribution to the understanding of pathological processes leading to the renal Fanconi syndrome in cystinosis and provides further directions for studying possible treatments of the disease aimed to restore the defective trafficking mechanisms.In our study we examined the direct consequences of cystinosin deficiency on proximal tubular reabsorption machinery in human proximal tubular cells after down-regulation of the Human recombinant GST-RAP was produced and isolated as previously described . ChemicaHuman kidney proximal tubular epithelial HK-2 cells originate from the American Tissue Type Collection  and were used to study the endocytosis previously . Human cCTNS gene were purchased from Dharmacon (ON-TARGET plus SMART pool J-019914-05–08). HK-2 cells were transfected with siRNA using Dharmafect4 (Dharmacon) transfection reagent according to the manufacturer’s instruction and incubated for 2 to 3 days before performing further experiments. Down-regulation was confirmed by real-time RT-PCR using primers, specific for CTNS. Actin was used as a housekeeping gene control.The siRNA oligonucleotides targeting http://rsb.info.hin.gov/ij/) and Adobe Photoshop CS4 software for cropping. The measurement of the lysosomal size was performed as described previously [2. Thresholds were kept the same for all images.For immunocytochemistry, cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature and washed once in PBS. Blocking reagent was added to the cells for 20 min, followed by a 1 h incubation with appropriate concentrations of the primary antibodies in blocking reagent . The cells were then washed with PBS and incubated with secondary antibodies (1:400) and DAPI (1:1000) for detection of nuclei, diluted in blocking solution. The cells were washed twice in PBS and once in ion-free water to remove salts. The coverslips were then mounted on glass microscope slides  with Mowiol . The samples were examined under either Zeiss LSM 710 or Zeiss Axiovert 100M confocal laser-scanning microscopes, in both cases equipped with a 63X (NA 1.4) oil-immersion objective. 5–10 randomly chosen fields were analyzed for each condition. For RAP uptake experiments, at least 150 individual cells were analyzed for each time point. Image processing and analysis were performed using ImageJ software. Fluorescence intensity was measured in green and red channels after subtracting background inside a selected area corresponding to an individual cell. Microscopic images were prepared for publication using ImageJ . Ultrathin sections were cut on an ultratome , and contrasted with 4% uranyl acetate for 45 min and subsequently with lead citrate for 4 min at room temperature. Sections were examined in a Jeol 1200 EX2 electron microscope .For electron microscopy, small pieces of cystinotic kidney biopsy described previously  and contCells were serum-starved for 1 h and washed twice in cold PBS containing 1% BSA. The cells were incubated for 30 min on ice with 2.5 μg/ml recombinant GST-RAP in serum-free medium. To measure surface binding of the ligand, cells were washed with cold complete medium and fixed in 4% paraformaldehyde. To assess the endocytosis, the cells were washed with complete medium and incubated at 37°C to allow internalization of the ligand at the indicated times. At the end of this incubation, the cells were briefly acid washed  and fixed.For the HA-Meg4 internalisation assay control and CTNS KD HK-2 cells were transiently transfected with HA-Meg4 and pcDNA3-RAP, serum starved for 2 h at 37°C in serum-free DMEM, washed twice in cold PBS with 1% BSA, chilled to 4°C, and incubated with the anti-HA monoclonal antibody for indicated times in an ice/water bath, to label the cell surface. The unbound antibody was rinsed off with cold HEPES-buffered DMEM, and the cells were incubated in preheated complete medium for indicated times at 37°C. After internalisation, the antibodies remaining on the cell surface were removed by a 30 s acid wash , with a brief wash in PBS, and then the cells were fixed.Lysosomal degradation was measured as described previously . BrieflyControl and cystinotic ciPTEC and control and CTNS KD HK-2 cells were loaded with 10 μM of acridine orange for 15 min at 37°C, washed in HEPES-buffered complete medium and analyzed under a confocal microscope. Acridine orange was excited at 450–490 nm and the signal recorded at 520–560 nm or >620 nm. Typically, 5–10 images were collected from 3 separate experiments and the red/green ratio was calculated.CTNS gene (CTNS KD). The efficacy of down-regulation was evaluated by real-time PCR prior to each experiment and was 80% or higher after 2 days. The cystine accumulation was not detectable after 2 days of down-regulation when the morphology and endocytosis assessments were performed. After a prolonged 4-day exposure, HK-2 CTNS KD cells demonstrated a ~7-fold (p<0.005) increase of cystine content in comparison to the control, confirming the disrupted function of the transporter. However, the formation of the dense cobblestone-like monolayer of HK-2 cells obstructed the imaging experiments at this time point. Next, we confirmed the results in ciPTEC that were established as described previously from living exfoliated cells extracted from urine of a cystinosis patient bearing the 57-kb deletion and of a healthy volunteer [We studied the changes in the endo-lysosomal compartments in cystinosis using two working models. First, we used human proximal tubular HK-2 cell line treated with siRNA specific to the olunteer .To study the endosomal compartments, control and cystinosis ciPTEC cells and CTNS KD HK-2 cells were stained with antibodies specific to markers of early endosomes and late endosomes/lysosomes (EEA1 and LAMP1 respectively), the ER (VAP-A), Golgi (GM130) and TGN (TGN46). To measure the diameter of individual lysosomes, we used the method, described previously . ClusterAs cystinotic ciPTEC are characterized by a significant cystine accumulation under normal culture conditions, we tested whether cystine-lowering treatment could rescue the abnormal phenotype observed. Control and cystinosis ciPTEC cells were treated with 100 μM of cysteamine for 24 h. Such treatment was previously shown to decrease the cystine accumulation by ~91% without causing toxic effects on cultured cells. Longer treatment did not result in a significant further decrease of cystine content .. We fouin vitro findings, we performed electron microscopy study of a biopsy material obtained from a cystinosis patient treated with cysteamine that was previously described [To confirm our escribed . We founTo identify different endosomal compartments, we transfected control and CTNS KD HK-2 cells with GFP-tagged Rab GTPases, commonly used for this purpose –33. The The perinuclear clustering of lysosomes suggested a possible involvement of the microtubule-based motor protein kinesin-1 responsible for the transport of endosomal vesicles towards microtubules plus-end. Visualization of kinesin-1 with anti-kinesin-1 heavy chain B (KIF5b) antibodies showed an altered subcellular localization of the protein in CTNS KD HK-2 cells as compared to control cells. Moreover, a ~8-fold increase of co-localization of kinesin-1 with LAMP1 was observed , p<0.001To estimate the surface expression of megalin, we used a recombinant GST-tagged RAP protein, which binds megalin with high affinity and has been used to study endocytosis in proximal tubular cells previously . HK-2 orTo study endocytosis, cells pre-incubated with GST-RAP ligand were transferred to 37°C to allow internalization of the ligand bound to the cell surface. At indicated time points, cells were fixed and stained for GST-RAP. At earlier time points, the staining was visible in vesicular structures corresponding to endosomes and then gradually disappeared. In CTNS KD HK-2 cells, GST-RAP remained visible for significantly longer time after internalization in comparison to controls .The observation was also confirmed in cystinotic ciPTEC . ImportaTo exclude the possibility that reduced megalin surface expression was caused by decreased expression of endogenous megalin in cystinosin deficient cells and to study megalin localization and trafficking, we used HA-Meg4 mini-receptor construct transiently expressed in HK-2 cells. This model has been successfully used for this purpose previously . While nWe next measured the expression of endogenous megalin in control and CTNS KD HK-2 cells by means of qPCR. The level of megalin expression was not reduced in cystinosin-deficient cells in comparison with the control . TogetheTo assess the endocytosis and processing of cargo, exclusively destined for lysosomal degradation, we performed a pulse-chase experiment in ciPTEC cells using fluorescently-labelled BSA as a ligand. We used Alexa-555-labelled BSA to trace the internalization and trafficking together with DQ-BSA green to visualize the lysosomal degradation sites. As expected, 555-BSA processing was delayed in cystinosis cells in comparison with the control, leading to a prominent accumulation after 2 hours. Strong DQ-BSA green fluorescence was observed in the same compartments where 555-BSA accumulation took place, identifying them as lysosomes . At the Together these results indicate delayed processing of endocytic cargo and suggest that cystinosin deficiency affects the surface pool of multiligand receptors that can explain reduced protein reabsorption.In this paper we describe a detailed study of receptor-mediated endocytosis in two models of human proximal tubular cells deficient for cystinosin and demonstrate an abnormal morphology of endosomal and lysosomal compartments and defective endocytosis.CTNS gene. Although using immortalized cell lines has several disadvantages, such as lower expression levels of proximal tubular receptors and transporters and difficulties to study cell proliferation rates, these models offered a possibility to explore the molecular events induced by the acute or chronic loss of expression of cystinosin in human cells. Indeed, the acute down-regulation of cystinosin induced in HK2 cells with specific siRNA allowed us to explore direct consequences of cystinosin deficiency limiting, on the one side, the effects related to inter-individual variability, which is intrinsic to the study of patients-derived  cells [-/- mice tissue. Furthermore, by comparing the two cystinosin deficiency cell models (acute KD vs cells from patients) we had an opportunity to discriminate between direct and indirect consequences induced by the loss of function of the protein. We found that cystinosin down-regulation leads to an acute disorganization of the endo-lysosomal compartments characterized by the decreased expression of the endocytic receptors on the cell surface, the delayed processing of the endocytic ligands, and the clustering of early and late endosomes and lysosomes in the perinuclear region. Only some of these endocytic dysfunctions could be rescued by cystine-lowering agent cysteamine. It therefore likely that cystinosin, next to its cystine transporting activity, plays a role in cellular vesicle trafficking.We used proximal tubular cells from adult kidney (HK-2 cells), in which cystinosin was down-regulated by cystinosin-specific siRNA, and conditionally immortalized proximal tubular cells derived from urine of a cystinosis patient bearing a homozygous deletion of the d) cells , and, on-/- mice [Being aware of the intrinsic limitations of using cell models to study complex pathogenic processes, we were reassured in verifying that many of our findings in human cystinotic cells are in concordance with those recently published in a mouse model of cystinosis , 21. The-/- mice , 21.The surface expression of multiligand receptors is strictly dependent on their level of expression and on their correct trafficking from the ER and Golgi apparatus and on the recycling processes . During -/- mice, a process associated with an activation of the transcription factor ZONAB (zonula occludens (ZO-1)-associated nucleic binding protein), which promotes cell proliferation and directly inhibits the expression of megalin and cubilin. However, it remained uncertain whether the activation of proliferation program is an initial pathogenic event triggered by the absence of cystinosin or a compensatory mechanism aiming to replenish cell losses. In our study we report that the acute down-regulation of the CTNS gene primarily reduced surface expression of megalin without affecting the total levels of this protein, thus suggesting a role of cystinosin in directing megalin trafficking.The reduced surface expression of these receptors has been reported by Raggi et al. as a consequence of the dedifferentiation of proximal tubular cells from CtnsInterestingly, the reduced expression on the cell surface could be partially rescued already after 24 hours of cysteamine treatment in cystinotic ciPTEC cells that were shown to accumulate significant amounts of cystine under normal culture conditions . In addiCTNS knock-down on motors and their regulators involved in late endosomal and lysosomal motility. Lysosomes undergo bidirectional trafficking along microtubules driven by minus-end and plus-end directed motors [The impaired degradation capacity of lysosomes had previously been reported both in patients with cystinosis  and in ad motors . As the d motors .-/- mice that also show lower Rab27 levels as compared to control [Different alternative possibilities can be considered. Firstly, the perinuclear clustering of lysosomes may be due to the inability to anchor them at the cell periphery. This anchoring is achieved by at least two different means: one involving the switch of lysosomes and of lysosome-related organelles from microtubule tracks to peripheral microfilament meshwork  and the  control . These rThe relationships existing between the functional impairment and the altered positioning of lysosomes in cystinosis remain an important subject for future studies.S1 FigCTNS gene.ciPTEC cells were incubated for indicated time points with 100 μM of cysteamine. Cysteamine-containing culture medium was refreshed daily. Cystine content of each sample was normalized by protein concentration and expressed as % of the initial. The graph represents averaged data from 2 different cystinosis ciPTEC lines deriving from patients bearing a homozygous 57 kb deletion of the (TIF)Click here for additional data file.


Chromatin immunoprecipitation (ChIP) experiments are now the most comprehensive experimental approaches for mapping the binding of transcription factors (TFs) to their target genes. However, ChIP data alone is insufficient for identifying functional binding target genes of TFs for two reasons. First, there is an inherent high false positive/negative rate in ChIP-chip or ChIP-seq experiments. Second, binding signals in the ChIP data do not necessarily imply functionality.It is known that ChIP-chip data and TF knockout (TFKO) data reveal complementary information on gene regulation. While ChIP-chip data can provide TF-gene binding pairs, TFKO data can provide TF-gene regulation pairs. Therefore, we propose a novel network approach for identifying functional TF-gene binding pairs by integrating the ChIP-chip data with the TFKO data. In our method, a TF-gene binding pair from the ChIP-chip data is regarded to be functional if it also has high confident curated TFKO TF-gene regulatory relation or deduced hypostatic TF-gene regulatory relation.E. coli genome. Finally, we provided an example showing the biological applicability of our notion.We first validated our method on a gathered ground truth set. Then we applied our method to the ChIP-chip data to identify functional TF-gene binding pairs. The biological significance of our identified functional TF-gene binding pairs was shown by assessing their functional enrichment, the prevalence of protein-protein interaction, and expression coherence. Our results outperformed the results of three existing methods across all measures. And our identified functional targets of TFs also showed statistical significance over the randomly assigned TF-gene pairs. We also showed that our method is dataset independent and can apply to ChIP-seq data and the  Cellular responses to external stimuli or environmental changes are usually conveyed through cellular regulatory networks consisting of different regulatory pathways -4. TransChromatin immunoprecipitation experiments (ChIP-chip or ChIP-seq) are now the most comprehensive experimental approaches for mapping the binding of TFs to their target genes ,3,5. HowIt was suggested that one can improve the confidence of the TF-gene binding pairs by integrating ChIP-chip data with data from other high-throughput technologies . AlthougPrevious studies had tried to extract functional binding target genes of TFs by integrating the ChIP-chip data with various kinds of high-throughput data. By the types of the integrated data, the integration processes could be roughly divided into two categories. The first type of existing methods relied on stepwise integration of the ChIP-chip data with the expression data and/or the TF binding motif data. Functionality of the TF-gene binding pairs was confirmed by some gene properties inferred from the mRNA expression profiles. For example, ChIP positives were classified into functional and non-functional TF-gene binding pairs by the regression analysis of the mRNA expression profiles . And othThe other type of existing bioinformatic approaches for extracting functional TF-gene binding pairs combined diverse biological data beside the mRNA expression profile data through the construction of different types of Bayesian classifiers. Some utilized the framework of probabilistic inference to predict the functional TF-gene binding pairs by TF binding site motifs, evolutionary conservation, regulatory potential, nucleosome data and DNase hypersensitive sites . Others While previous works had combined comprehensive sorts of high-throughput experimental data and biological data, these approaches did not consider the TF-gene regulatory relation when inferring functional TF-gene binding pairs. Expression data, TF binding motif data and other integrated biological data, such as nucleosome positioning and evolutionary conservation, did not directly provide the TF-gene regulatory relation. Nowadays, the TF knockout (TFKO) data are available for biologists to infer the TF-gene regulatory relation . TFKO daE. coli genome. Finally, we provided an example showing the biological applicability of our notion.In this study, instead of using the supervised or unsupervised learning tools as in the Bayesian approach and other methodologies, our method uses a network approach on the combination of the ChIP-chip data and the TFKO data to infer the functional TF-gene binding pairs. A TF-gene binding pair, or a ChIP positive, is called functional if we can also find evidence showing that the TF regulates the expression of the target gene. While direct overlapping of the ChIP-chip and TFKO datasets could give some possible functional TF-gene binding pairs, this only provided a very small number of such pairs because of the low overlap of the ChIP-chip data and the TFKO data ,17,18. ISaccharomyces cerevisiae TFs produced by the ChIP-chip technology were adopted from [p-values, on the hypothesis that the TF binds to the promoter region of the target gene. To show the data-independence of our method, we also adopted the ChIP-chip data generated from [Saccaromyces cerevisiae, transcription factor binding sites are positioned further upstream in the intergenic regions and vary over a wide range in promoters [et al. [Genome-wide in vivo TF-gene binding data of 204 yeast ted from . The TF-ted from . In Sacc [et al. .Saccharomyces cerevisiae TFs were retrieved from the Yeastract Indirect evidence [The TF knockout data of 156 yeast evidence . YeastraSaccharomyces cerevisiae from ExpressDB [Two different datasets were collected for use in the biological validations. For showing the prevalence of protein-protein interaction, we gathered the physical protein-protein interaction data from the Biogrid database, which had deposited comprehensive collections of protein-protein interactions . And forxpressDB . These 4xpressDB . DetailsxpressDB . These cxpressDB , yeast cxpressDB ,25, the xpressDB ,27 and eA set of 484 functional TF-gene binding pairs adopted from  were useX in the constructed regulatory relation network, this means that the given TF regulates the TF X and the TF X regulates the given gene. We said that there is deduced hypostatic regulatory relation (to the epistatic regulation cascade through TF X on the target gene) in the constructed regulatory relation network for this given TF-gene pair  algorithm . The algt-test to test against the null hypothesis that the length of the shortest regulation cascade found in the constructed regulation relation network is statistically equal to the average of the lengths of the shortest paths in the randomly generated regulation network. Multiple hypotheses test correction was done by using the method of FDR. And the regulation confidence score (RCS) is calculated by the formula, which takes the minus logarithm on the corrected statistic p-value:The deduced hypostatic TF-gene regulatory relation might be introduced by chance since there is still a large amount of random noises in the original TFKO data due to the inherent uncertainty in high-throughput technologies. These inherent random noises could cause the over-fitting problem when deducing hypostatic relations from analysing the network paths . We avoiThe RCS measures the non-stochastic confidence of the given regulation pair.To calculate RCSs, we constructed 10000 degree-preserving TFKO random regulation networks. The choice for sampling size of 10000 from the random distribution is to have a sampling precision of 95% confidence within 1% of error, according to the sampling theorem . To geneTFKO data conveys the experimental results showing the change in the expression of some target gene resulting from the deletion or mutation of certain TF-encoding gene and the ChIP-chip data conveys the TF-gene binding information Figure . To extrX [X may not directly bind the target gene. This innovated us to find the possible masked hypostatic TF-gene regulation. The compensated TF-gene regulation was said to be hypostatic to the epistatic regulation cascade through TF X since the knockout effect of this TF-gene pair may possibly be masked by the epistatic regulation cascade with a more confident regulation of an intermediate TF X on the given target gene. This meant that there might exist at least an epistatic regulation cascade, or a path from the TF to its target gene through an intermediate TF X in the regulatory relation network, for this TF-gene pair. Therefore, we constructed a regulatory relation network from the TFKO data and sought the hypostatic TF-gene regulation relation by checking the existence of a regulation path in the regulation relation network for the given ChIP postives. This was done by a previously published path finding algorithm (as described in the Methods section).For a given TF-gene binding pair, if there was no literature-curated TFKO TF-gene regulation for it, we then tried to see if there exists a possible hypostatic TF-gene regulation for it. It was shown that the low overlap between the binding data and the TFKO data may partly result from knockout epistatic mechanisms and a single TF knockeout effect on a target gene may be compensated by the epistasis regulation cascade through another paralogous partner TF X ,17 , which was scored through the comparison with random TFKO data (See Methods section). A regulation relation with RCS higher than 1000 was set to be confident. Finally, a TF-gene binding candidate pair was classified to be functional if it has a confident TF-gene regulation evidence, which may come from the curated TF-gene regulation or the hypostatic TF-gene regulation.First we validated our proposed method on a gathered literature-curated functional TF-gene binding set from . The litAn ROC curve plot is a graphical tool demonstrating the performance of the discriminating algorithm as its discriminating score varies. The curve is plotted for (1-specificity) against sensitivity. Specificity is defined as the fraction of true negatives out of the discriminated negatives and (1-specificity) is also known as the false positive rate. And sensitivity, also known as the true positive rate, is defined as the fraction of true positives out of the discriminated positives. In the ROC curve plot of our method, we can see that our method acted as a good classifier for discriminating functional binding pairs from non-functional binding pairs  due to its performance of low false positive rates with high true positive rates (to the leftmost of the ROC curve plot). Notice that the trembling phenomenon between 0.2 and 0.3 shows that most of the discriminating scores, which is the RCS, resulted in false positive rate of 0.2 to 0.3 with true positive rate of about 0.7 to 0.8. Since our method does not rely on any training process, this result is unlikely to suffer from over-fitting. Hence we conclude that our method can distinguish functional TF-gene binding pairs from non-functional binding pairs.Saccharomyces cerevisiae because of the comprehensibility and availability of the genome-wide TFKO data source. Harbison et al. have performed the most comprehensive genome-wide chromatin immunoprecipitation microarray (ChIP-chip) experiments on the yeast Saccharomyces cerevisiae [p-value threshold of 0.001 in the original error models to ensure a low false positive rate. But it has been shown that the TF-gene binding pair might already be functional with the p-value threshold of 0.01 [In this study, we demonstrated our algorithm on yeast revisiae . And fro of 0.01 . Hence iet al. were possible for our analysis because of the lack of the TFKO data. After applying our proposed algorithm, we further required that the percentage of the functional binding targets of a TF should reach above 25% since we observed a 'jump' from 23% to 60% in the percentage distribution of the extracted functional binding target genes  of the reported TFs in the study of Harbison s Figure . Since tSaccharomyces cerevisiae were selected for our comparison because of data availability. For the methods of combining diverse biological data sources to extract the functional TF-gene binding pairs, the log likelihood score (LLS) method [Saccharomyces cerevisiae, co-expression clustering, physical protein-protein interaction data and the phylogenetic profiles of gene pairs were used.We next compared the biological significance of the functional TF-gene binding pairs identified in this study and by three previously published methods. Only three approaches on yeast ) method  is availFor the methods relying on stepwise integration of the ChIP-chip data with the expression data, there are two approaches available for our comparison. One is the method of using the expression coherence score (ECS) and TF binding sites information  and the In the following sub-sections, we showed that our results conveyed better biological relevance than these three previous works by testing the identified functional binding target genes of TFs on functional enrichment, the prevalence of protein-protein interaction and co-expression. Details of the following validations can be found in Additional File p-value threshold of 0.05.When several genes are functionally bound by the same TF, one might expect that the gene products of these genes are prone to carry similar cellular functions ,31. GeneOf the 62 common TFs between our results and the results of the LLS method, 59 TF functional binding target gene sets (95.2%) extracted by our method showed significant functional enrichment while only 54 TF functional binding target gene sets (87.1%) extracted by the LLS method bore significant functional enrichment. And comparing the 46 common TFs between our results and the results of the ECS method, our results still outperformed the results of the ECS method . As for the 18 common TFs between our results and the results of the MA algorithm, our results showed better functional enrichment   extracted by our method showed prevalence of protein-protein interaction while only 42 TF functional binding target gene sets (67.7%) extracted by the LLS method did. And among 46 TFs between our results and the results of the ECS method, 34 TF functional binding target gene sets (73.9%) extracted by our method showed prevalence of protein-protein interaction, comparing with only 23 TF functional binding target gene sets 50.0%) extracted by the ECS method did. For the 18 common TFs between our results and the results of the MA algorithm, 14 TF functional binding target gene sets (77.8%) extracted by our method showed prevalence of protein-protein interaction in comparison with only 12 TF functional binding target gene sets (66.7%) extracted by the MA algorithm did  the means of the square of correlation coefficients for the TF functional targets mined out in this study are lower than those generated by other methods. Multiple hypotheses test correction was done by FDR correction and a threshold of α = 0.05 was adopted.It has been shown that functionally relevant target genes of TFs tend to have similar mRNA expression profiles . Using tIn different expression time series conditions, we first counted the percentage of the functional binding target sets of TFs with statistically higher expression coherence. Note that the percentages of more expression coherent functional binding target sets of TFs in two different methods may not add up to 100% since some of the TF functional binding gene sets may have statistically invariant average expression correlations between two methods. Then when one method gained more functional binding target sets of TFs than the other, we said that this method is more expression coherent than the other under this expression time series condition.We compared our results with those of LLS, ECS, and MA methods for all 40 different expression profiles. Compared with the LLS method, our method conveyed better expression coherence under 31 different conditions. Our results were more correlated in the expression profiles than the results of the LLS method under most of the conditions. And our method showed more expression coherent pairs than the ECS method did under all 40 different conditions. Finally, compared with the MA algorithm, our method stood out under 21 conditions while the results of MA algorithm got better expression coherence under 15 conditions Figure . Our metTo make statistical assessment of the results in this study, we made simulations against random assignments of functional/non-functional TF-gene pairs. In our study, we have shown that about 82% of the original TF-gene binding pairs suggests functionality. Hence we randomly removed 18% of the original binding targets from the 72 analysable TFs as the random assignment of non-functional TF-gene binding targets. We repeated this process for 50 times and gained 50 randomly assigned functional TF-gene binding pair lists. Then we performed the biological significance validation for the randomly assigned results as the stochastic lower limit of the performance of the validation methods.t-test to assess the significance of our result, compared to randomly generated TF-gene assignments for these 72 analysable TFs. The test was performed on the hypothesis that the average performance of the random results are statistically lower than the results in this study. As for the expression coherence validation, we performed the paired two sample t-test for our results and the random ones in every expression condition. In each condition-specific expression profile, we used the rank sum test as described earlier on the two stated hypotheses to compare the expression coherence between the result in this study and the randomly assigned TF-gene lists. The number of target gene sets satisfying the hypothesis of "results in this study is better than the random results" and the number of target sets satisfying the hypothesis of "results in this study is worse than the random results" for the comparison of our results to the 50 randomly assigned TF-gene lists formed the testing pairs. We said that our result is better than random assignments in the specific condition if we have the right-tailed p-value by the paired t-test on the 50 testing pairs below 0.05 in this condition.After that, for the functional enrichment validation and the prevalence of protein-protein interaction validation, we used the left-tailed one sample student p-value threshold of 0.05). For the 72 TFs, our results generated 62 functionally enriched functional binding target gene sets of TFs, compared to the performance of random assignments with mean and standard deviation equal to 44.8 and 3.53, respectively . For the validation of prevalence of protein-protein interaction, there were 53 functional binding target gene sets of TFs in this study showing prevalence of protein-protein interaction, while the random lists obtained a performance with mean and standard deviation of 17.68 and 3.78, respectively . Finally for the expression coherence validation, in 39 of the 40 different expression conditions our results were statistically more expression-coherent than the 50 random lists. Detail of the validation results can be found in Additional File As shown in Table et al. [et al., we compared the biological relevance of this result with those generated by the ECS method and the MA algorithm. In applicable biological validations, similar conclusion also held for these comparisons  related to cell fusion  and mating  were identified in the filtered functional TF-gene binding targets but not in the original target list  as an example. BAP2 is a gene encoding a permease in Saccharomyces cerevisiae for the uptake of branched-chain amino acids from media containing nitrogen source [BAP2 greatly reduced the up-take of leucine, isoleucine and valine. And Leu3p is a TF in yeast that regulates the transcription of a group of genes involved in leucine biosynthesis [et al., the promoter region of BAP2 was found to be bound by Leu3p . But there were no single TF knockout evidence showing the regulation of BAP2 by Leu3p. In our method, we found out that although there were no TFKO evidence for this TF-gene pair, we could find a regulation cascade (Leu3p → Msn4p → Rpn4p → Yap1p → Stp1p → BAP2) in the constructed regulation relation network through the TF Stp1p with RCS bigger than 1000. Hence we concluded that the ChIP positive  has hypostatic regulation evidence and is a functional TF-gene binding pair.We have listed the potential epistatic regulation cascade for every functional TF-gene binding pairs settled in this study  Prevalence of protein-protein interaction validation 2) Functional enrichment validation 3) Expression coherence validation. Detailed description of the raw files is written in the file 'ReadMe.doc' in Additional File 3.Click here for fileet alSupplementary validation for the biological significance on the results using the dataset generated by Lee . Additional file 4 contains Figure S1-S3 showing the biological significance validation on our results using the dataset generated by Lee et al. Figure S1 demonstrated the percentages of functional binding target genes of TFs with available TFKO data. Figure S2 showed the results of functional enrichment validation and prevalence of protein-protein interactions validation. Figure S3 showed the results of the expression coherence comparison.Click here for fileSummary of the comparison of our results to the 50 randomly assigned TF-gene lists. Additional file 5 contains the table showing the detailed summary for Table 1 to Table 3, both for the results generated using the dataset of Harbison et al. and the results generated using the dataset of Lee et al..Click here for fileValidation results of our results compared to 50 randomly assigned TF-gene lists. Additional file 1 zipped the raw files for the three biological validations on the comparison of our results, both for the results generated using the dataset of Harbison et al. and the results generated using the dataset of Lee et al., to the 50 random TF-gene lists. Detailed description of the raw files is written in the file 'ReadMe.doc' in Additional File 6.Click here for file


In the autoimmune disease systemic lupus erythematosus (SLE), our normal antiviral defenses are inappropriately activated, resulting in over-activity of the type I interferon (IFN) pathway. This increased activity of the type I IFN pathway is an important primary pathogenic factor in the disease. Emerging evidence has implicated the antiviral helicases in this process. The antiviral helicases normally function as nucleic acid receptors in viral immunity. Genetic variations in antiviral helicase genes have been associated with SLE, supporting the idea that helicase pathways are involved in the primary pathogenesis of SLE. Studies have documented functional consequences of these genetic variations within the type I IFN pathway in human cell lines and SLE patients. In this review, we summarize the function of helicases in the anti-viral immune response, and how this response is dysregulated in SLE patients. In particular, we will focus on known functional genetic polymorphisms in the IFIH1 (MDA5) and mitochondrial antiviral signaling protein genes which have been implicated in human SLE. These data provide fascinating evidence for dysregulation of helicase-mediated innate immunity in SLE, and may support novel therapeutic strategies in the disease. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multisystem inflammation commonly including the skin, kidneys, and joints, and other systems. While the pathogenesis of SLE is not completely understood, it seems likely that both genetic and environmental factors contribute to the disease. A number of genetic factors have been associated with SLE in recent years , providide novo SLE has developed .Several families of receptors that recognize pathogen-associated molecular patterns (PAMPs) have been described, such as the Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs). TLRs are transmembrane receptors expressed in specific immune cells, such as dendritic cells and macrophages. TLR7, 8, and 9 are expressed in the endosomal membrane, and can recognize viral nucleic acid. In anti-viral immunity, viral immune complexes are taken up via Fc receptors, and then delivered to the endosome resulting in TLR activation. RLRs, on the other hand, are cytosolic proteins that can recognize viral nucleic acid in the cytosol. Activation of either TLRs or RLRs results in IFN production and an anti-viral response .Activation of RIG-I and MDA5 by nucleic acid leads to binding of the mitochondrial adaptor IFN β promoter stimulater 1 (IPS-1) also known as MAVS . As the IFIH1 is the gene that encodes MDA5, and a common coding-change polymorphism in the IFIH1 gene has been associated with risk of SLE and other autoimmune diseases in humans . This A9N-ethyl-N-nitrosourea (ENU) resulted in constitutive activation of MDA5  generated by  of MDA5 . Mice wi of MDA5 . This gaGenetic studies in human SLE have also identified a functional coding-change polymorphism in MAVS, a key adapter of both the RIG-I and MDA5 helicases. The C97F polymorphism in MAVS substantially reduced the expression of type I IFN and other proinflammatory mediators in human cell lines . InteresDysregulation of anti-viral helicase immune responses represent a primary pathogenic factor in human SLE. This is demonstrated by the presence of coding-change polymorphisms in both the IFIH1 and MAVS genes which modulate function of the type I IFN pathway and risk of SLE in humans. While immune complexes formed by SLE autoantibodies and nuclear material seem to be the likely trigger for endosomal TLR stimulation in SLE, the exact triggers of the cytosolic anti-viral helicases in human SLE are somewhat less clear. Viruses may stimulate some of the early events in SLE patients, as strong epidemiological data implicates Epstein–Barr virus infection in the initial pathogenesis of SLE . It is pThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Biochemical non-adherence to antihypertensive treatment was the most common cause of non-eligibility for renal denervation—23.5% of patients were either partially or completely non-adherent to prescribed antihypertensive treatment. About 5.9% of those referred for renal denervation had admitted non-adherence prior to performing the screening test. Suboptimal pharmacological treatment of hypertension and ‘white-coat effect' accounted for apparently resistant hypertension in a further 17.7 and 5.9% of patients, respectively. Taken together, these three causes of pseudo-resistant hypertension accounted for 52.9% of patients referred for renal denervation. Only 14.7% of referred patients were ultimately deemed eligible for renal denervation. Without biochemical screening for therapeutic non-adherence, the eligibility rate for renal denervation would have been 38.2%. Non-adherence to antihypertensive treatment and other forms of therapeutic pseudo-resistance are by far the most common reason of ‘resistant hypertension' in patients referred for renal denervation. We suggest that inclusion of biochemical screening for non-adherence to antihypertensive treatment may be helpful in evaluation of patients with ‘resistant hypertension' prior to consideration of renal denervation.Renal denervation is a potential therapeutic option for resistant hypertension. A thorough clinical assessment to exclude reversible/spurious causes of resistance to antihypertensive therapy is required prior to this procedure. The extent to which non-adherence to antihypertensive treatment contributes to apparent resistance to antihypertensive therapy in patients considered for renal denervation is not known. Patients ( The major emphasis in the existing guidelines lies on potentially offering this therapeutic approach only to patients whose uncontrolled hypertension has no identifiable/reversible and/or potentially spurious cause. Hence, white-coat effect/white-coat hypertension, suboptimal pharmacological antihypertensive treatment and secondary hypertension have been proposed as important screening and potential exclusion criteria for renal denervation.12 Many centres assessing patients for renal denervation developed their own eligibility criteria based on those published by European Society of Hypertension (ESH) and/or replicated the inclusion criteria for the Symplicity HTN-2 trial.16 Based on those criteria only ≈10–50% of referred patients were eligible for renal denervation.16 However, the reported rates may be a significant overestimation of the ultimate suitability for renal denervation, as systematic and objective screening for non-adherence to antihypertensive treatment (one of the most common form of pseudo-resistant hypertension)19 was not a part of the evaluation process in a majority of the studies.16Percutaneous radiofrequency catheter-based renal sympathetic denervation  has recently been introduced and evaluated as a potential treatment for resistant hypertension.20 Using this objective method of screening for non-adherence to antihypertensive treatment, we previously reported that approximately one in four of hypertensive patients are partly or completely non-adherent and those referred for renal denervation seemed to show particularly high rates of complete non-adherence.20We have recently developed a highly sensitive and specific assay that utilises high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to detect antihypertensive medications in spot urine samples.In the present analysis, we have examined the extent to which integration of biochemical screening for non-adherence to antihypertensive treatment into the diagnostic pathway may affect the ultimate eligibility rates for renal denervation. We also compared the frequency of biochemical non-adherence to antihypertensive treatment with other causes of non-eligibility for renal denervation and evaluated the importance of HPLC-MS/MS in assessment of patients referred for this intervention.12 The pathway consisted of several diagnostic steps. First, in addition to clinic BP measurements, either 24-h ambulatory BP (24 h ABPM) or 7-day home BP monitoring was conducted to objectively measure BP and exclude the presence of a white-coat effect and/or white-coat hypertension. Clinic BP was recorded using a validated oscillometry device . The 24-h ABPM and 7-day home-based monitoring were performed using calibrated BP measuring devices . All BP measurements were performed as recommended with a cuff size adjusted to the size of the arm.21 White coat effect was identified as a difference of >20 mm Hg between the clinic systolic BP and mean daytime ambulatory systolic BP or the average systolic BP recorded on the 7-day home BP monitor.22 Patients without evidence of white-coat effect or white-coat hypertension in whom out-of-office daytime SBP was >150 mm Hg were eligible for further diagnostic steps in the pathway for renal denervation.11A total of 34 patients referred for renal denervation at a local specialist hypertension centre  from December 2011 to December 2013 entered the evaluation pathway for renal denervation. This pathway was developed based on ESH guidelines and Joint UK Societies Consensus Statement on Renal Denervation recommendations.20 Our HPLC-MS/MS urine-based screening for therapeutic non-adherence can detect 40 of the most commonly prescribed antihypertensive medications including all those that were prescribed to examined patients. The detailed list of these medications is published elsewhere.20 In brief, a single spot urine sample was collected after verbal consent from patients on the day of their clinic appointment. Samples were prepared by solvent extraction and by dilution technique before the analysis by HPLC-MS/MS . The results of the biochemical screening were then reviewed against the prescribed antihypertensive medications by a panel of hypertensive specialists to ascertain that the referred patients were both adherent to BP-lowering therapy and that they received the most optimal combinations of medications in the most appropriate dosages.Second, screening for non-adherence to treatment was conducted using HPLC-MS/MS-based urine analysis, as reported previously.23 Where deemed appropriate, patients underwent additional investigations to exclude secondary causes of hypertension. Screening tests included measurement of plasma renin activity , circulating concentrations of aldosterone (by radioimmunoassay: SAS Steroid Hormone Centre), 24-h urinary excretion of adrenaline, noradrenaline and metanephrines (by HPLC-MS/MS: in-house method), serum cortisol (by immunoassay: ADVIA 2400) after overnight dexamethasone suppression test and 24-h urinary excretion of free cortisol (by HPLC-MS/MS: in-house method). Abdominal ultrasound, echocardiogram and abdominal magnetic resonance imaging were conducted where appropriate. The anatomy of kidneys and renal arteries was assessed by magnetic renal angiography according to the previously suggested criteria ).12Appropriate basic biochemical investigations were also included in the protocol—serum creatinine was measured by the Jaffe method , and estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula.The final decision of patient eligibility for renal denervation was taken after review of each case by a multi-disciplinary team involving specialist clinicians in hypertension and vascular radiologists. For each patient, the main reason for non-eligibility was identified based on information on the results of screening collected using dedicated forms. These forms together with the clinical files were reviewed for the purpose of this audit project.The project was approved by the University Hospitals of Leicester NHS Trust as a retrospective analysis of patients referred for assessment of their suitability for renal denervation (audit registration number: 6930).The clinical characteristics of patients referred for renal denervation is summarised in −1 per 1.73 m2). All patients were on standard dosages of antihypertensive pharmacotherapy and did not require any alteration in their dosage schedule due to eGFR.All patients had stable eGFR prior to inclusion in this audit. About 20.6% (7 of 34) of them had reduced eGFR . Our data also indicate that collective exclusion of three well-recognised causes of pseudo-resistant hypertension  early in the diagnostic pathway may reduce the apparent eligibility for the intervention by >50%. Even if one considers that nobody should undergo renal denervation outside of the context of a properly designed randomised controlled trial, this data support the view that more robust screening for ‘pseudo-resistance' should be undertaken, especially for non-adherence to therapy before undertaking this procedure.26 However, screening for non-adherence to antihypertensive treatment was mentioned only in a minority of these studies.26 One of the largest to date analysis conducted by Persu et al.13 attempted to exclude non-adherence to antihypertensive treatment in their multi-centre survey but only ≈50% of the participating sites included some form of testing for non-adherence to antihypertensive treatment and only 1 of 11 sites used an objective biochemical method of screening for presence of BP-lowering medications in body fluids. Therefore, based on the data from this study it is difficult to conclude how frequently true non-adherence to antihypertensive treatment would have led to exclusion from renal denervation. This may explain significantly lower rates of non-adherence in their study when compared with our data.13 Rosa et al26 screened objectively for non-adherence to antihypertensive treatment among patients referred for renal denervation and found a much lower—12.5% non-adherence rate—around half of the figure reported in this study. However, in contrast to our analysis, they conducted the screening for therapeutic non-adherence after exclusion of secondary hypertension. The difference in order of these investigations between both studies may explain why we have captured a higher number of non-adherent patients than Rosa et al.26Nearly all previous studies reported that a majority of patients assessed for suitability for renal denervation were non-eligible for this procedure.16 Persu et al.13 reported that inappropriate pharmacological treatment of hypertension accounts for almost 50% of non-eligibility for renal denervation. Our data also shows a high rate of suboptimal BP-lowering therapy—approximately one in three patients considered for renal denervation required adjustment/changes in the medications. The lower percentage of patients on inappropriate medications in our study as compared with the data reported by Persu et al.13 may probably be explained by an early exclusion of non-adherent patients in our diagnostic pathway.The main reasons for exclusion from renal denervation differ between studies. These discrepancies are most likely driven by the differences in clinical characteristics of examined populations, the selection criteria used to exclude patients from renal denervation and the priority assigned to these criteria. Similar to previous studies, our data shows that insufficiently high BP is one of top two reasons for ineligibility for renal denervation.16 Collectively, non-adherence to treatment and other forms of pseudo-resistant hypertension were a much more common cause of non-eligibility for renal denervation than secondary hypertension. Therefore, we propose that screening for non-adherence to treatment is most cost-effective if conducted prior to tests to exclude secondary hypertension—a significant proportion of patients might not require expensive investigations to exclude secondary hypertension if the biochemical screening for non-adherence to antihypertensive treatment is performed early in the diagnostic pathway.Secondary hypertension was a relatively uncommon reason for exclusion from renal denervation in our analysis. Indeed, <10% of patients were ineligible for renal denervation based on this clinical criterion. Similar percentages of secondary hypertension-driven exclusion from denervation were reported by several previous studies.21 Thus, primary care physicians (who referred a large proportion of patients to our centre) might have preferred the former option. This could explain why less than half of the patients were on spironolactone when referred for consideration of renal denervation. The NICE guideline in UK also recommends that 24-h ABPM monitoring is conducted for the diagnosis of hypertension.21 This test is often arranged in primary care prior to referral to specialist centres. This, in our opinion may account for a relatively low prevalence of white-coat effect in our patient sample when compared with estimates from other studies27—individuals with white-coat effect may have been excluded prior to the referral to our centre.Our study was based on a real life retrospective analysis of patients considered for renal denervation because of difficulty to treat hypertension (based on assessment of referring clinicians) rather than one specific diagnosis of resistant hypertension (many of which require the presence of a diuretic in the treatment). A significant number of referrals were received from non-specialists from primary care. The UK National Institute of Clinical Excellence (NICE) guideline recommends that hypertensive patients who are considered as resistant to treatment be either referred to specialist clinics or have spironolactone added as a fourth line antihypertensive.26 We cannot exclude that variable information about renal denervation received, prior to the referral to our centre, may have driven the initial agreement to enter the diagnostic pathway by 4 out of 5 potentially eligible patients. It is likely that further in-depth information about renal denervation  provided later by the local specialist centre might have contributed to their change of mind.Our study revealed a high rate of potentially eligible patients who having completed the diagnostic pathway to renal denervation successfully have decided not to undergo the procedure. This rate appears higher when compared with other previously published data.25 Therefore, non-adherence to antihypertensive therapy would very likely remain a problem in such patients after renal denervation. We should also acknowledge the potential limitations of renal denervation as the therapeutic strategy in non-adherent patients from the healthcare economy point of view. Indeed, in our experience non-intentional non-adherence to treatment (driven by forgetfulness and/or polypharmacy) is the main single cause of deviations from the prescribed antihypertensive treatment.28 In such patients, non-adherence can be improved by simple and cheap measures such as simplifying the medication regime and providing tools to aid patients to remember to take their medications. Finally, non-adherence due to side effects can be managed by altering therapy. Any irrational patient beliefs about side effects of antihypertensive medications driving the non-adherence could also be addressed by targeted patient education that is clearly much more economically viable when compared with expensive renal denervation.32It can be argued that renal denervation may be an attractive therapeutic option in hypertensive patients who are non-adherent to BP-lowering treatment. Indeed, renal denervation is a one off procedure and based on the existing data would not require extensive follow-up. However, the available data, obtained from research that was not designed to study non-adherent patients, suggests that renal denervation does not cure hypertension and patients continue to require antihypertensive pharmacotherapy.Therefore, in our opinion, renal denervation at present is not a suitable approach to optimise BP control in patients who are non-adherent to antihypertensive treatment.36 The HPLC-MS/MS technique is used by many laboratories especially in forensic science for the detection of chemical compounds/pharmaceuticals in bodily fluids. Although the technology is widely available in UK university hospitals, only our centre provides HPLC-MS/MS-based urine analysis as a biochemical screening for non-adherence to antihypertensive treatment nationwide. The test is relatively inexpensive. A recent predictive modelling study of the use of screening for non-adherence to antihypertensive treatment using HPLC-MS/MS-based analysis of urine samples showed the cost-effectiveness of this method in management of resistant hypertension.29HPLC-MS/MS-based urine analysis to screen for non-adherence to antihypertensive treatment can be easily applied in real life clinical practice as a non-invasive reproducible method.37 This increases the duration of half-lives and therefore the duration of presence of the medications in the urine. Thus, the impairment of GFR would if anything extends the detection window for the medications and their metabolites in both blood and urine. In clinical practice, HPLC-MS/MS-based detection of antihypertensive medications can be performed using blood26 or urine.33 In our opinion the major advantage of using urine samples for the purpose of biochemical screening to non-adherence lies in the non-invasive nature of specimen collection—patients are less likely to refuse donation of urine sample than a blood sample. The possible exception is a group of patients with complete anuria in whom plasma/serum-based test is the obvious choice.The detection of antihypertensive medications in urine by HPLC-MS/MS is not negatively affected by patients' low GFR. Indeed, the clearance of medications from plasma (and their subsequent appearance in urine) is decreased in patients with chronic kidney disease with reduced GFR.39 This can translate into the individual differences in pharmacokinetics of antihypertensive medications and other therapeutics as reflected in the published half-lives references for many medications. These are defined in ranges .37 However, for a majority of antihypertensive medications the half-lives were in the range such that even if patients were genetically mediated fast metabolisers, they would not be classified as non-adherent if they took their medications. Future studies should focus on in-depth pharmacogenetic analysis of commonly prescribed antihypertensive medications to characterise the patterns of their elimination in relation to biochemical screening for non-adherence to treatment.Non-detection of a drug in urine or plasma is related to inter-individual variation in pharmacogenetics .40 Monitoring of prescription pick up rates may be informative but requires good electronic records.42 In addition, picking up a prescription does not always equate with taking prescribed medication. Directly observed administration of antihypertensive therapy is sometimes used43 but it is expensive, time-consuming and may be clinically hazardous. Indeed, there are anecdotal reports of seemingly adherent patients with resistant hypertension who after supervised ingestion of the prescribed antihypertensives developed hypotension and had to be admitted to hospital.The routine use of other methods of screening for non-adherence to treatment has several major limitations. Indeed, although questionnaires such as Modified Morisky Adherence Scale are simple to use, they tend to have limited specificity in ascertaining non-adherence.13 We acknowledge that a single biochemical screening for non-adherence to treatment as reported here cannot confirm that the patient is non-adherent (or adherent) in the long-term.41 It is also possible that some patients may improve their adherence directly prior to attending a clinic .44 The growing interest in persistence (a measure of long-term patterns of adherence) requires further studies that will assess the extent of correlation between single point detection of non-adherence with its long-term patterns. Finally, further studies on reasons why patients are non-adherent to treatment are important. Indeed, knowing the barriers to therapeutic non-adherence will make its management easier to target by elimination/reducing the impact of the factors that prevent patients to take their antihypertensive medications on a regular basis.We also appreciate several limitations of this analysis. First, the data comes from a retrospective single-centre analysis. However, the sample size reported here is typical for a majority of single European specialist centres involved in assessment of patients referred for renal denervation.Our data confirm that a vast majority of patients referred with resistant hypertension for consideration for renal denervation would not be eligible for this procedure according to current guidelines because their BP elevation is not driven by pharmacologically untreatable hypertension but primarily by therapeutic pseudo-resistance (of which non-adherence to antihypertensive medication is the most common from). Our study suggests that adherence to antihypertensive medications by HPLC-MS/MS urine-based analysis may need to be confirmed earlier in evaluation pathway than exclusion of secondary causes of hypertension to establish eligibility for renal denervation. Our data concur with notions that previously proposed wide-spread use of renal denervation, even if it worked, may have very limited clinical justification as the prevalence of genuinely resistant hypertension even in the specialist centres is low. While the initial enthusiasm for renal denervation driven by the first two Symplicity trials has been dampened by the results of Symplicity-HTN-3 trial, renal denervation remains available as an experimental therapeutic strategy for carefully selected patients in specialist centres in some countries. Use of biochemical screening for non-adherence treatment early in the evaluation pathway for renal denervation in future studies may help to establish the genuine eligibility of patients for the procedure. Further, larger studies are needed to clarify the best method of assessing adherence to treatment in resistant hypertensive patients referred for renal denervation.


Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 – an aberrant tyrosine kinase – in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs. BCR-ABL1 fusion gene resulting from a reciprocal translocation between chromosomes 9 and 22 (the Philadelphia (Ph) chromosome) that gives rise to an aberrant chimeric tyrosine kinase (TK) (reviewed in336Chronic myeloid leukaemia (CML) is clonal stem cell disorder driven by the 9/L, WBC 75.5 × 109/L) and splenomegaly. A peripheral blood (PB) and bone marrow (BM) morphological examination were consistent with the chronic phase of a myeloproliferative disorder. Conventional cytogenetic analysis of BM revealed a normal male karyotype with the presence of Ph chromosome in 21/21 metaphases. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the ipsogen BCR-ABL1 Mbcr IS-MMR Kit  detected existence of the p210 fusion transcript and the BCR-ABL1/ABL1 ratio of 106.25% was determined. The diagnosis of low-risk  Ph+ CML in chronic phase (CML-CP) was thus made. After an initial cytoreduction using hydroxyurea for two weeks, standard dose imatinib treatment was initiated. Within 3 months of treatment, complete haematological response anti-CML  were achieved albeit persistent lymphocytosis occurred  locus on chromosome 14. We confirmed by PCR amplification and subsequent sequencing the involvement of the two loci. Next, in order to track the origins of the lymphoma, we designed primers  mapping up and down-stream of the fusion junction and applied them to cDNA retrotranscribed from mRNA sample extracted at the time of CML diagnosis. The end point-qualitative PCR experiments showed that the transcript deriving from the t translocation fusion was present at the time of the first diagnosis  formation. The experiments with the Vysis LSI BCR/ABL DC/DF probe  disclosed the presence of an atypical hybridisation pattern consisting of one fusion signal, two green and one red signal  showed that the diagnostic CML sample contained about 4.6% (48/1028) of cells with the rearrangement in the TCRA/D locus (BCR and ABL being four times bigger than the TCRA/D Break-apart ones (600 kb vs ~150 kb). Cells with BCR/ABL fusion and no TCRA/D rearrangement as well as cells with TCRA/D split and no BCR/ABL fusion were identified in percentages equal to the ones seen for single-probe experiments. No cells with both abnormalities were encountered in the diagnostic CML sample  was also present. However, FISH experiments confirmed that there were no cells in which the two translocations co-existed, i.e. PTCL cells are BCR/ABL negative. This is inconsistent with the theory of a subclone of CML evolving into PTCL as one would expect that such clonal evolution resulted in a cell that continued to bear t and express BCR-ABL1. There is a slight discrepancy between the results of qPCR and FISH data when it comes to the evaluation of the CML burden at the time of PTCL diagnosis. The explanation for it includes several issues – the transcript levels/cell and the efficiency of PCR for the two primer pairs may be different in the two clones, the Cts were high and therefore outside of the range of accurate quantification. Nonetheless, there is no doubt as to the presence of residual BCR-ABL1 transcript at the time of PTCL diagnosis, as diagnostic and nested protocols showed its presence.In order to answer the question of whether the two diseases manifested in the case presented here were genetically related or a coincidence, we took advantage of the presence of disease-specific translocations as clonal markers and looked for the presence of sequences derived from the respective fusions in the reciprocal diagnostic samples. The end point-qualitative PCR experiments showed that the transcript deriving from the t translocation fusion was already present at the time of the first diagnosis suggesting that a small clone of lymphoma cells existed 10 months before the finding of an overt lymphoma or that a small subclone of The novelty of the case described here lies also in the finding of the t translocation in PTCL. This translocation, together with the more frequent inversion of the chromosome 14, has been previously described in T-cell prolymphocytic leukaemiaet al.Although secondary tumours have been described after imatinib therapy, the analyses of their frequency with respect to the age-adjusted general population yielded conflicting results341718ipsogen BCR-ABL1 Mbcr IS-MMR Kit  was used according to manufacturer’s instructions. Immunophenotypic/cytogenetic analyses of CML and PTCL were performed following standard diagnostic procedures and were carried out in accordance with the guidelines approved by Ethics Committee of the Istituto Europeo di Oncologia, Milan, Italy. Primers listed in We obtained informed consent from the patient prior to sample collection. The How to cite this article: Gruszka, A. M. et al. Molecular investigation of coexistent chronic myeloid leukaemia and peripheral T-cell lymphoma - a case report. Sci. Rep.5, 14829; doi: 10.1038/srep14829 (2015).