PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of  PRDM9 as the gene responsible for localizing recombination hotspots via recognition and binding to specific DNA motifs. Variation within PRDM9 can lead to changes to the recognized motif, and hence changes to the location of recombination hotspots thought the genome. Multiple studies have shown that PRDM9 is under strong selective pressure, apparently leading to a rapid turnover of hotspot locations between species. However, uniquely amongst mammals, PRDM9 appears to be dysfunctional in dogs and other canids. In this paper, we investigate how the loss of PRDM9 has affected the fine-scale recombination landscape in dogs and contrast this with patterns seen in other species.Recombination in mammalian genomes tends to occur within highly localized regions known as recombination hotspots. These hotspots appear to be a ubiquitous feature of mammalian genomes, but tend to not be shared between closely related species despite high levels of DNA sequence similarity. This disparity has been largely explained by the discovery of  Until recently the mechanisms controlling the localization of recombination hotspots in mammalian genomes were largely unknown. However, recent research has revealed that zinc-finger protein, PRDM9, binds to specific DNA motifs in the early stages of recombination initiation in order to direct such events PRDM9 in humans can alter the DNA motif to which the protein binds, and in turn alter the activity of recombination hotspots PRDM9 across species PRDM9 shows clear evidence of rapid evolution across the metazoan taxa covering an era of roughly 700 million years Prdm9 results in infertility in both male and female mice with arrest of spermatogenesis and oogenesis at pachynema, impairment of DSB repair, chromosome asynapsis, and disrupted sex-body formation in males Prdm9 has been shown to be involved with hybrid sterility, potentially implicating it in the process of speciation Variation in the zinc-finger encoding domain of PRDM9, it was therefore surprising to note that dogs (Canis familiaris) and other canids are the only known mammals to carry functionally inert versions of PRDM9 with multiple disruptive mutations PRDM9 is carried out by another gene, or that dogs have been able to avoid the loss of fertility associated with loss of PRDM9 while also ensuring that recombination continues to occur.Given the wide-ranging importance of In order to gain insight into recombination across the canine genome, we have constructed a genetic map using patterns of linkage disequilibrium (LD) estimated from next-generation sequencing of 51 dogs. Methods for estimating recombination rates from polymorphism data have been validated at both broad and fine scales The dogs were sequenced to 8–12× coverage with 101 bp paired end reads , allowinTo validate our recombination rate estimates, we compared our estimates to broad-scale experimental estimates obtained from pedigree studies Our estimates suggest a more uniform distribution of recombination in the dog genome than has been seen in human , as has et al. were obtained using a microarray with 170k markers Previous LD-based estimates of recombination in dog by Axelsson et al.We compared these hotspots to those identified by Axelsson PRDM9In humans and mice, specific DNA motifs have been implicated as the binding sites for CGCCGCG , which is found in 21.3% of hotspots but only 13.2% of coldspots, a relative enrichment of 61%. These highly CpG-rich motifs retain significantly high levels of enrichment in hotspots having masked either repeat or non-repeat DNA sequence.Using the sequences of the matched hotspots and coldspots, we performed a search of motifs showing enrichment in hotspot sequences. Our results indicate an extremely strong association with CpG-rich motifs , with thBoth GC and CpG content show a strong association with canine recombination at fine scales . HoweverThe influence of GC and CpG content can also be seen when considering the average recombination rate around DNA repeats. The most recombinogenic repeats are low-complexity with high levels of GC and CpG content . In contThe association between recombination and CpG-dense regions is suggestive of an association with gene promoter regions. Indeed, we observe highly elevated rates of recombination around transcription start sites TSS; , dwarfinPRDM9 is thought to have been disrupted early in canid evolution, as previous work has shown that the amino acid coding sequence contains multiple disruptive mutations across a diverse set of canid species PRDM9 disrupting mutations within the Canidae family by sequencing within exon containing the zinc-finger domain of PRDM9 in the Lycalopex and Urocyon genera. Within in the Andean fox, Lycalopex culpaeus . Using this data, we polarized the ancestral and derived alleles for polymorphisms observed in dog by assuming that shared alleles represent the ancestral allele. Using this data, we were able to polarize 3.2 million polymorphisms on the dog lineage with high confidence. Likewise, we performed SNP discovery in the fox data, and were able to identify and polarize 1.2 million polymorphisms on the fox linage.To investigate if BGC is active around canine recombination hotspots, we consider the ratio of AT→GC polymorphisms to GC→AT polymorphisms within local regions of the genome  of collagenase digested single cell suspensions. Chromatin immunoprecipitation (ChIP) of H3K4me3 was performed using standard procedures, and validated through qPCR . Librarihttp://autonlab.einstein.yu.edu/dog_recomb/Detailed methods are available in the Supplementary Information. Genetic maps and called hotspots are available for download from: Canine blood samples were collected under Cornell Institutional Animal Care and Use Committee (IACUC) approval  in accordance with applicable federal, state, and local laws, regulations, policies, and guidelines. For each animal, approximately 3–5 ml of blood was drawn from the cephalic vein into K2-EDTA blood collection tubes. Dogs were unsedated to minimize handling time and overall distress during the short blood drawing procedure. Culpeo blood was extracted from a captive female fox using a similar procedure during a routine physical by trained veterinary personnel at the Universidad de San Antonio Abad Zoo in Cusco.Figure S1et al.Decay of LD for village dogs compared to breed dogs. Genotype LD was calculated between the village dogs (red hued lines), breed dogs (blue hued lines), and wolves (green line). The genotype data for wolves and breed dogs was taken from Axelsson (EPS)Click here for additional data file.Figure S2Genetic maps for all autosomes estimated using LD (red) and pedigrees (EPS)Click here for additional data file.Figure S3et al Correlation at the 5 Mb scale between LD-based recombination rate estimates and those obtained from with Wong (EPS)Click here for additional data file.Figure S4A) Correlation between genetic map length and chromosome length for the LD-based map (black) and the linkage (red). Lines indicate least-squares lines of best fit. B) Shorter chromosomes tend to have higher recombination rates in both maps. Colors as for (A). Lines indicate a line of best fit estimated for a cubic function.(EPS)Click here for additional data file.Figure S5A) The distribution of recombination for each dog chromosome, compared to the human estimates from HapMap. B) Conclusions regarding differences between the human and dog distributions should be treated with caution due to sensitivity in the estimator to the effective population size. This figure shows the distribution of recombination for data simulated with three different effective population sizes (Ne).(EPS)Click here for additional data file.Figure S6et al.et al.Pearson correlation at various scales between recombination rate estimates from this study, Axelsson (EPS)Click here for additional data file.Figure S7et al. study around hotspots identified by this study. Estimates are shown for all hotspots (blue), those called by both studies (green), and those not called by both studies (red). B) Rate estimates from this study around hotspots identified in the Axelsson et al. study. Colors as for (A).A) Recombination rate estimates from the Axelsson (EPS)Click here for additional data file.Figure S8The association between recombination and GC and CpG content. A) GC content verse normalized recombination rate at 1 kb, 10 kb, and 100 kb scales. Recombination rates are shown for dog (red) and human (blue), having normalized the rates in each species by subtracting off the mean and dividing by the standard deviation. B) As for (A), but showing CpG content verses normalized recombination rate. C) Pearson correlation of GC content with  recombination rate, as a function of scale. D) As for (C) but showing correlation between CpG content and recombination rate at various scales.(EPS)Click here for additional data file.Figure S9st Exon, 1st Intron, Middle Exon, Last Intron, Last Exon, and 50 kb Downstream from the transcription end site (TES). Estimates within each intron and exon were estimated across 50 equally spaced bins, allowing a percentage to express the distance through the intron/exon.Recombination in each region of a gene. Shown are recombination estimates 50 kb Upstream of the transcription start site (TSS), within the 1(EPS)Click here for additional data file.Figure S10Recombination rates around CpG islands partitioned on the basis of distance to the nearest transcription start site.(EPS)Click here for additional data file.Figure S11A) No evidence of biased gene conversion around recombination coldspots. The plots show the ratio of the number of AT→GC mutations relative to the number of GT→AT mutations for polymorphisms identified in dog (red) and fox (blue). B) The same ratio around hotspots and coldspots in dogs, having excluded all polymorphisms for which either allele would create CpG dinucleotide.(EPS)Click here for additional data file.Figure S12Recombination rates for H3K4me3 peaks overlapping CpG islands (A) and not overlapping CpG islands (B). The three lines represent peaks shared between pachytene and L/Z (blue), those unique to pachytene (green), and those unique to L/Z (red).(EPS)Click here for additional data file.Figure S13nd chromosome. The subsequent repair following strand invasion results in mis-pairing between the G and T nucleotides. Biased gene conversion results from the biased repair of this mismatch, which favors the G allele over the T allele. B) Behavior of the ‘skew’ statistic in 5000 simulated hotspots, shown for simulations including biased gene conversion (red), and without biased gene conversion (blue).A) Cartoon representation of a possible biased gene conversion mechanism. A double-strand break in the vicinity of a G/A polymorphism results in the partial loss of the A/T base pair on the 2(EPS)Click here for additional data file.Table S1Locations of collected samples.(PDF)Click here for additional data file.Table S2Details of sequence coverage.(PDF)Click here for additional data file.Table S3Predictors of recombination rate. Regression of recombination with GC content, CpG content, and gene density at various scales.(PDF)Click here for additional data file.Table S4PRDM9 exon 7 DNA sequences. This table shows the ∼651 bp of DNA sequence from exon 7 of PRDM9 in 24 species or breeds. Sequences in black were taken from Axelsson et al.(PDF)Click here for additional data file.Table S5PRDM9 exon 7 Amino Acid sequences. Data is as for (PDF)Click here for additional data file.Table S6Primers used for qPCR validation of ChIP.(PDF)Click here for additional data file.Text S1Details of supplementary methods and analyses.(PDF)Click here for additional data file.


Traction Force Microscopy (TFM) is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D) imaging and traction force analysis (3D TFM) have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients. Traction Force Microscopy (TFM) is a powerful methodology of quantifying cellular forces during cell-material interactions. From the early work developed by Harris et al. in 1980 to the pioneering work by Oliver et al. and Dembo et al. in the mid 1990s, TFM studies have demonstrated the important role that mechanical cues play in cell biology by providing rigorous means of quantifying cellular traction forces TFM computes cell-generated surface tractions from a set of measured cell-induced displacement fields that are typically recorded by using either a single particle tracking or image correlation approach, such as digital image (2D) or digital volume (3D) correlation In almost all of the currently reported TFM methods, the underlying assumption is that the cell-generated material strains are small enough to be analyzed within a linear elastic continuum framework Utilizing a recently developed advanced high resolution digital volume correlation (DVC) technique The large deformation 3D TFM (LD 3D TFM) methodology presented in this paper consists of two basic components, analogous to our previous small deformation 3D TFM (SD 3D TFM) technique. First, a novel fast iterative digital volume correlation algorithm (FIDVC) Our newly developed FIDVC algorithm is capable of producing spatial resolutions and accuracies far beyond most current 3D methods, and is a significant advancement over our previously reported DVC displacement algorithm. Due to the improvement in spatial resolution, we found that cells are indeed capable of producing large deformations. This deviation is illustrated in more detail in To provide the reader with a more comprehensive analysis of using a small (SD) versus large (LD) deformation framework to calculate tractions, we present two specific examples. In the first example, an analytical, simulated displacement dipole is generated with a displacement gradient magnitude, To validate our LD 3D TFM methodology and to quantify the differences between a SD (linear) and LD TFM approach, we generate two analytical displacement dipoles applied along a free surface of the function  insert. observed .Comparison between To conclude our validation, we compare the total applied surface forces for both the SD and LD 3D TFM approaches to the analytically computed force. This result is shown in In the experimental cell example, Schwann cells are seeded on laminin-conjugated polyacrylamide substrates as detailed in the While total force and strain energy are convenient scalar quantities to classify cellular contractility, root-mean-squared and maximum traction measures present other useful metrics that are not dependent on the actual projected cell surface area. This paper presents an advanced high resolution 3D TFM technique capable of calculating fully non-linear cell-induced material stresses and traction fields. By utilizing a recently developed FIDVC technique, cellular displacement fields can be measured with unprecedented resolution and at computation times an order of magnitude faster than our previous 3D DVC algorithm. The increase in computational speed and accuracy is achieved through the extension of the well-known iterative deformation method (IDM) http://franck.engin.brown.edu).To illustrate the significance of the displacement gradient and its role in determining the most appropriate theoretical TFM framework, we provide a quantitative side-by-side comparison of the traditionally SD 3D TFM framework to our LD 3D TFM approach. As shown in In conclusion, our new LD 3D TFM algorithm presented here provides unprecedented spatial detail of 3D cellular traction fields at computation times of a few minutes on an average personal computer equipped with a GPU graphics card. We show that, depending on the cell-induced displacement gradient magnitude, the traditional linear TFM frameworks such as the Boussinesq theory can introduce significant error –7 in estx to location y can be represented by its material displacement, In continuum mechanics, a material point undergoing a deformation from location or as(4)where theThe observation presented in this paper, that cells can indeed generate non-linear material strains, should not only have implication for traditional TFM methodologies, but also for cell traction assays that utilize very compliant micropillars The two significant advances in improving our traction resolution capability are: (a) a completely new approach to measuring the cell-imposed displacement fields, via our recently developed Fast Iterative Digital Volume Correlation (FIDVC) algorithm Analogous to our previous 3D TFM technique, 3D volumetric image stacks are acquired using laser scanning confocal microscopy (LSCM). The overall dimensions of each stack are user-dependent, but are generally within the range of 512Motivated by the Gaussian-like material displacement field signature that cells generate While the mathematical and implementation details of this new FIDVC algorithm can be found elsewhere One of the key benefits of the IDM method is the spatial refinement of the local interrogation window size. In our previous SD 3D TFM method we used a fixed interrogation, or subset, size of 64 voxels, similar to many correlation-based algorithms. This particular choice in subset size is typically governed by the maximum intensity information that can be obtained within the subset, such as the fiducial marker size, density and distribution. Higher particle densities allow for smaller interrogation windows to be used. To provide maximum spatial resolution, the smallest possible subset size is sought while still maintaining high cross-correlation robustness, which is often found at a window size of 64 voxels. However, by employing an interrogation window refinement during the iterative IDM process, our FIDVC technique is capable of reducing its subset size to 32 voxels and in some instances even lower, without introducing significant correlation error. The result provides higher overall spatial resolution than previously possible, which is exemplified in After the cellular displacements, Similar to our SD 3D TFM approach, calculating cellular surface tractions first requires determination of the material's true stress tensor, or Cauchy stress Finally, the surface tractions are calculated via the Cauchy relations, i.e.,2 for all samples with a Young's modulus of 1.70 kPa. A similar analysis of the maximum resolution sensitivity for 2D and 3D TFM methodologies can be found elsewhere Since the determination of the surface tractions involves calculations of strains and experimental determination of material constants, the sensitivity of our LD 3D TFM technique in terms of traction forces needed to be assessed. This was accomplished by converting the measured displacement and strain fields of control samples (without cells) into surface tractions. Using standard statistical error analysis, we determined the noise floor to be similar to that for our SD 3D TFM technique. Specifically, we can accurately resolve any displacements and strains greater than 0.5 Analogous to our SD 3D TFM methodology, we compute the sum of all forces and moments acting on any given control volume inside each PA gel, to verify that static force and moment equilibrium are satisfied. The overall procedure is identical to our previous methodology The remainder of the Circular coverslips  are chemically modified to allow covalent attachment of polyacrylamide substrates, using previously described protocols Thin films of different PA gels are prepared from 40% w/v acrylamide  and 2% w/v N,N-methylene-bis-acrylamide (BIS)  stock solutions as described previously Mechanical characterization of all PA substrates is based on previously established testing protocols on a custom-built uniaxial compression apparatus. Briefly, gel samples are cast in circular nylon molds (16 mm in diameter and 10 mm in height). Following polymerization, the molds are removed and the samples are submersed in DI water. The custom-built compression device consists of a centrally positioned linear actuator  and a built-in linear encoder, that provides displacement information with a displacement resolution of 1 To promote cell attachment to polyacrylamide films, substrates are functionalized with laminin (Schwann cells) or fibronectin (neutrophils and fibroblasts) using the bifunctional crosslinker, sulfo-SANPAH  2 on PA gels. Cells were allowed to attach for 4 hours before initiation of the time-lapse microscopy.Schwann cells were cultured as previously described by Mitchel et al. After an initial seeding period, three-dimensional image stacks of individual cells on PA gels are acquired using a Nikon A-1 confocal system mounted on a TI Eclipse inverted optical microscope, controlled by NI-Elements Nikon Software. Red fluorescent microspheres are used as fiducial markers and excited with a Diode (561 nm) laser. 512Figure S1Analytically calculated Gaussian traction dipoles on the substrate surface. Contour plot of the analytically calculated traction vector magnitude due to the prescribed Gaussian displacement dipoles presented in (TIF)Click here for additional data file.Figure S2Measurement sensitivity thresholds in terms of Cauchy stresses and surface tractions as a function of substrate stiffness. A plot showing the noise floor of (A) the Cauchy stress (red) 0.1%, (green) 1%, and (blue) 10%; and (B) the tractions are calculated from the Cauchy stress via the Cauchy relation. The  normal vector was additionally corrupted with an appropriate level of Gaussian white noise. To compute the final traction noise floor, the largest standard deviation for each Cauchy stress component was chosen, thus representing the most conservative traction noise floor estimate.(TIF)Click here for additional data file.Figure S3Experimental example of a migrating Schwann cell on the surface of a 3D LSCM imaging volume (Cell Example 2). (A) Magnitude of the 3D Schwann cell surface displacement field, (TIF)Click here for additional data file.Figure S4Comparison of commonly reported metrics in TFM for cell example 2. Side by side comparison of the (A) total force, (B) root mean squared (RMS) tractions and maximum tractions, and (C) strain energy for both the linear elastic, small deformation (SD) and non-linear, large deformation (LD) models.(TIF)Click here for additional data file.Figure S5Experimental example of a migrating Schwann cell on the surface of a 3D LSCM imaging volume (Cell Example 3). (A) Magnitude of the 3D Schwann cell surface displacement field, (TIF)Click here for additional data file.Figure S6Comparison of commonly reported metrics in TFM for cell example 3. Side by side comparison of the (A) total force, (B) root mean squared (RMS) tractions and maximum tractions, and (C) strain energy for both the linear elastic, small deformation (SD) and non-linear, large deformation (LD) models.(TIF)Click here for additional data file.


Spinal cord injury (SCI) is a devastating event with a limited hope for recovery and represents an enormous public health issue. It is crucial to understand the disturbances in the metabolic network after SCI to identify injury mechanisms and opportunities for treatment intervention. Through plasma 1H-nuclear magnetic resonance (NMR) screening, we identified 15 metabolites that made up an “Eigen-metabolome” capable of distinguishing rats with severe SCI from healthy control rats. Forty enzymes regulated these 15 metabolites in the metabolic network. We also found that 16 metabolites regulated by 130 enzymes in the metabolic network impacted neurobehavioral recovery. Using the Eigen-metabolome, we established a linear discrimination model to cluster rats with severe and mild SCI and control rats into separate groups and identify the interactive relationships between metabolic biomarkers in the global metabolic network. We identified 10 clusters in the global metabolic network and defined them as distinct metabolic disturbance domains of SCI. Metabolic paths such as retinal, glycerophospholipid, arachidonic acid metabolism; NAD–NADPH conversion process, tyrosine metabolism, and cadaverine and putrescine metabolism were included. In summary, we presented a novel interdisciplinary method that integrates metabolomics and global metabolic network analysis to visualize metabolic network disturbances after SCI. Our study demonstrated the systems biological study paradigm that integration of 1H-NMR, metabolomics, and global metabolic network analysis is useful to visualize complex metabolic disturbances after severe SCI. Furthermore, our findings may provide a new quantitative injury severity evaluation model for clinical use. In the United States, 12,000 new SCI cases are annually reported, and $24 billion is annually spent on paralyzed SCI patients Human metabolism is a complex network with hundreds of cross-linked paths. During critical SCI illness, the metabolic network is dynamically disturbed at multiple points An interdisciplinary method that includes high-throughput quantitative techniques and effective mathematical and visualization tools is necessary because metabolic network disturbances after SCI are complex; hundreds of molecules and metabolic paths interact n = 7), mild SCI (n = 10), and severe SCI (n = 25). All the rats were fed the standard formula feed from 7 days before the operation until sacrifice. All rat forage was formulated according to the formulation established by the State Bureau of Quality Technical Supervision of China (GB Standards No: 14924.3-2010). Animal use was strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Experimental Animal Committee of Sichuan Academy of Medical Sciences approved the research protocol .Animal modeling procedures were performed according to an established protocol, as described previously Rats assigned to the SCI group were anesthetized by intra-peritoneal injection of sodium pentobarbital (40 mg/kg). We tried our best to minimize animal suffering. Rats were observed for at least 3 days after the start of the experiment. For severe SCI, we established a 7-day observation subgroup. In the mild SCI group, three rats died during the surgical modeling procedure and one died 2 days after the surgery. In the severe SCI group, five rats died during the surgical modeling procedure and four died 1 and 2 days after the surgery. Therefore, there were six animals in the mild SCI group, 16 in the severe injury group (seven rats were fed for 7 days after modeling), and seven in the blank control group. Plasma samples were collected for 1H-NMR metabolomic analysis.Plasma samples were centrifuged at 10,000 rpm for 10 min to remove particulate matter. Following this, 100 µL of deuterium oxide (D2O) was added to 400 µL of the plasma samples in 5 mm Wilmad NMR tubes to lock the field frequency for the NMR spectrometer.A high-solution Bruker AVIII-DRX600MHz NMR spectrometer  was used to obtain 1H-NMR spectra from the plasma samples. The probe temperature was 300 K. One 1D-NMR experiment was included. A standard protocol that included a one-dimensional pulse sequence and a Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence was followed Principle component analysis (PCA) and partial least-square (PLS) regression were performed on 1H-NMR data to reduce data dimensionality and for multivariate analysis. We used Matlab R2012b for computing and mathematical modeling . The variable importance for the projection (VIP) value was used to screen metabolites. The Human Metabolome Database (HMDB) was used to identify key metabolites related to enzymes and upstream genes. Metabolites and their enzymes were mapped, and the disturbed metabolic paths were located on a global metabolic map of the Kyoto Encyclopedia of Genes and Genomes (KEGG).A linear discriminant formula was established as a quantitative injury evaluation model. In this model, weighted PLS-DA values were used. The root mean square error (RMSE) was calculated to verify the validity of the equations according to injury severity.Metabolic disturbances after severe SCI were established according to two steps: 1) key metabolites were mapped onto the KEGG metabolic network (KEGG metabolic pathways PATHWAY: map01100) and 2) the shortest path for each key metabolite (node) was calculated and outlined onto the metabolic network using the KEGG pathway API for the Matlab bioinformatics toolbox.Compared with the control group, mild and severe SCI 1H-NMR spectra were perturbed at 0–8 ppm . This phPCA. PCA scores indicated that injured rat plasma metabolomes differed from controls. The first two principal components (PCs) explained 83% of the total variance and suggested that most metabolome differences between normal and SCI cases were because of injury (f injury .PLS was used to investigate correlations between severity of SCI and the metabolome. The VIP indicator can describe correlations between the variable (X) and response (Y). We used VIP to identify metabolites correlated with severe SCI and named these metabolites as “Eigen-metabolome” of severe injury. Segmental integrations in 83, 90, 92, 151, 152, and 153 were found to have the highest VIP values and represented metabolites with 4.72, 4.44, 4.4, 2, 1.98, and 1.96 1H-NMR spectra chemical shift values, respectively.We identified 15 metabolites that made up an “Eigen-metabolome” capable of distinguishing rats with severe SCI from healthy control rats. We also found that 16 metabolites impacted neurobehavioral recovery  and 2.2 = 0.284 . The accuracy ratio of PLS regression was 86.2%, R = 0.284 . Xi reprred rats .After metabolites related to severe SCI were identified, metabolite roles and interactions were investigated on the global metabolic network. A three-step method was developed and included the following: 1) HMDB-based identification of enzymes that catalyze metabolites; 2) enzyme ID conversion from HMDB to KEGG enzyme IDs; 3) the use of Matlab KEGG API to map the enzymes into metabolic pathways (KEGG metabolic pathway ID: PATHWAY: map01100). Following this, disturbed metabolic pathways and metabolic pathways related to neurobehavioral recovery after severe SCI were visualized .Seven metabolic pathways related to severe SCI and eight metabolic pathways related to neurobehavioral recovery were identified .We successfully use plasma 1H-NMR spectra to construct an Eigen-metabolome-based quantitative SCI severity evaluation model, which poses clinical application potential in future. Fifteen metabolites made up an Eigen-metabolome that distinguished severe SCI from mild injuries and healthy controls, whereas sixteen metabolites made up an Eigen-metabolome that distinguished neurobehavioral recovery rats from other treatment groups.We established a PLS linear formulation group to provide a severe SCI evaluation model. In our model, X represented the ppm value from 1H-NMR spectra and Y represented the severity of SCI. We found that we could distinguish control, severe, and mild injury rats in the PLS score plot by using SVM. To avoid the challenge by excessive clustering and small sample size, we used RMSE to evaluate the accuracy of our model. RMSE of our model (4.76) indicated it was appropriate for evaluating injury severity.et al. found that neurobehavioral recovery after SCI is related to a decrease in N-acetyl-aspartyl-glutamate and N-acetyl-aspartate in a liquid chromatography–mass spectrometry (LC–MS) metabolomic model Although few studies demonstrate that SCI disturbs plasma metabolites and metabolic patterns, SCI metabolic disturbances continue to challenge researchers. Consistent with our study, Fujieda Y We introduced a new approach that integrates metabolomic analysis with metabolic network analysis. Significantly altered metabolites were extracted from the Eigen-metabolome and mapped onto a metabolic network. Enzymes that produce these metabolites were also identified and included in the network. Hundreds of plasma metabolites are altered after major trauma because enzyme activity increases or decreases to effectively decrease or increase metabolites levels 1) Retinal metabolism The retinal metabolism metabolic pathway contains two important metabolites, retinaldehyde (vitamin A) and retinoic acid (RA). RA plays a role in neural differentiation during embryonic differentiation. Studies have shown that RA possesses a neuroprotective effect during central nervous system injury. Plasma vitamin A, vitamin E, and beta-carotene are decreased after SCI. Furthermore, a positive correlation between plasma vitamin A and the severity of the chronic SCI has been identified 2) Glycerophospholipid and arachidonic acid (AA) metabolism Phosphoglyceride and sphingomyelin could be used to distinguish the severity of injury in 1H-NMR spectra. Both phospholipids are regulated by superoxide dismutase (SOD) and glutathione (GSH). SCI is currently considered as a myelin sheath damage. The myelin sheath is a greatly extended and modified cell membrane that is wrapped around the nerve axon in a spiral manner. Sphingomyelin and glycerol phospholipid are important components of the cell membrane, and when their metabolic paths are disturbed, the cell membrane is damaged. The simple sphingolipid metabolites ceramide and sphingosine-1-phosphate are major signaling cascade mediators involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation 3) The NAD–NADPH conversion process The NAD–NADPH conversion process includes etheral metabolism, ethanol–ethylal conversion, and choline to betaine aldehyde metabolism. Many anabolic pathways such as lipid synthesis, cholesterol synthesis, cellular respiration via the electron-transport chain, and fatty acid chain elongation are related to NAD and NADPH. Metabolomic fingerprints change after SCI, partly because of alterations in dehydrogenase activity.4) Tyrosine metabolism We found that the disturbances in tyrosine metabolism contributed to injury severity and neurobehavioral recovery. Tyrosine metabolism involves various neurotransmitter synthesis pathways. Tyrosine can be converted to L-DOPA, which is converted to the nerve injury biomarkers dopamine, norepinephrine , and epinephrine . The production and elimination of nitrotyrosine (NT) are markers of nerve injury during tyrosine metabolism. NT is a product of tyrosine nitration mediated by reactive nitrogen species such as the peroxynitrite anion and nitrogen dioxide. NT is one of the most important markers for evaluating tissue and cell damage. Tyrosine nitration leads to enzyme inactivation 5) Cadaverine and putrescine metabolism The role of decarboxylase ornithine decarboxylase (ODC) as a catalytic product in SCI has gained interest. ODC and putrescine levels significantly increase after SCI 6) Disturbances in ether and alcohol metabolism We found that several metabolic pathways such as chloroalkene and chloroalkene degradation and aldehyde and ethanol transformation (not well studied in SCI) appeared in the disturbance pathways. These metabolites belong to etheral metabolism and are primarily generated by the gut microbiome. This disturbance certainly takes place in clinical practice; however, it is typically ignored in traditional trauma biomedical research.In summary, SCI can be understood as disturbances in cellular signaling regulatory networks in response to trauma. Furthermore, these disturbances commonly present a pattern of general cellular transduction, as illustrated in Our study demonstrated that 1H-NMR spectra can be used to quantitatively measure and visualize the metabolic network disturbances after SCI. We identified major metabolites and enzymes through unbiased screening of compounds identified by metabolomic analysis. Metabolites were mapped to a metabolic network, and their functions were interpreted by KEGG metabolic network visualization. This method has the advantage of integrating metabolic research with the trauma mechanisms to aid our understanding of the pathophysiological SCI process.Our model incorporated metabolomics, biological informatics, and pattern recognition to develop a new evaluation and outcome prediction tool for SCI that can provide substantial improvements to the diagnosis and intervention monitoring of SCI patients.Table S1The Basso, Beattie, and Bresnahan (BBB) score scale of all rats.(XLSX)Click here for additional data file.Table S2Raw data of 1H-NMR: All the raw data through binning process of the NMR spectrum after Fourier transformation as a 200×29 matrix; Each row describes the integral binning value of a spectrum for a sample.(XLSX)Click here for additional data file.Table S3Grouping of animals: The treatment that 29 samples received are as follows: “control” present, control group; “mild” present, the rat received a laminectomy without hemisection of spinal cord injury; “op” present, the rat received a hemisection of the spinal cord and was scarified 3 days after the operation; “op7” present, the rat received a hemisection of the spinal cord and was scarified 7 days after the operation.(XLSX)Click here for additional data file.


CYP2A6 L160H was associated with pancreatic cancer. In addition, DNA damage was found to be associated with TNFA −308G>A and ERCC4 R415Q polymorphisms. These results suggest that measurement of DNA damage, as well as select SNPs, may provide an important screening tool to identify individuals at risk for development of pancreatic cancer.Several risk factors have been identified as potential contributors to pancreatic cancer development, including environmental and lifestyle factors, such as smoking, drinking and diet, and medical conditions such as diabetes and pancreatitis, all of which generate oxidative stress and DNA damage. Oxidative stress status can be modified by environmental factors and also by an individual's unique genetic makeup. Here we examined the contribution of environment and genetics to an individual's level of oxidative stress, DNA damage and susceptibility to pancreatic cancer in a pilot study using three groups of subjects: a newly diagnosed pancreatic cancer group, a healthy genetically-unrelated control group living with the case subject, and a healthy genetically-related control group which does not reside with the subject. Oxidative stress and DNA damage was evaluated by measuring total antioxidant capacity, direct and oxidative DNA damage by Comet assay, and malondialdehyde levels. Direct DNA damage was significantly elevated in pancreatic cancer patients (age and sex adjusted mean ± standard error: 1.00±0.05) versus both healthy unrelated and related controls . Analysis of 22 selected SNPs in oxidative stress and DNA damage genes revealed that  Pancreatic cancer, the fourth leading cause of cancer deaths in the United States, is characterized by rapid metastasis and profound resistance to chemo- and radiotherapy. Detection late in the disease course and limited treatment options contribute to its poor prognosis In addition to chronic pancreatitis and diabetes, several lifestyle risk factors have been linked to the development of pancreatic cancer, including smoking, heavy alcohol consumption, and obesity CYP1A1, GSTM1, GSTT1 and GSTP1 alone did not correlate with pancreatic cancer risk, a significant interaction between smoking and the GSTT1 null genotype was reported in Caucasian pancreatic cancer subjects MMS19L gene with pancreatic cancer risk ERCC4 R415Q and LIG3 G-39A minor alleles, while an increased risk was observed for the ATM D1853N allele An individual's oxidative stress level depends on lifestyle determinants, such as smoking, drinking and diet, and is also influenced by genetics. Several case-control studies have investigated the correlation of single nucleotide polymorphisms (SNPs) in genes related to carcinogen metabolism, oxidative stress and DNA repair, with pancreatic cancer. While SNPs in the phase I and II metabolism genes This study was approved by the Indiana University Institutional Review Board. Written informed consent was obtained from participants.2) was calculated from self-reported height and weight or patient's charts. Blood samples were obtained from participants at the time of enrollment.A total of 31 patients (cases) with pathologically confirmed pancreatic cancer (Stages I–IV) and 40 healthy controls  were enrolled. Cases were excluded if they had a history of other malignancies, or had already received treatment with chemotherapy or radiotherapy. Cases were matched with either genetically related controls and/or genetically unrelated controls. These distinct control groups were recruited in order to discern the contribution of environmental and genetic factors in pancreatic cancer risk. Genetically related controls were included provided they did not live with their matched case while genetically unrelated controls had to be cohabiting with the case. All enrolled participants were Caucasian, ≥18 years at time of consent, and able to understand and sign a written informed consent. Information concerning subject demographics, behavioral factors , and personal and family medical history were obtained by self-report using a questionnaire at the time of enrollment. Cumulative smoking was calculated as pack-years [(packs/day)x(years smoked)]. Alcohol consumption was reported as days drinking in the past year. Body mass index  was measured in serum as described Whole blood (10 µl) was mixed with 0.5 ml RPMI 1640 containing 10% FBS, 10% DMSO, 1 mM deferoxamine, step-frozen and stored at −80°C until analysis. The Comet assay was performed as described previously MDA was measured in serum using high performance liquid chromatography (HPLC) with UV detection as described previously Genomic DNA was extracted from PBMCs in whole blood using QIAamp DNA Blood Midi kit (Qiagen). Determination of SNPs by allelic discrimination was performed using TaqMan validated probes (Applied Biosystems) on an ABI 7900HT instrument according to manufacturer's instructions. The SNPs were selected based on their previously reported associations with oxidative stress status, DNA damage, and cancer risk.Generalized and linear mixed models that accounted for the correlations between patients and controls were used to compare demographic, environmental factors, and chronic pancreatitis and other inflammatory conditions between the three arms. Oxidative stress and DNA damage measures were compared between the pancreatic cancer patients and healthy unrelated or healthy related controls and correlated with environmental factors and SNPs  using linear mixed models adjusting for age and sex due to group differences in these demographics. Adjusted means ± standard errors are reported from these models. Hardy Weinberg equilibrium test was conducted to check the genotype QC. Genetic risk of pancreatic cancer was assessed by comparing SNP frequencies between patients and unrelated healthy controls using Fisher exact tests (for dominant and recessive genetic models) or Mantel-Haenszel Chi-square exact tests (for gene dose models). All analyses were performed using SAS Version 9.3 . P-values of <0.05 were considered statistically significant. The purpose of this pilot study was to generate hypotheses, especially genetic signals. Therefore, the type I error was not stringently controlled in multiple-comparisons.Demographic characteristics of the case and control subjects are shown in An oxidative stress/damage profile was generated from blood samples of all enrolled participants . Total aThe alkaline Comet assay, that measures single and double DNA strand breaks, was used to assess direct DNA damage in PBMCs of cases and controls. Oxidative DNA damage was determined by a modified Comet assay which included incubation of the sample with formamidopyrimidine-DNA glycosylase (fpg) prior to electrophoresis. Fpg recognizes oxidatively modified purines and introduces additional DNA strand breaks, thus the difference between fpg-modifed and un-modified Comet measurements corresponds to oxidatively damaged DNA. As shown in s = −.29, p = .02). No significant associations were observed between days drinking and TAC, direct, or oxidative DNA damage. No correlations between oxidative stress markers and chronic pancreatitis were observed; however, overall, inflammatory conditions were associated with elevated levels of MDA (p = 0.048).Next, we examined potential correlations between known pancreatic cancer risk factors and oxidative stress biomarkers . All corCYP2A6 L160H major allele was associated with pancreatic cancer overall (p = 0.03), as well as exhibiting significance for dominance, recessive and gene dose effects.The genotype of 22 SNPs in 17 genes involved in oxidative stress, inflammation, DNA damage, methionine/folate metabolism and carcinogen metabolism was determined in pancreatic cancer cases and control groups . AnalysiTNF −308 G>A SNP exhibited a significant recessive effect (p = 0.003). The ERCC4 R415Q polymorphism demonstrated significant dominant and gene dose effects (p = 0.009). For both genes, the minor allele resulted in a significant elevation in direct DNA damage  versus the AG (0.81±0.06) and GG (0.88±0.04) alleles. While no QQ homozygous individuals were present in this study, heterozygous ERCC4 R415Q subjects exhibited elevated DNA damage versus RR individuals (1.12±0.10 and 0.84±0.04 respectively).Since direct DNA damage was significantly elevated in pancreatic cancer cases compared to both control groups, the correlation between SNP genotype and this biomarker were analyzed  and 6. TA damage . The AA The etiology of sporadic pancreatic cancer remains largely unknown; however, epidemiologic studies have identified risk factors that contribute to the development of pancreatic carcinomas. These factors include environmental and lifestyle factors, such as smoking, drinking and diet, and inflammatory conditions such as diabetes and pancreatitis, all of which share the ability to generate oxidative stress and DNA damage. The present pilot study was designed to examine the contribution of environment and genetics to an individual's level of oxidative stress and DNA damage and the subsequent risk for development of pancreatic cancer. To accomplish this objective, we utilized three groups of subjects; a pancreatic cancer cohort, a healthy unrelated control group living with the case subject, and a healthy genetically related control group which did not reside with the case. We found evidence to suggest that both environment and genetics contribute to the oxidative stress and DNA damage observed in cases and controls in our study.Oxidative stress and DNA damage were evaluated using four measurements: TAC, direct and oxidative DNA damage measured in circulating PBMCs, and MDA levels. These parameters have previously been investigated in human diabetic subjects and in an animal model of diabetes One of the most significant observations in this study was an increased level of direct DNA damage in the pancreatic cancer group compared to both control groups . This obCYP2A6 L160H polymorphism. Among its diverse substrates, CYP2A6 catalyzes the metabolism of nicotine and tobacco-specific procarcinogens, such as 4-(methylnitrosamine)-a-(3-pyridyl)-1-butanone or NNK CYP2A6 His variant would be unable to activate procarcinogens in cigarette smoke and thus would be protected against cancer development, a finding that has been shown in lung cancer CYP2A6 allele compared to 75% and 89% of the healthy unrelated and related controls, respectively , a pro-inflammatory cytokine, has been implicated in autoimmune diseases and cancers associated with an inflammatory component A damage  and 6; hA damage . These rERCC4 minor alleles (AA) have been associated with increased risk of breast cancer in several studies ERCC4 polymorphism on enzyme activity has not been firmly established; however, modeling programs have predicted that the R415Q change would negatively impact protein function and repair capacity ERCC4 R415Q heterozygotes contributes to development of pancreatic cancer. This is in agreement with a recent study showing that heterozygous R415Q ERCC4 was associated with benign breast disease, a known breast cancer precursor In our study, DNA damage in PBMCs was significantly elevated in pancreatic cancer patients . Repair While these results show associations of DNA damage with pancreatic cancer and an association of DNA damage with selective SNPs, several limitations exist in our study. The low frequency of rare alleles for some SNPs may lead to spurious results whereby recruitment of additional subjects will be required to validate these findings. Recall bias may have led to misclassification of subjects into environmental lifestyle groups, as many of the identified pancreatic cancer risk factors  were self-reported. Age was significantly lower in the healthy related control group compared to pancreatic cancer subjects. Since increased DNA damage has been observed with aging CYP2A6), inflammation (TNFA), and DNA damage and repair (ERCC4). Evaluating DNA damage in circulating PBMCs, as well as select genotyping strategies may thus provide an important screening tool to identify individuals at an increased risk for developing pancreatic cancer. Due to the small sample size in this pilot study, assessment of these endpoints in additional pancreatic cancer and control subjects is needed. In addition, mechanistic studies of individual SNPs in CYP2A6, TNFA and ERCC4 genes would be useful to assess their contribution to the development of pancreatic cancer.The use of two control groups in this study enabled the investigation of the contribution of environment to pancreatic cancer in a unique way; control subjects who reside with cases are more likely to share the same lifestyle exposures, as opposed to controls that are matched to cases by questionnaire data alone. In addition, these potential exposures are likely to have been shared for a considerable amount of time which enables a more accurate assessment of the contribution of individual factors to pancreatic cancer development. Overall, results from this pilot study support a role of both genetics and environmental/lifestyle factors in the development of pancreatic cancer. We report an association of pancreatic cancer with DNA damage and associations with specific polymorphisms in genes involved in metabolism (Table S1Genotype Frequencies in Selected SNPs.(DOCX)Click here for additional data file.


Observational studies suggest high prenatal vitamin D intake may be associated with reduced childhood wheezing. We examined the effect of prenatal vitamin D on childhood wheezing in an interventional study.We randomised 180 pregnant women at 27 weeks gestation to either no vitamin D, 800 IU ergocalciferol daily until delivery or single oral bolus of 200,000 IU cholecalciferol, in an ethnically stratified, randomised controlled trial. Supplementation improved but did not optimise vitamin D status. Researchers blind to allocation assessed offspring at 3 years. Primary outcome was any history of wheeze assessed by validated questionnaire. Secondary outcomes included atopy, respiratory infection, impulse oscillometry and exhaled nitric oxide. Primary analyses used logistic and linear regression.We evaluated 158 of 180 (88%) offspring at age 3 years for the primary outcome. Atopy was assessed by skin test for 95 children (53%), serum IgE for 86 (48%), exhaled nitric oxide for 62 (34%) and impulse oscillometry of acceptable quality for 51 (28%). We found no difference between supplemented and control groups in risk of wheeze . There was no significant difference in atopy, eczema risk, lung function or exhaled nitric oxide between supplemented groups and controls.Prenatal vitamin D supplementation in late pregnancy that had a modest effect on cord blood vitamin D level, was not associated with decreased wheezing in offspring at age three years.ISRCTN68645785Controlled-Trials.com  Several observational studies have suggested a protective effect of higher maternal vitamin D intake during pregnancy on risk of wheezing in vitro models to have potent immune effects There are plausible biological mechanisms for an association between prenatal 25(OH)D status and wheezing. Developmental programming of lung function and immune responses occurs during pregnancy The protocol for this trial and supporting CONSORT checklist are available as supporting information; see St. Mary’s Hospital Research Ethics Committee approved the follow up study (10/H0712/13) and women gave written informed consent to participation for themselves and their child. Where women only consented to interview over the telephone, verbal consent was taken in place of written consent and this was approved by the Ethics Committee. For each participant who gave verbal consent, this was taken and documented by 2 separate members of the research team.This was a prospective follow up study of the offspring of women who took part in an ethnically stratified, parallel group, randomised controlled trial of vitamin D supplementation in pregnancy at St Mary’s Hospital London, a university-affiliated hospital prenatal clinic, between April and November 2007. The original trial was conducted to determine the effect of supplementation on 25(OH)D status of mothers in 4 specific ethnic groups, and their babies, at delivery Women were randomised at 27 weeks gestation to no treatment (control), 800 IU ergocalciferol until delivery (daily), or a single oral dose of 200,000 IU cholecalciferol (bolus). The randomisation sequence was generated by an independent researcher using computer generated random number lists in blocks of 15, stratified by 4 ethnic groups in a 1∶1:1 ratio. The researcher gave participants a study number on entry to the trial, and treatment was allocated from the hospital pharmacy. Women were given instructions to swallow the tablets whole and to avoid other multivitamin supplements containing vitamin D. This trial was conducted before national guidance on routinely providing advice on vitamin D intake during pregnancy was introduced in March 2008 The primary outcome was prevalence of ‘wheeze ever’ as defined by the International Study of Asthma and Allergies in Childhood (ISAAC) Exhaled nitric oxide (eNO) was measured using an offline, tidal breathing technique adherent to ATS/ERS guidelines Allergic sensitisation was defined as a skin prick test wheal at least 3 mm greater than the negative control (Glycerine) to one or more of the aeroallergens tested at 15 minutes, in the context of an appropriate response to the positive control (Histamine 10%). House dust mite, alternaria, cladosporium, cat, dog, grass pollen, silver birch pollen, peanut, milk and egg were tested .Serum total IgE , 25(OH)D and eosinophil count were determined on the day of the child’s assessment. 25(OH)D was measured in mothers prior to randomisation, and in offspring at birth (cord blood) using a radio-immunoassay  in a clinical biochemistry laboratory that participates in the international Vitamin D external quality assessment program (DEQAS). Vitamin D deficiency was defined as 25(OH)D <25 nmol/L Primary health care records were obtained from participants’ general practitioners and reviewed by a single investigator (RJB), blinded to treatment allocation. Children were categorised as having ‘recurrent wheezing’ where ≥2 episodes of either wheezing, or respiratory distress treated with bronchodilator were recorded; ‘eczema’ if they had ≥2 attendances separated by ≥6 months where either topical corticosteroids were prescribed for treatment of an itchy skin rash, or a doctor’s diagnosis of eczema was made; and ‘food allergy’ if this diagnosis was recorded in any part of the primary healthcare record.In the original study it was calculated that at least eight women in each ethnic group for each arm of treatment would be needed to demonstrate a significant difference in vitamin D levels at delivery in the no treatment group vs. the supplemented groups . To account for drop-outs, preterm delivery, and delivery at another hospital, 15 women were allocated to each arm of treatment within each ethnic group (n = 180).With 180 participants and 80% successful follow up, this study had 80% power with 2-sided alpha of 0.05 to detect a reduction in wheeze from 34% in the children of non-supplemented mothers Adjustment for multiple testing was performed using the Benjamini and Hochberg method P<0.001; bolus dose 25 nmol/l (IQR 18–34); P<0.001)].Participant flow is shown in We found no significant difference in the primary outcome of ‘wheeze ever’ between treatment groups D levels were available. We found no difference between the natural logarithm (Ln) of cord blood 25(OH)D levels in children with and without a history of wheeze, atopy, eczema, any LRTI or >4 episodes of URTI per year (Table S5 in Fifty-one of 180 (28%) children provided acceptable IOS data for analyses. We found no significant difference between groups in baseline respiratory resistance at 10 or 20 Hz, resonant frequency, area under the reactance curve or percentage response of these parameters to bronchodilator, when data were analysed for the two forms of prenatal vitamin D supplementation combined , 62/180 (34%) and 80/180 (44%) of offspring. We found no significant difference between groups in these outcomes assessed at age 3, when analysed for the two forms of prenatal vitamin D supplementation combined D concentration, found relationships with allergic sensitisation and/or wheezing in early childhood The findings of our study are specific to a population of vitamin D deficient women D levels below 25 nmol/L) and two specific forms of prenatal vitamin D supplementation. The doses of supplementation were chosen pragmatically, were not stratified based on individual vitamin D status or genotype, and response to treatment was not monitored before delivery. The study population was not selected for allergy or asthma risk and, as a mixed race urban population, is representative of many populations of pregnant women worldwide In summary we found no evidence for a protective effect of prenatal vitamin D supplementation from 27 weeks gestation on childhood wheezing, allergic disease, lung function or markers of airway inflammation in the first 3 years of life. Given the modest effects on cord blood vitamin D achieved by these interventions, the safety and efficacy of higher dose prenatal vitamin D supplementation strategies need to be explored.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.File S1Supporting Information.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOCX)Click here for additional data file.


Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors  and negative regulators  and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants, affecting almost ten percent of all infants with a birth weight under 1500 g This laboratory has had a longstanding interest in the development of host defense in the human intestine and the extent to which immaturities in host defense influence the expression of age-related neonatal gastrointestinal disease states such as NEC. As stated above, the most important risk factor for this condition as reported from several large studies, is prematurity ex vivo fetal intestinal xenografts. The results of these observations suggest that multiple genes involved in the innate immune response may be developmentally regulated.Recently, we have reported that the excessive inflammatory response of the immature intestine is in part due to a developmental underexpression of IκB, an important regulator of NFκB signaling NFκB/MyD88 innate inflammatory genes are over-expressed and negative regulator genes are underexpressed in fetal and NEC intestine. Using laser capture microdissection (LCM) of ileal resections from 1) three children subjected to elective intestinal surgery, 2) three therapeutically aborted fetus and 3) three NEC patients, epithelial RNA was isolated and innate immune response genes quantitated by qRT-PCR. Due to a scarcity of epithelial RNA from NEC resections, mRNA levels are only shown in ex vivo model. In these studies, we have shown a striking difference in the inflammatory (IL-8) response to exogenous and endogenous stimuli (LPS and IL-1β) between xenografts at 20 wks posttransplant (designated immature xenografts) compared to xenografts at 30 wks posttransplant (designated mature xenografts) In previous publications using fetal intestinal xenografts ex vivo xenograft model to demonstrate the developmental regulation of innate immune response genes for several reasons. 1) The time needed to expose enterocytes to hydrocortisone (5 days) precluded using primary enterocytes particularly from NEC patients; 2) we have previously reported that hydrocortisone injection into SCID mice followed by an inflammatory stimuli (IL-1β) reduced the IL-8 response only in immature xenografts and qRT-PCR measurement of IκB mRNA from LCM of epithelial RNA one week after steroid treatment resulted in an increased IκB gene expression ex vivo model of human intestinal development, we determined the effect of cortisone on the expression of selective innate inflammatory response genes in human intestinal xenografts. Both immature (20-week-old) and mature (30-week-old) human intestinal xenografts were treated with cortisone acetate or a vehicle control, as described previously We chose to use the To further compare the functional difference in the effector, TLR and negative regulators in primary enterocytes isolated from fetal and NEC ileum, we determined the fold difference in mRNA and protein. To determine if the expression of negative regulators was in part responsible for the highly significant lower fold IL-8 mRNA and protein expression in response to inflammatory stimuli in fetal vs. NEC-cells  cells and these receptors are further unregulated with inflammation  but the 1) as well as the effector IL-8 genes was noted in immature fetal intestine compared to intestine from children. Because of the limitation of NEC epithelial RNA, only selective genes were noted to be further overexpressed in the epithelium of freshly resected NEC tissue (We have previously reported that human premature enterocytes respond excessively to exogenous and endogenous stimuli to produce an increased inflammatory (IL-8) response After birth, the intestinal epithelium, particularly the distal ileum and colon, are suddenly continuously exposed to excessive quantities of MAMPs from initial colonizing bacteria. In term infants, the maturing enterocyte has devised protective mechanisms to prevent chronic inflammation from this exposure. These mechanisms include a reduction in TLR surface expression in utero are in a relatively germfree environment and therefore are not as vulnerable to microbial inflammatory stimuli as are those in infants in the extrauterine environment. Why are they expressed in abundance and on the enterocyte surface in utero? Toll receptors originally reported in Drosophila were shown to have developmental functions mediating dorsal/ventral orientation during development TLRs on enterocytes Several recent studies in rodent models for NEC have implicated a persistently increased TLR expression on immature enterocytes and excessive NFκB signal transduction as the basis for inflammatory necrosis ex vivo fetal intestinal xenograft model in addition to primary enterocytes isolated from freshly resected fetal and NEC small intestine to demonstrate that an immaturity in innate immune inflammatory response genes are developmentally regulated and undoubtedly important in explaining the excessive NEC inflammatory response. We have had extensive experience with the fetal human intestinal xenograft model ex vivo model system to provide functional relevance to in vitro cellular studies. In addition to the developmental upregulation of TLRs, signaling molecules and transcription factor genes, underexpression of negative regulators of TLR signaling appear to be the key to the excessive inflammatory response in immature enterocytes. Again this underexpression of negative regulators was further reduced in resected NEC intestine  at appropriate times during gestation or after delivery could result in a prevention strategy for the care of premature infants. However, when the use of cortisone was recommended, it was determined that early cortisone exposure, particularly in the postpartum period, had adverse consequences on other organ systems in vitro and the right dose to be used in multi-center trials before recommendations for routine clinical care can be made.In All the chemicals were of analytical grade obtained from the Sigma . Reagents related to cell culture were obtained from GIBCO BRL . All reagents for ELISA and recombinant IL-1α and epidermal growth factor (EGF) were obtained from R&D Systems .fC or Children's Hospital. Control tissues were obtained from children under six years of age who had elective small intestinal resections for intestinal anomalies . Again marginal small intestine was taken from the operating room for primary enterocyte separation and LCM for RNA. Informed written consent was obtained from the parents/guardian for this study. In selected segments laser capture microdissection was used to obtain epithelial RNA.Resected ileal segments were collected after obtaining informed consent from Partners Human Research Committee . Selected segments of resected ileum for necrotizing enterocolitis were obtained from multiple institutions Partners for Human Research Committee  [Claud]) and handled similarly. Each of the NEC specimens were obtained from prematures, weighing between 1200 and 1500 gms exhibiting pneumatosis intestinalis approximately two to three weeks after birth and after approximately one week of oral feedings. Marginal normal ileal tissue adjacent to the diseased intestine was obtained. Primary enterocytes were only isolated from freshly resected NEC tissue at MGHHuman small intestine was obtained from elective prostaglandin/saline-induced therapeutic abortion of 12 to 16 wk fetuses after informed written consent from Partners for Human Research Committee . All experiments involving utilizing human tissues were approved by Partners for Human Research Committee  cytotoxicity assay, immunofluorescence, total RNA isolation and quantitative real time qRT-PCR were described previously and detailed in previous publications ®, then eight micron thin cryo-sections were cut and mounted on microscopy slides  and epithelial cells were isolated as described previously The epithelial cells were isolated by LCM from fresh resected intestinal tissue or from immature (20-week) and mature (30-week) intestinal xenografts. The tissue was frozen in OCTBriefly, thin intestinal cryosections were fixed in 70% ethanol, rinsed with RNAse free water and toluidine blue stained followed by dehydration in increasingly concentrated ethanol and xylene before laser capture microdissection of the epithelial cells (LCM). Approximately 600 toluidine blue stained epithelial cells from each section of the mucosa were captured onto polyethylene collecting caps  In general, protein samples will be lysed and quantitated by the BCA method (Pierce) using BSA as a standard Specific siRNA oligonucleotide were designed and purchased from Invitrogen® . Epithelial cells at 50% confluence were transfected with 10 nM of specific siRNA or negative control using RNAiMAX kit according to manufacturer's recommendations. Transfection efficiency was assessed by transfecting the cell with BLOCK-iT™ Alexa Fluor Red fluorescent control and quantifying total number of cells by DAPI staining and transfected cells by red fluorescence to calculate the efficiency. Specific knockdown efficiency for each gene was measured by qRT-PCR and/or by western blots.6 cells. Isolated cells were washed once with FACS media  and blocked with FACS media containing 5% mouse serum for 30 min on ice. After washing, cell suspension were stained with PE-conjugated mouse anti-human TLR4 mAb  at 1 in 20 dilution for 30 min on ice in the dark, washed three times with FCS media and fixed in 1% paraformaldehyde. Negative controls were prepared by incubating the cells with PR-labeled isotope control mAb.Cell surface expression of TL4 was determined by flow cytometric analysis. The expression n the immature (H4 cells) The cell surface expression of TLR4 expression was analyzed by FACS analysis. The H4 cells were used as a model for immature enterocytes and CaCo-2 cells were used as a model for mature enterocytes. One million H4 cells or Caco2 cells were re-suspended and washed once with FACS buffer (PBS containing 1% BSA and 0.1% NaN3) and blocked with FACS buffer containing 10% mouse serum for 30 min on ice. The cells were incubated with anti-TLR4 mouse mAb conjugated PE  or IgG2a specific isotype control antibody  for 30 minutes at 4°C in the dark at the concentration recommended by the manufacturers. Then the cells were washed for three times with FACS buffer and then fixed with 1% Paraformaldehyde. Then the stained samples were analyzed on a Accuri C6 Flow Cytometer (BD Bioscience FACScan) with Cell Quest software. Dead cells and debris were excluded from analysis by gates set on forward and side angle light scatter. The results shown are histograms of flow cytometric analysis of H4 and Caco2 cells.Results were presented as the mean ± SE. The effects of age and treatment on chemokine secretion were analyzed by a two-way ANOVA and with post-hoc two-tailed unpaired t test. Differences with a P value of <0.05 were considered significant.


MGMT, IDH1, 1p19q, Ki67, MYCC, MYCN and β-catenin, only immunohistochemical IDH1 mutation testing in patients with diffuse gliomas is supported by sufficient evidence in order to be unequivocally qualified for clinical use. For the other candidate biomarkers lack of published evidence of sufficiently high analytical test performance and, in some cases, also of clinical performance limits evidence-based confirmation of their clinical utility. For most of the markers, no common standard of laboratory testing exists. We conclude that, at present, there is a strong need for studies that specifically address the analytical performance of candidate brain tumor biomarkers. In addition, standardization of laboratory testing is needed. We aim to regularly challenge and update the present classification in order to systematically clarify the current translational status of candidate brain tumor biomarkers and to identify specific research needs for accelerating the translational pace.  A large number of potential tissue biomarkers has been proposed for brain tumors. However, hardly any have been adopted for routine clinical use, so far. For most candidate biomarkers substantial controversy exists with regard to their usefulness in clinical practice. The multidisciplinary neurooncology taskforce of the Vienna Comprehensive Cancer Center Central Nervous System Unit (CCC-CNS) addressed this issue and elaborated a four-tiered levels-of-evidence system for assessing analytical performance (reliability of test result) and clinical performance (prognostic or predictive) based on consensually defined criteria. The taskforce also consensually agreed that only biomarker candidates should be considered as ready for clinical use, which meet defined quality standards for both, analytical and clinical performance. Applying this levels-of-evidence system to  In clinical medicine biomarkers are defined as objectively measurable/determinable patient-related factors that provide clinically meaningful disease-related information with regard to diagnosis, prognosis, therapy decisions and patient follow-up , 2, 3. IMGMT) promoter methylation status, isocitrate dehydrogenase 1 gene (IDH1) mutation status, chromosome arms 1p19q co-deletion status, Ki67 tumor cell proliferation index, MYCN status, MYCC status and β-catenin expression. We selected these biomarker candidates, because they are considered to be close to routine clinical use, but their translational status is still subject to controversy and discussion. Our multidisciplinary neurooncology task force within the CCC-CNS has defined the criteria for a four-tiered levels-of-evidence system and an adjunct scoring system for assessing the clinical utility of prognostic and predictive candidate brain tumor biomarkers in a continued process of discussion and consensual agreement . The levFor each candidate biomarker, we separately assessed the analytical performance and the prognostic and predictive clinical performance. Our ratings are based on review of published data and rely on consensual agreement within our multidisciplinary task force. We defined analytical performance as the reliability of the results yielded by a particular assessment or test. To this end, we evaluated published data with regard to repeatability of test results  and reproducibility of testing . As clinical performance, we defined the prognostic and predictive value of a given candidate biomarker. Prognostic markers were defined by their association with patient outcome, and predictive markers by their association with response to a given therapy. As the definition of prognostic and predictive markers in (neuro)oncology has been subject to continued debate and controversy, we provide – for illustration purpose – a generic example of a biomarker in medicine with prognostic, predictive and diagnostic properties, depending on the issue of interest see . We percOnly those factors reaching an A or B level for both analytical and cliniical performance were considered to have adequate justification for recommendation in routine clinical use as prognostic or predictive biomarkers . IDH testing – analytical performance IDH1-R132H mutation [IDH sequencing procedures showed inconsistent results among different laboratories [A recently published ring trial (round robin test) and several studies including large patient cohorts support the high analytical performance of immunohistochemical detection of the mutation , 11, 12.IDH testing – prognostic and predictive clinical performance IDH mutations in diffuse gliomas was confirmed in several large cohorts [IDH1 mutations, there is currently only little evidence based on few and inconsistent results from small studies [The high prognostic clinical performance of  cohorts , 14, 15. studies , 17, 18.Interpretation of the clinical utility of IDH testing IDH1-R132H mutation status by immunohistochemistry has sufficient evidence for clinical use as a prognostic marker in diffuse gliomas. IDH testing by DNA-based methods, which is in principle suitable to detect also other and rarer forms of IDH mutations, has promising clinical utility but is not yet ready for routine clinical use. Standard test protocols with the potential of sufficiently high analytical performance need to be elaborated and validated. Assessment of 1p19q testing – analytical performance Published data indicate promising analytical performance of the following methods for 1p19q co-deletions testing in oligodendroglial tumors: fluorescence in situ hybridization (FISH), PCR-based loss of heterozygosity (LOH) analysis and multiplex ligation-dependent probe amplification (MLPA). FISH seems to be the most robust method allowing also visual control of test results. However, a published ring trial formally documenting the high inter-laboratory reproducibility is missing [1p19q testing – clinical performance There is a high level of evidence for the prognostic value of 1p19q co-deletion in oligodendroglial tumors coming from several studies, including two large prospective trials , 20. The1p19q testing – clinical utility There is strong evidence that 1p19q co-deletion has a prognostic and predictive value in anaplastic oligodendroglial tumors. Ring trials remain to be performed in order to objectively validate the high reproducibility of 1p19q testing and thus making it recommendable for routine clinical use. Ki67 proliferation index analysis – analytical performance Several methods for Ki67 proliferation index analysis exist, such as direct counting, semi quantitative estimation, computer-based image analysis and direct microscope-assisted counting with a graticule. These assessments are associated with relatively high reproducibility among trained observers , 24, 25.Ki67 tumor cell proliferation index analysis – clinical performance A robust association of high Ki67 tumor cell proliferation index with unfavorable survival times in patients with ependymoma has been shown in several independent retrospective series , 28, 29.Ki67 tumor cell proliferation index analysis – clinical utility The Ki67 tumor cell proliferation index is associated with a high prognostic clinical performance in ependymoma. However, inter-laboratory variability of tissue processing and immunohistochemical staining protocols limit its clinical utility. There is a need for better standardization of tissue processing and Ki67 immunostaining procedures. MGMT methylation testing – analytical performance Various DNA-based methods for testing of MGMT promoter methylation status have been developed, but for none of them intra- and interlaboratory reproducibility has been sufficiently analyzed , 49, 50.MGMT methylation testing – clinical performance The high prognostic value of MGMT methylation in glioblastoma has been confirmed by many studies including prospective trial data , 52. A pMGMT methylation testing – clinical utility In glioblastoma, a high prognostic clinical performance of MGMT promoter methylation status has been robustly confirmed. In the subgroup of elderly patients there is also evidence for a high predictive value. However, there is insufficient evidence for a high analytical test performance. In particular, intra- and interlaboratory reproducibility remains to be confirmed by adequate scientific data. This lack of evidence impedes recommendation of MGMT testing for routine clinical use. MYCC amplification testing – analytical performance Promising data in terms of analytical performance exist for FISH-based analysis in medulloblastoma , 57, 58.MYCC amplification testing – clinical performance MYCC amplification is limited by sample size issues, as medulloblastomas are relatively rare and only ~ 5% of the cases harbour MYCC amplification. However, several retrospective studies consistently show an association of poor patient outcome and MYCC amplification status [MYCC amplification will be excluded from the average risk medulloblastoma group and included into the high-risk patient group. The investigation of the clinical performance of n status , 59, 60.MYCC amplification testing – clinical utility MYCC gene amplification status in medulloblastoma. Currently, there is still a lack of data confirming a high analytical performance of MYCC amplification testing. Data indicate a significant prognostic value of MYCN amplification testing – analytical performance MYCC gene amplification testing, currently available data suggest that FISH has the highest analytical performance for identification of MYCN amplifications [Like in ications , 57, 58.MYCN amplification testing – clinical performance MYCC only ~ 5% of medulloblastomas harbor MYCN amplification. A prognostic value of MYCN amplification has been reported in several studies [MYCN amplified medulloblastomas comprise two different molecular subgroups with different clinical characteristics and prognosis [MYCN gene amplification status in the whole medulloblastoma cohort, and refinement of the definition of MYCN amplified tumors by using additional clinical and molecular parameters is needed. In the forthcoming SIOP PNET5/6 clinical trial for average and low-risk medulloblastomas, patients with tumors harboring a MYCC amplification will be excluded and treated with a high risk protocol which is currently developed. Similar as  studies , 59, 60.rognosis . Thus, tMYCN amplification testing – clinical utility MYCN gene amplification status testing in medulloblastoma. Therefore, it does not fulfill the criteria for being implemented in the routine clinical setting. There is a need to systematically address both, analytical and clinical performance of MYCN amplification testing in adequately designed studies. Currently, there is insufficient evidence, both for analytical and clinical performance of β-catenin testing – analytical performance The β-catenin status in medulloblastomas can be tested by immunohistochemistry alone or in combination with gene sequencing , 64, 65.β-catenin testing – clinical performance CTNNB1) have been associated with improved patient outcome in several retrospective medulloblastoma studies including large patient cohorts [β-catenin protein expression and mutations within the β-catenin encoding gene , several independent studies have shown that β-catenin mutation is neither a prognostic nor predictive marker . β-catenin testing – clinical utility There is evidence for a prognostic value of β-catenin testing in medulloblastomas. However, there is a need for ring trials and elaboration of consensus guidelines for standardization of laboratory protocols for β-catenin testing. The prognostic value of the β-catenin status in medulloblastoma needs to be confirmed in adequately designed studies. Among the biomarker candidates that were evaluated according to our levels-of-evidence system, only immunohistochemical testing for IDH1-R132H mutations unequivocally meets the criteria that indicate sufficient evidence for routine clinical use. The most common and important reason why the other candidate biomarkers failed to meet the criteria for routine clinical use was lacking evidence of sufficiently high analytical test performance. This result indicates that there is a strong need of studies that specifically address the issue of test reproducibility, e.g., by means of repeatability testing, ring trials and interlaboratory comparison, as has been done recently in the case of IDH mutation testing , 69. IndAnother necessity specific to analytical performance is the standardization of testing procedures which constitutes an important prerequisite for interlaboratory comparability of test results. In the case of the widely used Ki67 immunostaining for example, a consensus-based standardized immunohistochemical staining procedure does not exist so far, which limits interlaboratory comparability of Ki67 tumor cell proliferation index threshold values. In the future, Euro-CNS may serve as a common platform for the definition of test standards as has recently been taken place in the case of the 1p/19q FISH protocol [MYCC and MYCN amplification, β-catenin testing), the prognostic value has not yet been sufficiently clarified because of the rarity of these tumors. This limitation underscores the need for sharing of tissue specimens within multicenter research collaborations, in order to increase both the total case numbers and – in further consequence – the statistical power of patient outcome studies. The prognostic clinical performance for most of the candidate biomarkers included in this study has been clarified by means of adequately designed and sufficiently powered clinical trials. However, with regard to certain biomarkers relevant for pediatric brain tumors  seem to be the major obstacles that impede the fast translation of candidate biomarkers into common clinical use. Future studies should be aware of and specifically address these limitations. We intend to regularly update the current translational status of candidate brain tumor biomarkers. Such periodic reassessments assessment may also be helpful to identify and exclude inappropriate biomarker candidates, and should be beneficial to identify specific research needs that may help to accelerate the translational pace. We thank Dr. Christine Haberler for helpful discussion and comments. The authors have no conflict of interest to declare.