Weight loss reduces risk factors associated with obesity. However, long-term metabolic improvement remains a challenge. We investigated quantitative gene expression of subcutaneous adipose tissue in obese individuals and its relationship with low calorie diet and long term weight maintenance induced changes in insulin resistance.Three hundred eleven overweight and obese individuals followed a dietary protocol consisting of an 8-week low calorie diet followed by a 6-month ad libitum weight-maintenance diet. Individuals were clustered according to insulin resistance trajectories assessed using homeostasis model assessment of insulin resistance (HOMA-IR) index. Adipose tissue mRNA levels of 267 genes selected for regulation according to obesity, metabolic status and response to dieting was assessed using high throughput RT-qPCR. A combination of discriminant analyses was used to identify genes with regulation according to insulin resistance trajectories. Partial correlation was used to control for change in body mass index.Three different HOMA-IR profile groups were determined. HOMA-IR improved during low calorie diet in the 3 groups. At the end of the 6-month follow-up, groups A and B had reduced HOMA-IR by 50%. In group C, HOMA-IR had returned to baseline values. Genes were differentially expressed in the adipose tissue of individuals according to groups but a single gene, CIDEA, was common to all phases of the dietary intervention. Changes in adipose tissue CIDEA mRNA levels paralleled variations in insulin sensitivity independently of change in body mass index. Overall, CIDEA was up-regulated in adipose tissue of individuals with successful long term insulin resistance relapse and not in adipose tissue of unsuccessful individuals.The concomitant change in adipose tissue CIDEA mRNA levels and insulin sensitivity suggests a beneficial role of adipose tissue CIDEA in long term glucose homeostasis, independently of weight variation.NCT00390637ClinicalTrials.gov  Insulin resistance (IR) and type 2 diabetes are strongly associated with excess fat mass. This is reversible with weight loss The adipose tissue (AT) is a tissue devoted to energy storage as triglycerides. An overload of the buffering capacities of AT leads to a pro-inflammatory, diabetogenic and atherogenic status Carefully monitored weight-control diets favorably affect parameters of the metabolic syndrome and delay the onset of diabetic complications. As a main target tissue during dietary interventions, adaptations occurring in AT are likely to have a profound impact on the whole body response.Up to now, gene expression remains the most powerful method to comprehensively explore molecular adaptations from minute amounts of tissue Here, we investigated whether individuals respond differently to the same dietary intervention regarding IR. We hypothesized that AT gene profiling would help identifying novel biomarkers that underline long term changes in IR with weight loss.The project Diet, Obesity and Genes (DiOGenes) is a pan-European study which was approved by the ethics committees of each of the 8 European centers participating to the program. Written informed consent was obtained from each patient according to the local ethics committee of the participating countries: 1, Medical Ethics Committee of the University Hospital Maastricht and Maastricht University, The Netherlands; 2, The Committees on Biomedical Research Ethics for the Capital region of Denmark, Denmark; 3, Suffolk Local Research Ethics Committee, UK; 4, University of Crete Ethics Committee, Greece; 5, the Ethics Commission of the University of Potsdam; 6, Research Ethics Committee at the University of Navarra, Spain; 7, Ethical Committee of the Institute of Endocrinology, Czech Republic; 8, Ethical Committee to the National Transport Multiprofile Hospital in Sofia, Bulgaria.ad libitum diet (WMD) consisting in one of four reduced fat diets that differed in glycemic index of carbohydrates (low or high) and protein content (low or high), or a control diet according to National Dietary Guidelines in the participating countries. Target intakes in the low protein diets were 10–15% energy intake for proteins and 57–62% for carbohydrates and, in high protein diets, 23–28% energy intake for proteins and 45–50% energy intake for carbohydrates. During WMD, in 2 centers, the individuals were provided dietary instruction plus free foods. At the remaining 6 centers, they received dietary instruction. Dietary intake was assessed at screening (prior to baseline), four weeks after the beginning of WMD and at the end of WMD. The subjects were asked to complete a 3-day weighed food record, including 2 week days and 1 week-end day. All dietary records were validated by a nutritionist. Despite low glycemic index diets were targeted to a reduction of 15 glycemic index points compared with the high glycemic index diets, the mean glycemic index in the low glycemic index groups was only 5 units lower than that in the high glycemic index groups DiOGenes is a multicentre dietary intervention study. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The 267 genes, the corresponding biological functions and the strategy used for selection are summarized in GUSB mRNA, using the comparative cycle threshold (Ct), 2−ΔΔCt method. The present study was based on a subgroup of 311 obese individuals (107 men and 204 women) with bio-clinical and adipose tissue gene expression data available at each CID  were performed as previously described each CID .2 tests) were carried out with SPSS Statistics version 17.0 for windows .R software (version 2.14.0) was used for hierarchical clustering, Random Forests (RF), sparse Partial Least Square Discriminant Analysis (sPLS-DA) and partial correlations, along with different R packages. Conventional statistical analyses  to get more homogeneous clusters resulting in a final analysis of 216 individuals.2, one-way ANOVA , Kruskal-Wallis, Wilcoxon (paired data) and Mann and Whitney (unpaired data) tests  with Bonferroni correction. Significance cut-off was set at 0.01 which is lower than the Bonferroni adjusted p value (0.0167).Gaussian distribution of data was tested using the Kolmogorov–Smirnov test. Group comparisons were made using the χrandomForest) and sPLS-DA (R package : mixOmics). RF is an ensemble of classification trees calculated on random subsets of the data, using a subset of randomly selected variables for each split in each classification tree. Mean Decreased Gini (MDG) is used as an indicator of importance of the variables. The sPLS-DA is based on the PLS approach in which there is only one dependent variable chosen to represent the class/group membership. The variable influence on projection (VIP) indicates the influence of each variable on the discrimination between the different groups. These two methods were chosen because of their complementarity: sPLS-DA is a method taking place in a linear framework whereas RF is a nonlinear one. To improve classification power, when comparing baseline to the end of the dietary intervention, groups A and B  were compared to group C. During LCD and WMD, because the variations in HOMA-IR were different in the 3 groups, no group aggregation was made. For each phase of the dietary intervention, the most important transcripts in the classification were ranked according to decreasing VIP or MDG. A classification score was calculated as the sum of the rank for each gene. Partial correlations were computed to assess direct correlations independently of other confounding variable, i.e. change in body mass index (BMI) Two multivariate discriminant analyses were used to identify the important variables (i.e. mRNA levels) for group classification: RF  which showed different profiles of change in HOMA-IR during the whole dietary intervention .At baseline, there was no significant between group difference in bio-clinical parameters except for blood pressure, fasting plasma glucose and insulin, and HOMA-IR . Groups Changes in bio-clinical characteristics during the 2 phases of the dietary intervention according to groups are displayed in CIDEA was found in top 5 ranking in all phases. In order to control for body weight changes, partial correlations were computed. Only genes with significant class prediction potency independent of changes in BMI were taken into account. When adjusting for change in BMI, CIDEA was the only gene with change in mRNA levels allowing distinction of individuals with alleviated IR from those who did not.The relationship between AT gene expression and the HOMA-IR response in the 3 groups was investigated using sPLS-DA and RF during each phase of the dietary intervention. Genes with the top 5 classification scores were considered . CIDEA wCIDEA mRNA levels according to groups during dietary intervention mirrored the evolution of insulin sensitivity  and 80 had no decrease in HOMA-IR between baseline and the end of WMD (fold change HOMA-IR >1). As shown on CIDEA mRNA levels by 47%  while those who returned to the original HOMA-IR value had no significant change in CIDEA gene expression .In the whole data set of 311 individuals, we could explore the relationship between changes in AT CIDEA as associated to changes in IR independently of weight change during a two-phase longitudinal dietary program including a low calorie diet-induced weight reduction and subsequent follow-up.Hypocaloric weight-reducing diets improve the metabolic profile of obese patients. However, the reasons for the limited longstanding success of dieting remain poorly understood. Here we focused on AT gene profiling and show regulation of The present analysis is an ancillary study to the DiOGenes trial which focused on body weight control after caloric restriction First, 3 groups were defined according to HOMA-IR profiles during the two phases of the dietary intervention, resulting in the study of 216 individuals sourced from the 311 obese patients group . There wCIDEA as the most consistent transcript related to groups. The changes in CIDEA mRNA levels mirrored changes in insulin sensitivity. To further extend and validate the relationship of AT CIDEA expression to insulin sensitivity the whole data set of 311 individuals was investigated according to change in HOMA-IR among the entire dietary intervention (from baseline to the end of WMD), independently of groups.Then, the use of discriminant analyses to find the transcripts most related to subject's grouping showed CIDEA encodes a member of the cell death–inducing DNA fragmentation factor 45-like effector (CIDE) proteins, which is expressed in both brown and white fat CIDEA mRNA level was found in the AT from obese individuals compared to lean ones CIDEA as the most up-regulated gene after calorie restriction induced weight loss CIDEA expression after dieting when calories are reintroduced while body weight is stable, shown in a subgroup of 40 obese women, indicates that during diet-induced weight loss, calorie restriction per se, rather than weight reduction, has the major impact on CIDEA expression A higher CIDEA in AT and change in IR both during (LCD) and after calorie restriction (WMD). The link between AT CIDEA and insulin sensitivity in obese humans has been described in a cross-sectional study showing higher CIDEA mRNA levels in AT of insulin-sensitive compared to BMI-matched IR obese individuals ad libitum follow-up corresponding to a program with clinical relevance to obese individuals.Here we found a negative relationship between regulation of CIDEA mRNA level in AT, compared to baseline. During the second phase 6-month follow-up, only the group who increased HOMA-IR again showed a decreased CIDEA gene expression. The independence of the relationship between change in insulin sensitivity and AT CIDEA regulation from body mass change is noteworthy.At the end of the calorie restriction phase, groups with largest IR improvement of had higher CIDEA in insulin sensitivity but does not establish a cause and effect relationship. However, previous investigations provide some evidence on the functional effect of CIDEA regulation in AT.The observation that CIDEA is an AT marker of insulin sensitivity raises the question of the potential role of Cidea null mice have smaller adipocytes and are resistant to diet-induced obesity CIDEA increased basal lipolysis, supporting the idea that CIDEA protects lipid droplet CIDEA regulation during dietary intervention was independent of body mass. Altogether, the increase in AT CIDEA mRNA levels is likely to explain IR improvement through a decreased AT lipolysis or fatty acids oxidation. The AT lipolytic capacities of the individuals enrolled in the present dietary program has not been studied during the course of the present trial. The investigation of a potential link between IR and AT CIDEA deserve future research.CIDEA is a multifunctional protein probably depending on intracellular localization. Overexpression in 3T3-L1 cells induced an increase in fatty acid oxidation and decrease in glucose transport CIDEA regulation is causative or consequence of changes in IR. Nevertheless, this is the first report of a link between human AT CIDEA regulation and insulin sensitivity, independently of weight change. Collectively, these results provide new data that CIDEA regulation in AT is related to insulin sensitivity. The potential of CIDEA induction in the treatment of obesity-related complications deserve future research.One can hypothesize that change in IR observed during dietary intervention is due to CIDEA overexpression-induced increase in fatty acid oxidation and/or decreased lipolytic capacities. The present study cannot answer the question whether Table S1Description of the 267 target genes.(DOCX)Click here for additional data file.Protocol S1Detailed study protocol of the DiOGenes trial.(PDF)Click here for additional data file.Checklist S1CONSORT checklist of the DiOGenes trial.(DOC)Click here for additional data file.


EBioMedicine aims to foster.2014 has been a remarkable year of scientific achievements, from advances in our understanding of molecular pathways of human diseases to the development of new interventional tools to tackle them. Here, we highlight some promising initiatives and success stories from the past year with clear translational relevance. The list is by no means exhaustive but hopefully it provides a perspective on some of the achievements that are good examples of the types of research Although not an entirely new concept, a powerful new approach called CRISPR has been catapulted to center stage this year as a means of highly specific gene targeting and modification. Clustered regularly interspaced short palindromic repeats (CRISPR) are sequences naturally found in bacterial DNA that work with CRISPR-associated (Cas) nucleases and guide RNAs to protect the bacterial genomes from attacks by targeting specific sequences detected in invading bacteriophages. Proof-of-principle research has emerged recently, which harnesses the high sequence specificity of the system to cure genetic disorders in animal models as well as creating disease-free stem cells from affected patients. CRISPR also allows the creation of mouse models with specific gene mutations for research purposes within much shorter timeframes than existing methods. Earlier this year, it was reported that the CRISPR-Cas system has successfully been applied as a tool for performing efficient and highly specific genome-wide screening in human cells, and vast improvements have been made in 2014 on increasing target specificity, which opens up limitless possibilities for interrogating gene function relevant to human health and diseases.Regenerative medicine and stem cell research have also seen multiple advances and improvements in 2014. Stem cell therapies have entered human clinical trials for diabetes, multiple sclerosis, and macular degeneration, and this year we saw the first demonstration that human embryonic stem cells can have real-life therapeutic benefit in restoring vision in a small cohort of legally blind patients. Achievements have also been made in the research of neurodegenerative diseases such as early-onset Alzheimer's and Parkinson's diseases, where the growth of patient-derived stem cells in vitro will facilitate genetic modeling of these diseases. These “in a dish” models of neurodegenerative disorders may also be used to screen and develop targeted therapies, which should speed up the translational process toward benefiting real patients.This year, the United States Food and Drug Administration (FDA) approved the first monoclonal antibody against programmed death protein 1 (PD-1) – pembrolizumab – for unresectable or metastatic melanoma. Based on early basic research regarding the role of PD-1 in the negative regulation of T-cell responses (which can render them ineffective in an anti-tumor response), research efforts have been focused on blocking the interaction of PD-1 with its two ligands PD-L1 and PD-L2, primarily for cancer treatment but also for other immunotherapeutic applications. Pembrolizumab is also currently under expedited FDA review as a breakthrough therapy for advanced non-small-cell lung cancer, and in clinical trials for several other cancers. Another anti-PD-1 monoclonal antibody under authority review for advanced non-small-cell lung cancer and melanoma is nivolumab, which is also going through numerous clinical trials for various other cancers including renal cell carcinoma and head and neck cancer. Agents targeting the interactions between PD-1 and its ligands are anticipated to be one of the most valuable therapeutics in the next few years, and two decades of laborious basic and clinical research on the PD-1 pathway have finally come to fruition.2014 also welcomed two new once-weekly, long-acting glucagon-like peptide 1 (GLP-1) receptor agonists for type 2 diabetes mellitus – albiglutide and dulaglutide – following the success of once-weekly exenatide. These drugs activate GLP-1 receptors expressed in many tissues, including the pancreas and brain, and lower blood sugar concentrations by promoting glucose-dependent insulin release from pancreatic beta cells and by slowing glucose absorption into the bloodstream. The once-weekly regimen is expected to improve treatment compliance compared with other daily regimens such as once-daily liraglutide and twice-daily exenatide. Since the first demonstration of GLP-1 receptor cloning and functional expression 20 years ago, enormous research efforts have been focused on developing GLP-1 receptor agonists to treat diabetes, resulting in first-in-human proof-of-concept studies in the early 2000s. GLP-1 receptor agonists are yet another example of translating decades of research activity into interventional tools that benefit human health.A new standard of care for the treatment of chronic hepatitis C virus (HCV) infection has also been realized this year. HCV is a major healthcare burden, even in developed countries, with millions of newly infected cases each year worldwide. The addition of sofosbuvir (a first-in-class HCV NS5B polymerase inhibitor), and its combination with the NS5A protein inhibitor ledipasvir, to the existing standard treatment of interferon and ribavirin have greatly improved HCV treatment choices, and for the first time certain groups of chronic hepatitis C patients can benefit from an entirely oral, tablet-based therapy without the need of interferon injection. The new fixed-dose sofosbuvir/ledipasvir combination is formulated in a single tablet, to be taken orally once a day, and can allow shorter treatment durations compared with interferon/ribavirin treatment. The sustained viral response rates were as high as 99% in some treatment arms, and the addition of ribavirin to this new sofosbuvir/ledipasvir combination did not improve response rates.EBioMedicine to help communicate those needs by translating science to improve health.All told, 2014 has been a great year for translational research, and we congratulate those basic and clinical researchers whose achievements have brought us closer to addressing long-standing challenges to human health. These achievements illustrate that continuous and persistent efforts to understand molecular mechanisms of human diseases and to develop innovative interventions can perfectly align with unmet medical needs in human healthcare, and with the mission of


The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole  Study of micro- and macroscopic structure of biological tissue provides essential information required both in medical diagnostics and research. Existing techniques for observation of tissue morphology, morphometry and phenotyping at the microscopic scale rely on histologyA histological method producing true volumetric information was introduced as High Resolution Episcopic 3D Microscopy (HREM) or Surface Imaging Microscopy (SIM). HREM and SIM have much in common with conventional histology, but utilizing a different scanning approach isotropic spatial resolution down to 0.5 μm can be achieved234With the rapid development of technology in the field of X-ray imaging, high resolution information about the inner structures of objects can be obtained even non-destructively. The spatial resolution of X-ray micro-tomography systems is becoming almost comparable to conventional histology  have been developed in the last decade which are today commercially available (Medipix and Timepix2 (array of 100 Timepix chips). Such large-area semiconductor pixel detectors combined with modern table-top sources (e.g. micro-focus X-ray tubes) open the way for extended and new applications in X-ray imaging.Despite the significant progress in imaging parameters provided by the photon counting technology a crucial limitation in the practical use of these devices for imaging has been the small sensitive area of only few square centimetres. This technological barrier has been fully overcome by the development of large-area photon counting semiconductor detectors such as the WidePIX deviceex-vivo ethanol-only preserved murine organs. The effect of ex-vivo soft tissue contrast enhancement after ethanol-preservation has been demonstrated in studies related to phase contrast X-ray imaging238910111213ex-vivo soft tissue visualisation.In this work we demonstrate performance of this recently developed large area hybrid pixel photon counting detector for high resolution X-ray microradiography and micro-tomography of Compared to water, ethanol evaporates more effectively promptly leaving all kinds of cavities such as ventricles, vessels, alveoli, bronchi etc. Structures normally filled by liquid, therefore, become hollow and produce detectable absorption contrast. Since ethanol stiffens the tissue, investigated organ samples can easily withstand time consuming tomographic scans (from tens of minutes up to hours) without significant changes of the tissue shape and inner structure. The demonstration of the observed contrast improvement of the ethanol preserved tissue sample compared to the native sample is shown in From three different ways of ethanol fixation which were tested , we evaluated as most appropriate the approach based on the series of increasing ethanol concentration. The 50% ethanol solution has maintained the native look of the tissue, but it did not stiffen and improved the contrast of samples sufficiently. On the other hand, the use of the high ethanol concentration (97%) directly to a native tissue sample has resulted in severe tissue deformation or even occurrence of ruptures.Experimental measurements indicate that the contrast develops with respect to the degree of drying of the sample. The ethanol evaporation is the fastest from the surface of the sample and from larger cavities. Contrast in radiographic images changes consequently as a function of resting time  within the same type of tissue, as demonstrated for the case of lungs in In a similar manner also other hollow systems like veins, arteries and various ventricles provide extensive contrast enhancement following alcohol staining. Contrast enhancement in a murine kidney is demonstrated in The proposed approach provides enhanced CNR imaging with 3D information about investigated soft tissue structures at the micrometre scale without physical sectioning of the sample. Quick, non-destructive and non-distorted 3D visualisation of soft tissue structures with spatial resolution approaching the cellular level opens new possibilities in so-called virtual histology8Very good soft tissue contrast together with non-destructive volumetric information can be provided also by highly sophisticated methods such as X-ray phase-contrast23242526272931ex-vivo soft tissue absorption imaging utilize high-Z contrast agents which are delivered to the specific structure by elaborate, time demanding and irreversible staining techniques which depend also on the knowledge of skilled staff. Using the proposed method, high quality micro-radiographs and micro-CT scans of basically all murine organs can be acquired just after simple ethanol preservation of tissue. The method, moreover, keeps the samples fully usable for further measurements and research. The significant simplification of the sample preparation protocol and compatibility with established techniques makes consequently the method easily accessible to the general scientific community and opens the use of micro-CT as a new standard tool for inspection of soft tissue biological samples.Currently used X-ray micro-CT staining techniques for A set of comparative measurements was performed using a commercially available micro-CT scanner Bruker 1172 equipped with 11 Mpixel CCD detector (9 um pixel pitch) and micro-focus X-ray tube. The comparison has showed that the contrast improvement of soft tissue structures based on the ethanol staining is detectable even with state-of-the-art conventional X-ray cameras. Nevertheless, the profit of a noiseless PCD lies in the possibility to detect even extremely small variation in density/thickness formed, for example, by few micron thin veins. For charge-integrating devices such sensitivity is a difficult task due to various intrinsic sources of noise  negatively affecting the CNR of radiographic images. Results of comparison between the large area PCD and CCD camera are demonstrated for the case of ethanol-preserved murine liver in Another important advantage of the approach based on the large-area photon counting detector is the capability to image whole murine organs with very high magnification. The sensitive area of state-of-the-art Charge-Coupled-Devices (CCDs) which are a promising detector technology currently used in micro-CT scanners is still limited to few square centimetres and their use is therefore limited to imaging of small objects. Thanks to development of large area photon counting detectors even larger organs, such as human, can be also visualized with moderate spatial resolution given basically by the pixel size of the detector (55 μm). The approach therefore provides from a single tomographic scan very complex 3-D model of relatively large tissue volume. This feature together with the high contrast provided by the ethanol staining opens new possibilities in understanding of the structure and functionality of soft tissue organs.ex-vivo soft tissue samples based on the use of the large area photon-counting detector and simple ethanol preservation. The method provides substantial improvement of image quality of various soft tissue structures in absorption X-ray micro-radiography and micro-tomography applied without the use of higher-Z contrast agents. The achieved contrast provides different type of information compared to conventionally used contrast agents. Moreover, ethanol preservation stabilizes the samples structurally and improves the stability of sample imaging during long lasting tomographic scans. As ethanol preservation is one of standard steps of tissue fixation for histology, the presented approach can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft tissue visualisation.We have developed and demonstrated a flexible imaging method for 3D imaging of ex-vivo mouse organs was performed. Use of laboratory animals was approved by Ethical Committee of the Third Faculty of Medicine, Charles University in Prague. The animals were treated with accordance to guidelines defined by Ethical Committee in decisions no. 246/1992 and no. 419/2012. Genetically modified mice C57BL/6 (weight 17 g) were terminated by ether inhalation overdose and their organs  were extracted out for purposes of this study. All samples were inserted into ethanol solution immediately after extraction. As fixative we used 50% and 97% ethanol solutions and an ethanol series with gradually increasing concentration (50–97%). The samples were scanned after 24 hours, 72 hours, 1 week and 2 weeks after inserting into the fixative. Before data acquisition each sample was rested on the air for certain time (5–60 minutes) to let the redundant ethanol evaporate from the surface of the sample and from cavities. For the purposes of 2D projection imaging the sample was placed in a sample holder keeping the sample in place between two thin self-adhesive stretch foils which minimised the occurrence of motion artefacts and slowed down the further drying-out of the tissue (see A set of measurements with ssue see  right. I2). Thanks to the rotating gantry construction with a short source-to-detector distance and high beam intensity, rapid measurements of unstable samples and acquiring of tomographic datasets consisting of many projections are feasible. The gantry housing the X-ray source and detector rotates around the sample laying stationary on the holder in the horizontal direction. The configuration minimises the possibility of changes within the soft biological sample during the scan.For the first evaluation, samples were scanned using a compact small animal X-ray micro-CT scanner10×5 composed of 50 Timepix chipsIn order to obtain high-resolution radiographs and micro-CT scans of whole murine organs the upgraded high-resolution X-ray imaging setup5 and 1.5·104 events per pixel behind the object for 2D radiography and tomographic projection, respectively. All presented 2D radiographies and the projections used for tomographic reconstruction were processed by beam hardening correctionThe detected intensity in acquired data was over 10How to cite this article: Dudak, J. et al. High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector. Sci. Rep.6, 30385; doi: 10.1038/srep30385 (2016).


Peripheral deafferentation downregulates acetylcholine (ACh) synthesis in sensory cortices. However, the responsible neural circuits and processes are not known. We irreversibly transected the rat infraorbital nerve and implanted neuroprosthetic microdevices for proximal stump stimulation, and assessed cytochrome-oxidase and choline- acetyl-transferase (ChAT) in somatosensory, auditory and visual cortices; estimated the number and density of ACh-neurons in the magnocellular basal nucleus (MBN); and localized down-regulated ACh-neurons in basal forebrain using retrograde labeling from deafferented cortices. Here we show that nerve transection, causes down regulation of MBN cholinergic neurons. Stimulation of the cut nerve reverses the metabolic decline but does not affect the decrease in cholinergic fibers in cortex or cholinergic neurons in basal forebrain. Artifical stimulation of the nerve also has no affect of ACh-innervation of other cortices. Cortical ChAT depletion is due to loss of corticopetal MBN ChAT-expressing neurons. MBN ChAT downregulation is not due to a decrease of afferent activity or to a failure of trophic support. Basalocortical ACh circuits are sensory specific, ACh is provided to each sensory cortex “on demand” by dedicated circuits. Our data support the existence of a modality-specific cortex-MBN-cortex circuit for cognitive information processing. As early as the 1970s it has been known that sensory deafferentation provokes changes in the somatotopic maps at any relay station of the somatosensory pathway Kaas, . FunctioIn a previous work we quantified cortical plasticity phenomena in young adult rats submitted to infraorbital nerve transection and subsequent artificial electrical neurostimulation  because it is heavily involved in plastic reorganization of the sensory and motor cortices and because it downregulates after peripheral deafferentation. ACh is mainly provided to the cortex by the “diffuse” corticopetal cholinergic system originated in the basal forebrain in magnocellular basal nucleus-MBN, the analogous to Meynert nucleus in primates  cortical ACh depletion is triggered by the interruption of the sensory input; (2) the interruption of sensory input reduces ACh in MBN, consequently reducing ACh provision to the cortex; (3) The depletion of ChAT in MBN is due to the decreased activity in sensory input or to reduced trophic support. Additional questions addressed in this paper are to which extent basalocortical cholinergic system is sensory modality-specific and how deafferentation exercises a strong influence on MBN that doesn't receive direct sensory input.Our work shows that interruption of the sensory input: reduces cortical ACh in a sensory-specific manner; acts on the cortex through MBN corticopetal neurons; is not dependent on the intensity of MBN afferent activity or on the supply of trophic factors. We discuss a possible mechanism by which peripheral deafferentation acts on MBN who doesn't receive direct sensory input.Our experimental model is the irreversible transection of the infraorbital nerve associated to a neuroprosthetic microdevice for stimulation of the proximal stump  divided into three experimental groups: Control or C-group, with intact peripheral nerves  along the horizontal plane. Consecutive series were collected for the study of different biomarkers. For the present study Nissl staining, CyO histochemistry, and ChAT immunostaining were considered.For the anatomical tracing between deafferented somatosensory cortex and MBN, retrograde fluorescent tracer was injected in 4 additional deafferented rats . Under general anesthesia animals were placed in the stereotaxic frame, scalp was removed and an a hole was opened in the bone at the coordinates of barrel C3, approximate center of the barrel field, at 4.5 mm lateral and 2.3 mm posterior to Bregma  in aseptic conditions. Animals were freely moving with free access to food and water, with 12h/12h day/night cycles. Animal handling, housing, surgery, and euthanasia were carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments, national legislation (R.D. 223/88), and EU directives on this matter (86/609/EC).Sections were dipped in distilled water and stained in 0.1% cresyl violet for 15–30 min at 45°C. They were differentiated in water for 3–5 min and then dehydrated through ascending alcohols.Sections were incubated in a solution containing 0.04% cytochrome C , 0.05% 3.3′ DAB , and 4% sucrose in sodium phosphate buffer 0.1 M, pH 7.3, at 37°C according to the original protocol of Wong-Riley .We performed indirect immunolabeling with a goat anti-ChAT monoclonal antibody  revealed by the ABC-peroxidase technique . In most cases, the final reaction was intensified with Nickel . After staining, sections were mounted serially on chrome alum coated slides, dehydrated, defatted, and coverslipped.Staining procedures were standardized and carefully performed, in order to minimize any bias or variability due to experimental procedure. In each animal the two hemispheres were simultaneously processed with the same reagents under the same conditions, so staining intensities of the two hemispheres are comparable. Moreover, to guarantee staining homogeneity animals from each group  were processed all together.Anatomical identification of relevant areas was carried out on Nissl- and CyO-stained sections.The affected primary somatosensory cortex corresponds to the Posteromedial Barrel Subfield  was identified caudally to the PMBSF and the secondary somatosensory cortex  corresponding to Au1 level in Paxinos's atlas  in horizontal sections was identified as the most caudal pole at dorsal levels,  and binocular (V1B) levels in Paxinos's atlas  at amygdala and ventral pallidum level  and its immunoexpression is linearly correlated to ACh levels  and regional metabolic activity (CyO-expression) in barrel, auditory, and visual cortices were estimated by the same unbiased quantitative optical densitometry (OD) procedure followed in Herrera-Rincon et al. . QuantifTo establish iperipheral deafferentation induces a focused loss of cortically-projecting MBN neurons and to identify the possibly affected zone we took into account the rostro-caudal topographic organization of the MBN. Rostral division is formed by neurons located dorsally and medially to the globus pallidus while caudal division is formed by the more lateral neurons, located ventro-medially with respect to the globus pallidus Figure . MBN neuIn cortices OD measures were obtained from layers II/III and layer IV separately as well as the II-IV field as a whole. In detail (1) all stained tissue sections were digitized using the same bright field microscopy with planapochromatic 2.5×, 4×, 10×, 20×, and 40× dry objectives and stored to a hard disk; (2) each tissue section was sampled using a gray scale gradient of 1–256 (white to black) at a resolution of 3000 dots per inch; (3) a systematic random sampling procedure was performed both at section (approximately fraction of 1/3) and at counting frame level ; three sections were randomly selected from each brain series; in each section 5 samples of 10 × 10 pixels were obtained using a systematic random sampling procedure (4) for each sample the mean OD was calculated using ImageJ  image processing software; (5) OD measurements were averaged for each animal; (6) in each tissue section three additional random samples were obtained from the background -unstained areas-; (7) OD correction was performed for each image by subtracting the corresponding mean background OD from the original OD value. So, for each experimental group from each animal fifteen unbiased measurements were obtained from the left and fifteen from the right hemisphere. For both CyO and ChAT staining DO values were assigned in a gray scale between 0 and 255. In order to avoid the possible inter-individual variability due to the staining procedure, for inter-group comparative analyses we employed the intra-group change percentage . For all measurements coefficients of error due to the sampling procedure  and we counted the number of neurons in layers II-III, layer IV, and the totality of the neurons in PMBSF. Estimation of the coefficient of error for systematic random sampling was used to assess the precision of the estimates of N  and we counted the totality of the neurons under a 10× objective. Estimation of ChAT-positive neurons was done by considering two anatomically different regions, rostral, and caudal Figure ; ChAT-imt-tests. For inter-group comparisons ANOVA tests followed by post-hoc LSD test (when P < 0.05) were used. The same statistical analyses were applied in the estimation of the number of PMBSF and MBN ChAT- and FG-positive neurons. Significance level (α) was set to 0.05 in all cases. Statistical values are reported as mean ± standard error of the mean (SEM).Intra-group (inter-hemispheric) and inter-group analysis was performed for both, CyO and ChAT expression. In the first case we compared mean ipsilateral to mean contralateral OD calculating the percentage changes 100 * To test possible neuroprotective and plastic effects exerted by the neurostimulation on the cholinergic activity of the deafferented cortex and to compare these affects to those observed on the metabolic activity we evaluated bilateral CyO and ChAT levels and number of cholinergic interneurons in the barrel cortices of deafferented and prosthetic animals and we compared them to the cortices of the control ones. Control, amputated and prosthetic animals are denoted with C, A, and P, respectively.To test the effectiveness of artificial stimulation and to assess the physiological conditions of the barrel cortices we recorded evoked potentials from the somatosensory cortex of C-, A-, and P- animals under electrical stimulation of 200 μs-long pulses  Statistical tables for OD CyO- reacted barrel cortex (A) and for OD ChAT- reacted barrel cortex (B).Electrical stimulation of the transected infraorbital nerve prevented CyO-intensity from dropping in the affected hemisphere and maintained its metabolic activity at levels similar to the contralateral ones; contrary to CyO neuroprosthetic stimulation did not prevent ChAT downregulation in the deafferented cortices Figures .Inter-group comparisons for CyO optical density showed significant differences between C- and A-animals as well as between A- and P-animals for all three analyzed zones; no statistical differences were detected between C- and P-animals Table . Inter-gNext we examined the role of cortically projecting MBN neurons in such processes. The number of MBN ChAT-immunostained neurons was estimated in control, amputated, and prosthetic animals at rostral and caudal level .P < 0.01) and −17 ± 9% in the rostral (P = 0.06). Caudal zone corresponded to the region with maximum drop of ChAT-positive neurons  while amputation resulted in a significant decrease in the number of ChAT-positive interneurons in the affected hemisphere in all three layers II/III  and for OD ChAT- reacted visual cortex (B).Table 5Statistical tables for OD CyO- reacted auditory cortex (A) and for OD ChAT- reacted auditory cortex (B).Here we investigated the involvement of ACh in cortical plasticity and reorganization after peripheral deafferentation and subsequent prosthetic stimulation of the severed nerve. In a previous paper we showed that sensory neuroprosthetic input to an amputated peripheral nerve has direct and measurable neuroprotective effects on the deafferented cortices; it halts downregulations and avoids atrophies  have also been considered responsible for the decrease of MBN cholinergic activity (Knusel et al., DNF have Cortical cholinergic innervation from the MBN has been traditionally considered as a “diffuse” innervation to the whole cortical mantle and contributing to the general state awareness Mesulam, . HoweverTaking into account that loss of MBN neurons after the damage of the trigeminal nerve is limited to the barrel cortex-projecting region we suggest that each sensory pathway has separate access to the cholinergic MBN system. This should explain why loss of the somatosensory input or its replacement by an artificial one almost exclusively affects the “somatosensory” cholinergic circuit. In turn it should imply individual MBN neurons to be predominantly modulated by a single sensory modality and to mainly or exclusively project to the corresponding sensory areas. Our results and inferences are in agreement with (Collier and Mitchell, How deafferentation exercises such a strong influence on the MBN which does not receive direct sensory input from the whiskers? Why artificial stimulation of the transected peripheral nerve does not normalize the down-regulated basalocortical cholinergic system? ACh down-regulation could be due to a failure in the broader neural circuit involving connections with subcortical circuits. Under normal conditions such circuits should mediate MBN activation either through intermediate nuclei (i.e., amygdala and/or hypothalamus; Irle and Markowitsch, Taken together, here we have shown that cortically projecting MBN neurons provide topographically specific, functionally independent inputs that are modality specific. Our data support the existence of a modality-specific cortex-MBN-cortex circuit for cognitive information processing. Functional or context related activity may be necessary to maintain the ascending cholinergic circuit.ad-hoc” protocols of transcranial magnetic or current stimulation could be developed to improve learning performance or outstrip cognitive bottlenecks.Due to its involvement in both cognitive processes and neuroplasticity, the cholinergic circuit represents the main target of innovative therapeutic neuroprotection-neurorehabilitation strategies. In order to be effective, neuroprosthetic-based therapeutic approaches should necessarily incorporate contextual or cognitive meaning. This might lead to the definition of new stimulation protocols based on the involvement of the cholinergic system. These stimulation protocols may be more effective for stroke or traumatic brain injury therapy and may be helpful in designing novel sensory neuroprostheses to perform in a behaviorally relevant manner in coding and transmitting sensory information to the central nervous system. Furthermore, considering the involvement of ACh in decision making and learning processes in the prefrontal cortex, new “The 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.


Personalized cancer treatments depend on the determination of a patient's genetic status according to known genetic profiles for which targeted treatments exist. Such genetic profiles must be scientifically validated before they is applied to general patient population. Reproducibility of findings that support such genetic profiles is a fundamental challenge in validation studies. The percentage of overlapping genes (POG) criterion and derivative methods produce unstable and misleading results. Furthermore, in a complex disease, comparisons between different tumor subtypes can produce high POG scores that do not capture the consistencies in the functions.p-value of the rank-sum of the overlapping genes (PRSOG) to evaluate the quality of reproducibility. Though the POG scores were low in different studies of the same disease, the PRSOG was statistically significant, which suggests that sets of differentially expressed genes might be highly reproducible.We focused on the quality rather than the quantity of the overlapping genes. We defined the rank value of each gene according to importance or quality by PageRank on basis of a particular topological structure. Then, we used the Evaluations of eight datasets from breast cancer, lung cancer and four other disorders indicate that quality-based PRSOG method performs better than a quantity-based method. Our analysis of the components of the sets of overlapping genes supports the utility of the PRSOG method. BRAF mutant allele selective inhibitor is related to the BRAF V600E mutational status in the cancer cells p-value[rank(g) > rank]p-value[rank(g) > rank]p-value[rank(g) > rank]p-value[rank(g) > rank]p-valuep-value[rank(g) > rank]p-value[rank(g) > rank(g.We performed the resampling procedure 10,000 times to obtain the mean and standard deviation of the


Arabidopsis thaliana to cytokinin has been investigated by different research groups as soon as large-scale transcriptomic techniques became affordable. Over the last 10 years many transcriptomic datasets related to cytokinin have been generated using different technological platforms, some of which are published only in databases, culminating in an RNA sequencing experiment. Two approaches have been made to establish a core set of cytokinin-regulated transcripts by meta-analysis of these datasets using different preferences regarding their selection. Here we add another meta-analysis derived from an independent microarray platform (CATMA), combine all the meta-analyses available with RNAseq data in order to establish an advanced core set of cytokinin-regulated transcripts, and compare the results with the regulation of orthologous rice genes by cytokinin. We discuss the functions of some of the less known cytokinin-regulated genes indicating areas deserving further research to explore cytokinin function. Finally, we investigate the promoters of the core set of cytokinin-induced genes for the abundance and distribution of known cytokinin-responsive cis elements and identify a set of novel candidate motifs.The genome-wide transcriptional response of the model organism  Arabidopsis thaliana, they are perceived by three closely related receptors localized mainly at the ER membrane GAC(C/T) was identified as the ARR1-binding DNA motif  under the accessions E-MEXP-670, E-CAGE-111, E-MEXP-689, and E-CAGE-49.CATMA microarray hybridization and data processing were described before  The meta-analysis of CATMA microarray results presented in this study; (2) a meta-analysis of publicly available datasets performed with the Affymetrix ATH1 gene chip focusing on studies with a similar experimental layout . Detailed GO term enrichment was carried out using the AmiGO tool (http://amigo1.geneontology.org/cgi-bin/amigo/blast.cgi).Quantification of the major GO categories was carried out using the GO annotation search, functional categorization and download service at TAIR (http://www.arabidopsis.org/tools/bulk/sequences/index.jsp). Motif mapping was carried out using the Toucan2 suite of sequence analysis tools (https://gbiomed.kuleuven.be/apps/lcb/toucan/toucan.jnlp)  . GSE6719 contains data from root and leaf samples of the cultivar Nipponbare treated with 5 μM trans-zeatin for 30 or 120 min, respectively. GSE37557 is a series of different hormone treatments, from which we selected only the data from cytokinin treatment which were obtained from 7-day-old seedlings of the cultivar IR64 treated with 50 μM benzyl adenine (BA) for 3 h. The data of GSE55902 are from samples of the third leaf of 30-day-old plants of cultivar Nagina22 treated for 72 h in darkness with 2 mg/mL BA. The data were normalized with the RMA algorithm using the Bioconductor affy package  were also identified in Excel. One dataset (GSE37557) gave no meaningful data for the cytokinin-induced samples. Therefore, the analysis was continued with the two remaining datasets and no meta-analysis was performed with the rice data.The raw data of three transcriptomic datasets from rice with the accessions GSE6719, GSE37557, and GSE55902 were downloaded from the GEO database (http://www.rothamsted.ac.uk/ppets/modperl/ets2/at_template_form.pl?config=Wheat) at PpETS (http://www.rothamsted.ac.uk/ppets/modperl/index.pl)  and have a small overlap (<11%) whereas the two datasets from meta-analyses of Affymetrix ATH1 data have a much larger overlap  and contain only 18 and 33% unique genes, respectively. The mismatch between the results of the two microarray platforms is only to a small part due to a lacking overlap in the gene sets: 61% of the genes found regulated in the analysis of the Affymetrix data also have a probe on the CATMA array, and 90% of the genes found regulated on the CATMA array have a probe set on the Affymetrix array. The mismatch may rather demonstrate a weakness of quantitative hybridization-based methods, in which the sensitivity for detecting a given transcript is partly dependent on the sequence of the probe.The outcome of this comparison demonstrates that the choice of technology is an important determinant of the final outcome of a large-scale transcriptomics experiment. Furthermore, it shows that the preselection of primary datasets for a meta-analysis—reduction of the data to a set of comparable growth conditions, treatments, and time points  were found to be induced in at least three of the four independent analyses    at least one rice ortholog was identified . Remarkably, the molecular functions and biological roles of more than half of the cytokinin-regulated genes do not belong to one of the major categories Figure . The thrTo investigate the functional categories of cytokinin-induced genes in more detail, we carried out a statistical analysis of the abundance of the GO terms associated with the corresponding genes using AmiGO : A conspicuous number of expansin genes is regulated by cytokinin in individual experiments (data not shown), but only expansin A1 is consistently upregulated throughout all of them. Expansins, encoded by a family of at least 36 genes in Arabidopsis   whose transcript was consequently decreased. Unfortunately, the analysis presented here is lacking useful information on cytokinin-repressed transcripts, so a reverse search for potential microRNAs targeting these genes cannot be carried out.One aspect of gene regulation that is missing from this analysis is the regulation of microRNAs. This important regulatory mechanism for decreasing gene activity is inevitably absent in this type of analysis because microRNAs are not represented on the microarrays considered here. The only information comes from the RNA sequencing analysis data (Bhargava et al., in vitro experiment or a randomly degenerated set of octamers has led to the identification of a number of enriched sequence motifs in cytokinin-responsive promoters. The AT-rich consensus of the motifs is consistent with the previously identified nature of type-B response regulator binding elements. As our analysis is pure bioinformatics, the true binding capacity of type-B response regulators to the novel candidate sequence motifs must ultimately be determined experimentally.Given the fact that in most cases the binding motifs for transcription factors are clearly and significantly overrepresented in their target promoters, the search for enriched motifs is an accepted way to identify new potential binding motifs in the promoters of a set of co-regulated genes. However, it was shown that the experimentally identified binding motifs of the type-B response regulators ARR11 and ARR14 are surprisingly not overrepresented in the promoters of cytokinin-regulated genes (Franco-Zorrilla et al., Wolfram G. Brenner designed and carried out the research, evaluated the results, and contributed to writing the manuscript. Thomas Schmülling contributed to evaluating the results and writing the manuscript. Both authors finally approved the manuscript.The 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.


Parasite biology, by its very nature, cannot be understood without integrating it with that of the host, nor can the host response be adequately explained without considering the activity of the parasite. However, due to experimental limitations, molecular studies of parasite-host systems have been predominantly one-sided investigations focusing on either of the partners involved. Here, we conducted a dual RNA-seq time course analysis of filarial worm parasite and host mosquito to better understand the parasite processes underlying development in and interaction with the host tissue, from the establishment of infection to the development of infective-stage larva.Brugia malayi–Aedes aegypti system, we report parasite gene transcription dynamics, which exhibited a highly ordered developmental program consisting of a series of cyclical and state-transitioning temporal patterns. In addition, we contextualized these parasite data in relation to the concurrent dynamics of the host transcriptome. Comparative analyses using uninfected tissues and different host strains revealed the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription. We also critically evaluated the life-cycle transcriptome of B. malayi by comparing developmental stages in the mosquito relative to those in the mammalian host, providing insight into gene expression changes underpinning the mosquito-borne parasitic lifestyle of this heteroxenous parasite.Using the The data presented herein provide the research community with information to design wet lab experiments and select candidates for future study to more fully dissect the whole set of molecular interactions of both organisms in this mosquito-filarial worm symbiotic relationship. Furthermore, characterization of the transcriptional program over the complete life cycle of the parasite, including stages within the mosquito, could help devise novel targets for control strategies. Brugia malayi interacts with its vector's transcriptome temporally during its complex life cycle and also simultaneously provides information on how Aedes aegypti responds to filarial worm infection. These data are extremely valuable for future studies of the underlying mechanisms of this mosquito-filarial worm relationship.In a parasitic relationship, both host and parasite genotypes influence the parameters of their relationship. Previous studies examining host-parasite systems have examined the effects of the genotype of the host or the parasite on the relationship, but due to limitations of technology, have rarely examined interactions between the two genotypes. Here, we utilized a dual RNA sequencing (RNA-seq) approach to examine the entirety of the known transcriptomes and their interactions in the dynamic process of filarial worm development in the mosquito. In addition, the unprecedented sequencing depth achieved with this technology allowed us to compare, for the first time, parasite gene expression of larval developmental stages within the intermediate host with those life cycle stages found within the mammalian definitive host. These data provide a strong foundation for understanding how  Wuchereria bancrofti, Brugia malayi, or Brugia timori; but W. bancrofti is responsible for 90% of LF infections worldwide. Mosquitoes belonging to a number of different genera, including Culex, Aedes, Mansonia, Anopheles, and Armigeres, can serve as competent vectors. In susceptible mosquitoes, ingested microfilariae (mf) traverse the midgut epithelium and migrate to the indirect flight muscles. Migration to the head and proboscis occurs after a series of larval molts to the infective third stage (L3), which occurs in thoracic muscle cells. The L3 is then passed to the vertebrate host when the infected mosquito takes a bloodmeal Human lymphatic filariasis (LF) is one of the most debilitating of the neglected tropical diseases, causing severe morbidity as a result of stigmatizing and disabling clinical manifestations  The ability of certain mosquito species to ingest mf of filarial worm parasites and to support their development after ingestion is an important determinant of filarial parasite transmission It is well accepted that the study of inter-species interactions are fundamental to understanding the biology of vector-borne parasites in vivo infections B. malayi-Aedes aegypti interactions, which investigated the temporal organization of transcriptional events in both nematode and mosquito tissue from the establishment of infection to after infective-stage parasites had completed development in the mosquito. We report that parasite gene transcription dynamics exhibited a highly ordered developmental program consisting of a series of cyclical and state-transitioning temporal patterns, and we contextualized this parasite developmental program in relation to the concurrent dynamics of the host tissue transcriptome. Furthermore, we critically evaluated parasite gene expression changes during the heteroxenous life-cycle of B. malayi by comparing mRNA abundance in larval stages within the intermediate host relative to developmental stages within the mammalian definitive host in order to provide information on the transcriptomic features underpinning the mosquito-borne parasitic lifestyle.Accordingly, we initiated transcriptome profiling studies to assess the whole set of molecular interactions of both organisms involved in the dynamic symbiotic processes of filarial worm development in the mosquito. Advances in sequencing-based approaches to quantitative transcriptome profiling (RNA-seq) facilitated direct, integrated analysis of mixed-species samples obtained from Guide for the Care and Use of Laboratory Animals. All animals and animal facilities were under the control of the School of Veterinary Medicine with oversight from the University of Wisconsin Research Animal Resource Center. The protocol was approved by the University of Wisconsin Animal Care and Use Committee .This study was carried out in strict accordance with recommendations set forth in the National Institutes of Health Ae. aegypti used in this study were maintained at the University of Wisconsin-Madison as previously described Ae. aegypti, black-eyed Liverpool (LVP) strain supports the development of B. malayi mf to L3 B. malayiB. malayi to L3 B. malayi  by feeding on ketamine/xylazine anesthetized gerbils, Meriones unguiculatus. The same animals were used for both biological replicates. Microfilaremias were determined, using blood from orbital punctures, immediately before each feeding and ranged from 90–204 mf per 20 µl of blood. Control mosquitoes were exposed to anesthetized, uninfected gerbils. Mosquitoes that fed to repletion were separated into 30×30×30 cm colony cages and maintained on 0.3 M sucrose in an environmental chamber at 26.5°±1°C, 75±10% relative humidity, and with a 16∶8 hour light∶dark photoperiod with a 90 minute crepuscular period at the beginning and end of each light period.Mosquitoes were exposed to The sample groups were defined by the time post exposure (PE) to the infective blood meal, and thoracic tissues were collected every 24 hours from mosquitoes exposed to an infected or uninfected blood meal. Thoracic tissues were collected for four (RED) and eight (LVP) consecutive days, and also from non-blood-fed mosquitoes at the time of exposure . For each time point, 30 thoraces were collected per strain per condition. Dissected tissues were immediately flash-frozen in microfuge tubes on dry ice and stored at −80°C until RNA isolation. At 8 days PE, an additional 30 mosquitoes for each strain were dissected, and the head, thorax, and abdomen were examined microscopically as described previously Ae. aegypti thoraces using the MasterPure RNA Purification Kit , which included built-in Proteinase K and DNase treatments. A Labgen7b electric tissue grinder  was used for tissue homogenization, and RNA purity and concentration were determined spectrophotometrically . For all samples, RNA integrity was visually assessed by denaturing gel electrophoresis using GelRed staining  and further confirmed by Agilent 2100 bioanalyzer . Only quality intact RNA was used for RNA sequencing.Total RNA was isolated from female Multiplex sequencing libraries were generated from 10 µg total RNA (per library) using Illumina's mRNA-seq sample prep kit and multiplexing sample preparation oligonucleotide kit  following the manufacturer's instructions. Separate libraries were constructed for each replicate sample from infected mosquitoes. Equal amounts of total RNA were pooled from replicates prior to library construction for uninfected blood-fed and non-blood-fed samples. Cluster amplification was performed on the Illumina cBOT following the manufacturer's protocol. Sequencing-by-synthesis on the Illumina Genome Analyzer IIx generated 2×50 bp paired-end reads. CASAVA v1.7 was used for de-multiplexing, and sequence quality was assessed based on %GC content, average base quality and sequence duplication levels.To minimize the confounding effects of lane-to-lane or run-to-run technical variations, instead of sequencing each library using a single lane, libraries were sequenced over a number of lanes and runs by the use of Illumina's multiplexing capability Ae. aegypti [VectorBase: AaegL1] and B. malayi [GenBank:DS236884-DS264093] was performed using TopHat v1.0.14, a splice junction mapper built upon the short read aligner Bowtie de novo junction mapping algorithm. Low quality alignments with mapping quality scores less than five were removed before downstream analyses www-huber.embl.de/users/anders/HTSeq). A set of paired-end reads were counted as one fragment to represent a single sampling event, and reads overlapping more than one gene model were counted as ambiguous with the mode parameter set as “union”. In addition, previously published B. malayi sequence reads (ArrayExpress accession number: E-MTAB-811) were aligned and quantified using an identical analysis pipeline to facilitate comparative analysis of mosquito vs. mammalian life-cycle stages Sequence reads from technical replicates  were combined per library prior to mapping as they shared the same insert-size distribution. Alignment to the combined genomes of The RNA-seq data were prepared according to “minimum information about a high-throughput sequencing experiment” (MINSEQE) recommendations, deposited in the Gene Expression Omnibus (GEO) database, and can be accessed via the web [accession number GSE53664]. Statistical analysis of count data was performed with edgeR in Bioconductor 2 fold-change, and because log fold-change (logFC) estimates for genes with small read counts can be highly variable, we moderated logFC estimates using the ‘preFC’ function in edgeR. Thus, undefined values were avoided and poorly defined fold-changes for low counts were shrunk towards zero. We then used logFC values between sequential time points for each gene as a basis for clustering. After median-centering, K-means clustering was performed using a (uncentered) Pearson correlation as a distance metric to group and summarize expression profiles into common temporal patterns. Figure of merit (FOM) plots were utilized in determining the appropriate number of clusters to ensure informative partitioning All data were transformed using a logMDS plot (a type of unsupervised clustering) was generated in edgeR to analyze sample relationships. Distances between each pair of RNA-seq profiles corresponded to the average (root-mean-square) of the top 500 largest absolute logFC between each pair of samples.Ae. aegypti (LVP) exposed to either a B. malayi-infected or uninfected bloodmeal. Thoracic tissue samples were collected every 24 hours after exposure for eight days, and also from non-blood-fed mosquitoes at the time of exposure. LVP is a genetically selected susceptible strain that supports the complete development of B. malayi to the infective stage Ae. aegypti (RED) to comparatively evaluate the transcriptome dynamics in an incompatible parasite-host association  mapped unambiguously to the reference genomes (Poly(A)-selected mRNA samples isolated from these tissues were subjected to Illumina sequencing (38 libraries across 48 lanes), and the resulting paired-end reads (2×50 bp) were aligned to the combined genomes of  genomes . In the  genomes . Interes genomes .Next, in both parasite and host, we examined genome-wide temporal changes in transcript expression patterns in an effort to better understand the dynamic progression of infection processes  and 5. GB. malayi during successful development from mf to L3 indicated a progressive and complex turnover in transcriptional contents  that is formed around the pharyngeal thread of the mf 7 and P8 , enriche7 and P8 , in whic7 and P8 . Given oB. malayi, like all nematodes, progresses through its life stages via molts, each of which involves synthesis and secretion of a new cuticle, followed by separation and shedding of the old cuticle. From intracellular signaling to extracellular execution, molting requires a series of complex molecular reactions under tight spatiotemporal regulation at the level of the hypodermis. Also, equally critical to successful molting is a precise coordination of tissue development throughout the animal Genes with pulsatile transcription dynamics identified in the present study included a number of structural and regulatory components  that have been predicted to participate in various aspects of the molting process Ae. aegypti, filarial worm-infected muscle fibers show a large decrease in the amount of glycogen granules In addition to the initial wave of acute reactions to the invading parasites, the mosquito host's transcriptional responses to developing larvae also contained relatively moderate but sustained temporal patterns of induction, as illustrated in cluster HR4 . These eB. malayi, a diverse set of genes in clusters P2, P3 and P9, with oscillatory expression patterns, exhibited their second peak of transcription . These may play a crucial role in the repair process of the host cell by serving as molecular chaperones, and thereby possibly suppressing or delaying necrosis during intracellular parasite development B. malayi gene expression changes discussed thus far underlie successful developmental progression to the infective stage, which is dependent on the mosquito host tissue environment. Changing the genetic background of the host to an incompatible strain (RED) results in a failure of the parasite to develop and survive. Using a negative binomial generalized linear model, we assessed time dependent transcriptional changes between days one and four that were different in B. malayi depending on host environment, i.e., compatible (LVP) versus incompatible (RED). The most significant transcriptional alterations, as judged by statistical evidence, are presented in Caenorhabditis elegans, a mutation in β-spectrin resulted in retarded growth and paralysis, suggesting that it is required for normal nematode development B. malayi has not been elucidated, these could be involved in the control of key developmental pathways that are initiated during early larval development in the mosquito. Among the transcripts that showed an increasing trend in non-developing or dying worms, was a calpain gene that encodes a member of the Ca2+-dependent cysteine protease family. Calpain activation is an integral component of necrosis, and has been implicated in apoptotic cell death To further investigate the transcriptomic features of this heteroxenous parasite in the context of host transitions, we extended our analysis to include our previously reported RNA-seq profiles see  and compin vivo transcriptome dynamics of this metazoan parasite during its obligatory intracellular developmental phase in the mosquito host, which is critical for the maintenance of the disease cycle and transmission. Integrative analysis of the host transcriptome in a dual RNA-seq approach provides a high-resolution overview of the parasite-host system in which each partner's transcriptional state is dependent on the other partner. Studies of this nature will grow increasingly important in investigating the unique and common biological mechanisms that enable parasites to maintain their symbiotic association with the host, as well as the host strategies to counter parasitic infections, within a unified framework.Transcriptomic approaches have been instrumental in studying the developmental regulation of gene expression underlying filarial worm life-cycle progression Dataset S1Aedes aegypti and Brugia malayi time course transcript abundance profiles and over-represented gene ontology (GO) terms; comparative analysis of B. malayi time course transcript abundance profiles during infection of compatible (LVP) versus non-compatible (RED) mosquitoes; life cycle transcript abundance profile and mosquito stage enriched expression patterns in B. malayi; Over-represented GO terms among the mosquito stage-enriched genes of B. malayi; Over-represented KEGG pathways among the mosquito stage-enriched genes of B. malayi; and putative secreted proteins among the mosquito stage-enriched genes of B. malayi. All time points and parasite stages are represented.A tabular representation of (XLSX)Click here for additional data file.


Colorectal cancer (CRC) is the third most common cancer in California among both men and women and the third most common cause of cancer-related death . CRC morSocioeconomic status is a strong predictor of colorectal cancer screening . Studiesz-test to determine whether the top and bottom quintiles were significantly different in the proportion of their populations within a 30-minute drive of an FQHC. We also compared the proportion of households without access to a private vehicle in the top quintile versus the bottom quintile of late-stage CRC diagnosis by using data from the 2008–2012 American Community Survey (www.census.gov/programs-surveys/acs/). We considered P ≤ .05 significant. We used Esri’s ArcGIS version 10.2 for desktop (Esri) to create maps by applying the Network Analyst Extension. We used Esri’s Smart Data Compression network data set to perform the drive-time analysis. All other analyses were completed in SAS version 9.4 .We selected for our analysis the 682 FQHCs and FQHC look-alikes (centers that serve the same population as FQHCs but do not receive the same grant funding) that were active in California in 2012. We measured geographic access (availability and accessibility of services) to FQHCs by using a spatial, street-level network analysis to calculate 30-minute drive-time buffers . This peP <.001), but this difference may not be practically important because the difference between them was only 3 percentage points. Compared with the bottom quintile, the top quintile had a greater percentage of people living below the poverty level, a higher percentage of people without access to a private vehicle, and a higher percentage of people living in rural MSSAs (Most (96%) of California’s population was within a 30-minute drive of an FQHC . Ninety-six percent of the population of MSSAs in the top quintile of late-stage CRC diagnosis and 93% of the population in the bottom quintile of MSSAs were also within a 30-minute drive of an FQHC . The difal MSSAs . These dP < .001)  . In addi


In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements. About half of the mammalian genome is comprised of repeat elements of different types Agouti and Axin. Both of these loci contain one type of retrotransposons, IAP , and their LTRs  are partially repressed by DNA methylation. Furthermore, the methylation levels of these two IAP LTR are variable between individual mice with visible phenotypic consequences, such as coat color variations for the viable yellow agouti (vyA) allele and tail kinkedness variations for the axin-fused kinky (fusedAxin) allele, and are thus named ‘epialleles’ Although the majority of repeat elements are repressed by DNA methylation, a small fraction of these elements are also known to escape the DNA methylation-mediated repression. Two well-known cases include the mouse genomic loci Despite the significant roles played by DNA methylation in the repeat elements, many important questions have not been addressed so far, such as what fraction of the repeat elements escape the DNA methylation-mediated repression and which individual repeat elements escape this repression. To address these questions, we developed and tested the feasibility of a new protocol named High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS). This new protocol is designed to provide genome-wide, single-base resolution, and highly enriched DNA methylation data of any subset of repeat elements. Using this protocol, we successfully analyzed the methylation status of individual loci of the mouse IAP LTR family. The results indicate that a minor fraction (about 5%) of IAP LTR are hypomethylated, and also that the methylation levels of majority of IAP LTR are variable between tissues and also between individuals.and HT-TREBS is based on adaptations of the two high throughput bisulfite sequencing techniques: Reduced Representation Bisulfite Sequencing (RRBS) 6 reads were successfully mapped to approximately 7,000 IAP LTR loci, representing, on average, >100X sequencing depth for each IAP LTR locus. The lengths of the sequence reads were long enough to analyze the methylation levels of an average of 7 CpG positions for a given IAP LTR. According to the results, the sequenced IAP LTR are evenly distributed over the entire lengths of all the chromosomes without any bias for any particular region of the chromosomes . The sequenced IAP LTR also cover more than 80% of each of the initial 5 subtypes in almost all the samples based on the total number of each subtype in the mouse genome .The DNA methylation pattern of a given IAP LTR was characterized with three different values Fig. 2A.Inspection of these sprinkler plots of the brain of Female#1 provided the following conclusions. First, the majority of IAP LTR (90%) are positioned within the 80–100% methylation level, consistent with the fact that the majority of retrotransposons are usually repressed by DNA methylation Table S1). For the subsequent analyses, we retained only those IAP LTR that had been represented by all 12 samples, producing a representative set of 5,233 IAP LTRs. We also calculated average methylation levels and standard deviation values for the 5,233 IAP LTRs using the individual methylation values derived from the 12 different samples. Based on the results from this initial analysis, the representative set could be further divided into the following categories: less than 80%, 80-90%, and 90–100% methylation level categories with 4.6% (242), 40.0% , and 55.4%  representation, respectively  and also between individuals  ectively Fig. 3A.Fig. S3) and loci with less than 80% methylation level  and epigenetic modifications (data not shown), but we have not found any shared features that are closely associated with this group. Interestingly, however, two subtypes (IAPLTR2 and IAPLTR2a) are represented more frequently than expected in this group given their relative compositions in the mouse genome in terms of their numbers compared to the other subtypes . For the intra-individual variations, a total of 4,231 IAP LTR displayed statistically significant variations in the DNA methylation level: the number of variable IAP LTRs ranged from 1,135 to 3,594 among the four individuals  show variations in the DNA methylation levels, intra- and/or inter-individually. Also, the DNA methylation variations of IAP LTR are more prevalent in the brain than in the other two tissues examined, kidney and liver.As described earlier, the remaining IAP LTRs (mostly in the 70–100% average methylation range) also display inter- and intra-individual variations in their DNA methylation levels, but the ranges of these variations are much narrower (10–30% differences) than those of the hypomethylated group. Thus, to confirm the statistical significance of these variations, the entire representative set including the hypomethylated group was analyzed again using a series of statistical tests . The current study further strengthens this initial observation by providing the total numbers and actual locations of individual IAP LTR with DNA methylation variations. The current study also derived two additional findings regarding the DNA methylation variations of IAP LTR. First, the relatively large number of IAP LTR are shown to have DNA methylation level variations, intra- and/or inter-individually. The estimated number of variable IAP LTR could be easily more than 80% of the entire tested IAP LTR Fig. 4. All the experiments related to mice were performed in accordance with National Institutes of Health guidelines for care and use of animals, and also approved by the Louisiana State University Institutional Animal Care and Use Committee (IACUC), protocol #10-071.CCATCTCATCCCTGCGTGTCTCCGACTCAG) was designed to bind to the 5′-end of the ‘A’ adaptor region whereas the reverse primer was designed to bind to the 24-bp conserved region among the consensus sequences of the IAP LTR subtypes . The 5′-end of the reverse primer also had the sequence of the regular Ion Torrent ‘P1’ adaptor (CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT∧CTCCCTAATTAACTACAACCCATC). The break in the reverse primer sequence indicates the joining point of the P1 adaptor sequence and the IAP LTR-specific sequence. The amplification cycle number of PCR was individually determined for each of the twelve samples so that the minimum possible cycles were used to generate just enough the amount of the PCR product for sequencing. The PCR product was size-selected for a range of 250–300 bp in length using agarose gel electrophoresis. The size-selected PCR product was verified for its length and quantity using the Agilent 2100 Bioanalyzer. Finally, each of the twelve PCR products was individually sequenced in the Ion Personal Genome Machine (PGM) Sequencer using Ion 318 Chips .Genomic DNA has been isolated from the brain, liver and kidney of four 1-week-old littermates  of the C57BL/6N mouse strain (Jackson Lab). For each of the twelve samples, 1 µg of the isolated genomic DNA was sonicated to fragments with their average size being 700 bp in length . Since the average lengths of IAP LTR are 300-350 bp in length and the desired sequences need to contain non-repeat flanking sequences, the optimum size of the fragments are empirically determined to be around 700 bp in length. The fragmented DNA was immediately end-repaired using the NEB Next End Repair Module (New England BioLabs), and then ligated to custom-made duplex Ion Torrent ‘A’ adaptors using T4 DNA ligase (New England BioLabs). In these adaptors, all the Cs have been methylated (Integrated DNA Technologies). The adaptor-ligated DNA fragments were size-selected to remove any fragment smaller than 300 bp in length using the Agencourt AMPure XP beads (Beckman Coulter). The size and quantity of the selected fragments were analyzed using the Agilent 2100 Bioanalyzer. The adaptor-ligated DNA library was modified using the bisulfite conversion reaction according to the manufacturer's protocol . The bisulfite-converted ‘A’ adaptor-ligated library was used as a template for a round of PCR . In this PCR, the forward primer (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49222).The sequence reads generated from the twelve Ion PGM runs were individually mapped to a curated reference genome using the aligner Bowtie 2 The methylation level variations among the representative IAP LTR loci were calculated using the following combinations: i) intra-individual variation , and ii) inter-individual variation . For each specific locus, the CpG methylation values of the reads of the relevant samples were compared with each other to find variations by using the non-parametric Kruskal-Wallis test (p<0.001). Each positive test of variation was followed by a post hoc Mann-Whitney test with Bonferroni corrections to isolate the pair of samples that actually gave rise to the variation.Figure S1The genome graphs of the twelve sequenced samples as visualized using the UCSC Genome Browser website shows that the sequenced IAP LTRs are distributed over the entire lengths of all the chromosomes.(TIF)Click here for additional data file.Figure S2The two-dimensional read-based and CpG position-based sprinkler plots of the all the twelve samples sequenced . The sprinkler plots have been described in (TIF)Click here for additional data file.Figure S3Heatmap of the 12 sequenced samples showing the difference in CpG methylation of all the representative IAP LTR loci. The dendrogram on top shows the clustering of the samples while that on the left shows the clustering of individual loci based on their CpG methylation difference. The color key on the top left depicts the colors representing each value of CpG methylation while the histogram in it shows the number of loci present in the heatmap at those respective methylation values.(TIF)Click here for additional data file.Figure S4(A) A plot of the average methylation of the 5,233 representative IAP LTR loci against their distance from the nearest transcription start site (TSS). (B) A plot of the average methylation of the representative IAP LTR loci against their absolute distance from the nearest TSS. A very low coefficient of determination (2R) indicates the absence of any particular relation between the distance of the IAP LTR elements and their methylation status.(TIF)Click here for additional data file.Table S1This supplemental data contains table a through g. Lists of IAP LTRs with detailed information regarding genomic positions, DNA methylation levels derived from 12 samples using HT-TREBS, and their inter and intra methylation variation patterns based on statistical analyses. .(XLS)Click here for additional data file.