A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level.trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species.Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.A combination of the non-coding  A DNA barcode is an aid to taxonomic identification which uses a standard short genomic region that is universally present in target lineages and has sufficient sequence variation to discriminate among species trnH-psbA intergenic spacer region and the multicopy nuclear Internal Transcribed Spacer (ITS) are two of the leading candidates trnL intron has been suggested as a possible plant barcode and does have conserved priming sites accD, matK, ndhJ, rpoB2, rpoC1, and ycf5) also have been recommended as putative plant barcodes (see http://www.rbgkew.org.uk/barcoding/index.html), but no comparisons of their effectiveness have been published. Finally, even though the plastid rbcL gene has been discounted as a species-level discriminator A variety of loci have been suggested as DNA barcodes for plants, including coding genes and non-coding spacers in the nuclear and plastid genomes. For flowering plants the non-coding plastid The objectives of the current study are two-fold: 1) to quantify universal application (PCR and sequencing) and sequence divergence among a phylogenetically diverse set of species pairs for nine putative barcode loci and, 2) to determine which loci, if more than one locus is required, will maximize species identification when combined as a barcode.trnH-psbA and rbcL-a, exhibited high PCR success with standard primers by amplifying 95.8% (46 of 48 genera) and 92.7% (43 of 48 genera), respectively, of the test species , which was significantly more divergent than rpoB2 (2.05%), rpoC1 (1.38%), and rbcL-a (1.29%). Due to the low PCR success of matK, and hence the small number of available comparisons, this locus was not shown to be significantly different than any of the other loci, except ITS1. The coding loci rpoB2, rpoC1, and rbcL-a exhibited statistically equal sequence divergence values for the data set  than the other seven loci, none of which had a value higher than 70%. If universal application is incorporated and all genera are considered, then the overall proportion of genera in which species in a pair were differentiated dropped considerably in ITS1 (45.8%) while trnH-psbA maintained the highest resolution (79.1%) and rbcL-a the second highest (62.5%) with values for all other loci at 50% or less. Six genera were invariant between species in a pair for all of the candidate loci .The proportion of genera in which species in a pair could be differentiated also varied widely among loci . The trntrnH-psbA was successful at returning a correct match. These tests using BLAST were employed as a complement to the primary results on barcode loci derived from the empirical comparative sequence data set. Many of the putative loci had too few sequences in GenBank to conduct a robust test  or were ruled out due to limitations in universal application (ITS1 and matK). For these reasons the in silico tests were not exhaustive and only focused on trnH-psbA and rbcL. Of the 103 genera tested, 75.7% (78 genera) of the searches identified the target sequence as the single best match with the BLASTn search. Similarly rbcL, which is a gene noted for its utility as a phylogenetic marker at the rank of family and genus, also demonstrated utility as a species-level identifier in the comparative data-mining tests trnH-psbA, 59 had rbcL sequences available in GenBank; of those 59 genera 76.3% (45 genera) of the searches identified the target sequence as the single best match with a BLASTn search  or correct family (five cases). The repeated trials for the trnH-psbA spacer with this reduced data set resulted in a slightly higher percentage of success (83.0%) at identification at the species level; the remaining cases identified to the correct genus  or rbcL . For the trnH-psbA spacer there was also no statistical difference between using partial sequences versus complete sequences in the searches: partial sequences resulted in 28.6% multiple matches while complete sequences resulted in 26.5% multiple matches .The results from data-mining sequences in GenBank, notwithstanding the drawbacks of using such data  and the highest proportion of differentiated species pairs  and rbcL-a+matK (82.6%). The PCR success for these two combinations was 99% and 95.8%, respectively. The remaining combinations of loci showed differentiation of species in a pair in less than 82% of the genera.The various combinations of two loci in the multi-locus tests were all more powerful at differentiating between species than either locus individually . The trns 87.5%; . The othrbcL and trnH-psbA showed that together the two loci provided correct matches at the species level in 95.0% of the trials The results suggest that the non-coding trnH-psbA spacer is the most promising single locus for a land plant barcode according to the criteria of universal application and high sequence divergence among species. The intent of the present study was to use these criteria to compare the trnH-psbA spacer with other suggested barcode loci across land plants. Several of the plastid genes  as well as the nuclear ITS region exhibit some features that would make each a possible candidate for a plant barcode , but poor PCR success in non-angiosperms ; rpoC1 had better PCR success (83.3%) than rpoB2, but a lower mutation rate (1.38%). The locus matK, which has been shown to be quite variable in numerous phylogenetic studies matK and the other loci may improve amplification success, but none of these genes have highly conserved sites near the most variable parts of the locus and hence it is not likely that sufficiently universal primers will be developed. Interestingly, rbcL-a in some cases proved better than other coding loci as a barcode. The mean percent sequence divergence for rbcL-a ranked sixth, but it exceeded all other loci except ITS1 and trnH-psbA in the percent of genera in which species pairs could be differentiated (69.8%). PCR success in rbcL-a was also very high (92.7%). ITS1, which was earlier suggested as a possible barcode for flowering plants accD absent in grasses, ndhJ absent in pines, ycf5 absent in bryophytes The  barcode . HoweverCitrus, Encephalartos, Ludisia, Magnolia, Raphanus, and Sabal) were invariant at each of the nine loci in the species pairs tested. Some of these genera are members of families that are known to show low levels of interspecific sequence divergence  can minimize type I errors (such that sequences are robustly assigned to the correct genus at least) and type II errors . Thus rbcL with its proven ease of amplification with broadly applicable primers across land plants and its proven ability to identify taxa at the level of genus and family make it the most appropriate choice for a two-locus barcode coupled with trnH-psbA.A two-locus combinatorial method has been suggested previously rbcLtrnH-psbAThe balance of within- and between-species sequence variation is an important aspect of barcode identification rbcL (rbcL-a) with the non-coding trnH-psbA spacer is recommended. rbcL-a provides a strong recognition anchor that will place an unidentified specimen into a family, genus, and sometimes species; the highly variable trnH-psbA spacer will further narrow the correct species identification where rbcL-a lacks discriminating power, especially in species-rich genera of angiosperms. Both of these loci have standard primers currently available that make them universally amplifiable with the least effort in the broadest range of land plants. This two-locus plant barcode is now being applied to build a library of over 700 species of the world's most important medicinal plants . This barcode library can then be used to test the identity and purity of plant-based medicines and herbals, such as ginseng, ginkgo, echinacea, and St. John's wort, sold in commercial markets and used by consumers. The results of this effort will contribute to the suite of uses of DNA barcodes with substantial economic and social value.In conclusion a two-locus barcode that combines a subunit of the coding locus a priori expectation of relatedness, hence the congeneric pairs do not necessarily represent nearest neighbor species. Because the experiment was focused on comparing the discriminating power of loci the inclusion of at least some species pairs that could be resolved by all loci increases the statistical power to differentiate among loci. Only a single individual per species was included in the analysis (see comments on intraspecific variation in Discussion). Tissues  were collected fresh and dried in silica-gel, or recovered from preserved herbarium specimens of various ages; vouchers with institutional accession numbers were prepared for each sample and are stored at the United States National Herbarium at the Smithsonian Institution's National Museum of Natural History. In addition some tissue samples were obtained from the United States Department of Agriculture germplasm resource network and are identified by a discrete USDA accession number . Dry plant material was disrupted in individual lysing tubes with a bead-mill. DNA extraction was conducted following manufacturer's protocols. For all taxa and loci, we conducted PCR amplification in a two stage trial. The first stage used a standard (non-hot-start) DNA polymerase  in 25 ul reactions following the protocols of Kress, Wurdack, Zimmer, Weigt and Janzen http://www.rbgkew.org.uk/barcoding/index.html. A 550–600 bp subset of the rbcL molecule (termed rbcL-a) located at the 5′ end of the large subunit that exhibited maximal sequence variation and universal amplification was also included in the analysis. All available combinations of primers for each of these seven loci were tested on a subset of 4 divergent taxa to select the primer sequences that were subsequently used throughout the experiment . Six of the loci are described at periment .trnH-psbA), were tested along with the coding loci. The two components of the nuclear internal transcribed spacer (ITS 1 and 2) were compared across 13 of the test genera for size and variability. The ITS1 subset produced a consistently smaller amplicon with fewer artifactual amplification products and exhibited higher levels of sequence divergence relative to ITS2  and one in the plastid genome .Each locus was quantified for PCR amplification success, which is defined as the recovery and successful sequencing of each locus for each species. The phylogenetic range  over which each locus would amplify with standard procedures was recorded. Two measures of the power of a locus to discriminate among species were calculated: 1) percent sequence divergence between pairs of species and 2) the proportion of the 48 genera for which species in a pair could be differentiated. The first measure was calculated for each barcode by summing the number of mutations separating the two species of a pair relative to the total sequence length, which was then averaged over all genera. The second measure was calculated at two levels: first, for only those genera in which both species amplified and second, for all genera regardless of amplification, thereby incorporating both the level of sequence divergence and universal application into this measure. Significant differences in percent sequence divergence between loci were tested using a Wilcoxon-Signed ranks test contrasting all possible 2-way combinations of loci  were included in contrasts between pairwise combinations were evaluated for their ability to discriminate between species across our sample of land plants. Three coding regions  were eliminated from these tests because they are absent in at least one important group of land plants  for which six or more full length or partial sequences for trnH-psbA were identified and recovered from Genbank. A species sequence representing each genus was used then used as a query sequence in a BLASTn search (short nearly exact search)trnH-psbA was used to test the utility of rbcL as a complementary locus. Of the 103 genera used in the trnH-psbA trials, 59 had corresponding rbcL sequences available in GenBank. These 59 genera were queried in the same fashion as above for the portion of rbcL and as a repeat trial for the trnH-psbA spacer. These 59 genera were then used to test the success of a combined two-locus approach using the BLASTn search. T-tests Click here for additional data file.Table S2Taxa sampled in tests of nine putative plant barcode loci.(0.40 MB DOC)Click here for additional data file.Table S3Primer sequences for test loci.(0.04 MB DOC)Click here for additional data file.


The aim of this study was to investigate the role of 99mTc-DTPA scan in patients with Idiopathic Pulmonary Fibrosis (IPF). Our hypothesis is that the rate of pulmonary 99mTc-DTPA clearance could be associated with extent of High Resolution Computed Tomography (HRCT) abnormalities, cell differential of bronchoalveolar lavage fluid (BALF) and pulmonary function tests (PFTs) in patients with IPF.Clearance of inhaled technetium-labeled diethylenetriamine pentaacetate  of median age 67yr (range 55–81) with histologically proven IPF. HRCT scoring included the mean values of extent of disease. Mean values of these percentages represented the Total Interstitial Disease Score (TID). DTPA clearance was analyzed according to a dynamic study using a Venticis II radioaerosol delivery system.t1/2<40 min) was found in 17/18 (94.5%) [mean (SD) 29.1 ± 8.6 min]. TID was weakly correlated with the DTPA clearance  and with % eosinophils . No correlation was found between TID score or DTPA and PFTs in IPF patients.The mean (SD) TID score was 36 ± 12%, 3 patients had mild, 11 moderate and 4 severe TID. Abnormal DTPA clearance half-time (99mTc-DTPA lung scan is not well associated with HRCT abnormalities, PFTs, and BALF cellularity in patients with IPF. Further studies in large scale of patients are needed to define the role of this technique in pulmonary fibrosis.Our data suggest that  Abnormal DTPA clearance half-time  Figure . No corrThe total and the differential cell counts of BALF are shown in Table 99mTc-DTPA clearance prospectively. We found that the 99mTc-DTPA clearance time is abnormal in almost all (94.5%) of IPF patients. On the contrary, we failed to detect a strong correlation between HRCT TID score and 99mTc-DTPA clearance. An additional finding of this study is a weak linkage between TID score and eosinophils in BALF.This is the first study to systematically evaluate newly diagnosed IPF patients with biopsy proven histology of usual interstitial pneumonia (UIP), excluding the other clinical-pathologic entities of idiopathic interstitial pneumonias, comparing quantitatively the extent of abnormalities in the HRCT and 99mTc-DTPA scanning in predicting survival in a large scale population of 106 IPF patients. On the other hand, only 30% of them had histologically proven disease. The authors showed that the predictive value of this technique  was lower than that of total lung capacity and DLCO. They also detected that the clearance values did not correlate with the HRCT scoreby using a both mono-exponential and bi-exponential analysis [99mTc-DTPA is not of value in following the progress of IPF [99mTc-DTPA and functional tests or HRCT score [In a recent prospective study Mogulkoc et al.  evaluateanalysis , in accos of IPF . Mura anCT score . DTPA scCT score -25 but aCT score .The specific mechanism of increased clearance time in IPF is still unclear but has been thought to be secondary to the stretching of epithelial junctions in the alveolar wall as a result of fibrotic traction or lymphocytic infiltration ,28. We sWe found no correlation between aerosol clearance and pulmonary function parameters, in agreement with the findings of previous studies ,18-20. SWells et al. reported that depression of TLCO and FVC were positively related to the extent of disease on the CT scan and that there were no independent relationships between functional indices and the pattern or distribution of disease on the CT scan ,15,30. WWe found a moderately significant correlation of DTPA clearance with the visual score in HRCT in 18 patients with IPF. We could not overestimated these results because are based only on a limited number of subjects and on a correlation factor of -0.47; it should be supported by a follow-up study in order to investigate if the clearance of 99m-DTPA can actually predict the clinical course of IPF. Furthermore, a recent study demonstrated that clearance of 99m-DTPA, although abnormal in all subjects in presentation, was not a predictor of disease progression in IPF patients . AdditioWe also found a moderate correlation between BAL eosinophilia and HRCT TID score. It has been shown that excess neutrophils and/or eosinophils (>5%) in the BALF have been associated with a higher likelihood of disease progression and a failure to respond to immunosuppression .99mTc-DTPA lung scan in the follow-up of IPF patients. Further prospective studies in a large scale and well-defined IPF population are necessary to define the role of this modern technique in the evaluation of pulmonary fibrosis.Our data suggest that at the moment there are not sufficient evidences to recommend the implementation of 99mTc-DTPA: inhaled technetium-labeled diethylenetriamine pentaacetateIPF: Idiopathic Pulmonary FibrosisHRCT: High Resolution Computed TomographyPFTs: pulmonary function testsBALF: bronchoalveolar lavage fluidTID: Total Interstitial Disease ScoreThe author(s) declare that they have no competing interests.KMA, NMS and DB participated in the design and coordination of the study. KMA prepared the manuscript. KM carried out the HRCT scoring, NT carried out the Bronchoalveolar Lavage, KP and NK carried out the DTPA scans. NT and EKS carried out the statistical analysis. DB and NMS were invo;ved in revising the article for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We first performed combined ultrasound and histology examinations of the same tissue in two subjects undergoing surgery: in one subject, we examined the relationship of ultrasound to histological images in three dimensions; in the other, we examined the effect of a localized tissue perturbation using a previously developed robotic acupuncture needling technique. In ten additional non-surgical subjects, we quantified changes in tissue spatial organization over time during needle rotation vs. no rotation using ultrasound and semi-variogram analyses.In humans, connective tissue forms a complex, interconnected network throughout the body that may have mechanosensory, regulatory and signaling functions. Understanding these potentially important phenomena requires non-invasive measurements of collagen network structure that can be performed in live animals or humans. The goal of this study was to show that ultrasound can be used to quantify dynamic changes in local connective tissue structure 3-D renditions of ultrasound images showed longitudinal echogenic sheets that matched with collagenous sheets seen in histological preparations. Rank correlations between serial 2-D ultrasound and corresponding histology images resulted in high positive correlations for semi-variogram ranges computed parallel  and perpendicular  to the surface of the skin, indicating concordance in spatial structure between the two data sets. Needle rotation caused tissue displacement in the area surrounding the needle that was mapped spatially with ultrasound elastography and corresponded to collagen bundles winding around the needle on histological sections. In semi-variograms computed for each ultrasound frame, there was a greater change in the area under the semi-variogram curve across successive frames during needle rotation compared with no rotation. The direction of this change was heterogeneous across subjects. The frame-to-frame variability was 10-fold (p < 0.001) greater with rotation than with no rotation indicating changes in tissue structure during rotation.in vivo.The combination of ultrasound and semi-variogram analyses allows quantitative assessment of dynamic changes in the structure of human connective tissue  This network also includes superficial subcutaneous connective tissue (pannicular fascia) which forms interconnecting longitudinal fibrous sheets intermixed with adipose tissue and linked to the skin by string-like "skin ligaments" -9. Recen(PS-OCT) ,19. Howein vivo tissue measurements [3, speed of sound = 1,410–1,480 m/s), regions with predominantly collagen , and regions with predominantly glycosaminoglycans and water . Several parameters, including the ratio of specular-to-diffuse backscatter coefficients and the ultrasound intensity signal-to-noise ratio, have been used to assess tissue organization by quantifying the presence of organizational scatterers within tissues [in vivo, specifically regarding the structural continuity of longitudinal collagenous sheets, as well as changes in this structural organization over time.For many organ systems (e.g. cardiovascular), ultrasound is increasingly used to visualize human anatomy non-invasively and obtain urements -24. In aurements . Elastogurements ,27, prosurements ,29, bloourements , heart [urements , skin [3urements and cartiurements . During urements -37. It iurements ,38, as wurements ,40. Howe tissues ,41-45. T tissues -50. Statin vivo induced using a previously developed robotic acupuncture needling technique [The goals of this study were 1) examine the degree of correspondence between the three dimensional pattern of echoes generated by ultrasound imaging of connective tissue and the three dimensional pattern formed by fibrous connective tissue in histological serial sections of the same piece of tissue and 2) show that ultrasound can be used to quantify dynamic changes in connective tissue structure echnique ,52. We hechnique . In this3 cube of tissue at the bone graft site, then excised a corresponding subcutaneous tissue biopsy specimen that was fixed and processed for histology  during rotation compared with no rotation among the ten subjects. For semi-variograms calculated parallel to the skin, VAUC was 0.0686 during rotation compared with 0.0050 no rotation. Similar results were obtained in the perpendicular direction (VAUC was 0.0228 vs. 0.0012 for rotation and no rotation respectively). When the order of frames was taken into consideration, the mean absolute change in area under the semi-variogram curve (AUC) across successive frames was approximately two-fold greater during rotation compared to no-rotation both parallel  and perpendicular  to the skin.In order to further investigate whether needle rotation consistently induces change in tissue structure that is measurable with ultrasound, we tested ten non-surgical subjects. In each subject, we acquired ultrasound images during needling at the same point on the thigh bilaterally, with right and left sides of the body randomized to needle rotation vs. no rotation. Figure Together, our results show that 1) needle rotation produced changes in connective tissue structure and 2) individuals responded differently from one another in response to the same mechanical input (i.e. needle rotation). This heterogeneous response of tissues observed among subjects was interesting and may be due to differences in initial tissue structure. Increased or decreased structural continuity over time may correspond to collagenous bundles either becoming more straight and tightly organized (i.e. less wavy) or "breaking up" due to the mechanical pull resulting from the tissue winding around the needle. Whether collagen bundles straighten or break in response to a given mechanical input may depend on a number of factors including collagen bundle size, cross-linking, tissue glycosaminoglycan concentration and water content -57. FurtA potential limitation of structural tissue analysis based on ultrasound imaging is that the strength of reflected ultrasound signals is dependent to some degree on the orientation of a given structure relative to the direction of the incident wave. In this study, because the ultrasound transducer was placed perpendicular to the skin, organized structures oriented parallel to the skin would be expected to generate stronger echoes than structures perpendicular to the skin. Figure The complementary techniques used in this study show that the laminar collagenous structures seen with ultrasound reorganized during rotation of an inserted needle. These changes were localized to the area of the needle and were accompanied by a "whorl" of collagen in corresponding biopsy sections. The combination of ultrasound and semi-variogram analysis used in this study is novel in biology. These imaging methods may be used along with dynamic visualization of molecular markers and computational modeling to investigate connective tissue as a functional network system capable of responding to local perturbations.Twelve human subjects  were recruited and signed informed consent for this study. The Institutional Review Board at the University of Vermont approved the study methods and procedures. Two subjects were patients undergoing spinal fusion surgery . In the first surgical subject, we examined the degree of correspondence between serial ultrasound and histology images of the same tissue sample. In the second surgical subject, we examined structural changes occurring during robotic needle rotation using both ultrasound and histology, . In the ten non-surgical subjects, we performed robotic acupuncture needling at the same anatomical location on the thigh bilaterally to compare the effect of needle rotation vs. needle insertion alone without rotation.3 cube of tissue with pixel intensity ranging from 0–255. After ultrasound imaging, a 4 cm × 2 cm × 4 cm tissue sample was excised at the site of the surgical incision, centered on the middle of the area scanned by ultrasound  was imaged by ultrasound Figure . An ultrd Figure . The tisd Figure . The sevd Figure . In ordeth frame); next, x coordinates were matched by lining up the medial-lateral midpoint of the middle histology section with the medial-lateral midpoint of the ultrasound image; finally, y coordinates were matched by lining up the deep border of the dermis in the middle histology section to the deep border of the dermis in the middle ultrasound image. Thus, histology sections were stacked relative to each other before matching the whole histology data set with the serial ultrasound images. This avoided the potential bias associated with using connective tissue layers themselves as landmarks. Three-dimensional visualizations of ultrasound and histology image stacks were performed using Environmental Visualization EVS-Pro V (8.0) software.The ultrasound cine-recording included 160 individual 2-D frames. Each ultrasound frame was cropped to match the area of the histology image. Histology slides were stacked sequentially using major landmarks of the tissue (e.g. blood vessels) for orientation. However, tissue landmarks were not used to match ultrasound and histology images with each other. Rather, ultrasound and histology data sets were matched as follows: the histology section corresponding to the middle of the biopsy (middle histology section) was first identified based on serial section numbers; this histology section was then matched with the ultrasound image corresponding to the middle of the ultrasound recording  data were collected and stored following digitization by the ultrasound system at a sampling frequency of 20 MHz.After ultrasound data acquisition, the needling instrument was disconnected from the needle, leaving the needle in place within the tissue. A 4 cm × 2 cm × 4 cm tissue sample was then excised as above, centered on the needle. After fixation, the position of the needle in the tissue was marked by dipping the tip of the needle (protruding from the end of the tissue) into green ink before pulling it out of the tissue, thus dragging the ink through the tissue as the needle was being pulled back. The tissue sample was then cut into five blocks perpendicular to the needle as shown in Figure Ultrasound cine-recordings were acquired at 13.2 frames/sec as described above on the anterior thigh (18 cm superior to the middle of the superior edge of the patella) bilaterally. The two testing locations were randomized to needle rotation  vs. no rotation. Needle insertion depth was calculated for each individual so that the needle tip extended 5 mm past the peri-muscular fascia, and the needle insertion angle was 45° to the transducer. Both raw radio-frequency (RF) ultrasound data and B-scan image were saved for quantification of tissue displacement (elasticity imaging) and changes in tissue structure .The displacement occurring between each of the seventy successive frames acquired during needle rotation was calculated using cross-correlation techniques with a 3 mm window and a window overlap of 90% as previously described ,52. The γ(h), defined as the spatial dissimilarity in intensity between image pixels separated by a range of distances, h, in a chosen direction:Semi-variogram analyses statistically evaluate the spatial structure  in a particular data set using a semi-variogram plot. In this study, we have computed the semi-variance, N(h) is the number of pixel pairs separated by the distance, h; and u(a) and u(a+h) are the respective pixel intensities at locations (a) and some distance away (a+h). The difference in pixel intensity between each data point and every other data point in a given direction was calculated and these differences were binned according to separation distance (h)  (the variability in the difference in pixel intensity between pairs of points) reaches a plateau. Larger ranges correspond to less spatial variability and therefore greater spatial correlation (i.e. points located large distances apart are still correlated) while smaller ranges imply more spatial variation and therefore weaker spatial correlation. Spearmans's rank correlation was used to examine the concordance between estimates obtained from ultrasound and histology.In this study, we used semi-variograms to 1) compare B-scan ultrasound images with histology based on quantitative measures representing tissue structure and 2) quantify changes in tissue structure over time during needle rotation. For the ultrasound/histology comparison, we used a standard semi-variogram parameter (the range) derived using non-linear regression models fit using JMP statistical software  -60. The The area under the semi-variogram curve (AUC) was used to examine structural changes in tissue during needle rotation. The AUC was calculated using the trapezoidal rule from the raw semi-variogram data corresponding to each B-scan ultrasound image acquired during needle rotation (or no rotation). We used the AUC as a surrogate measure for the range as it provides a quantitative measure of spatial structure without necessitating non-linear curve fitting for each of the 300 ultrasound frames (15 frames × 2conditions × 10 subjects) in parallel and perpendicular directions. The AUC is sensitive to change in data structure, and was found to be highly correlated with the range in the ultrasound/histology comparison .AUC) for rotation and no-rotation conditions. Variance estimates for the two conditions were compared based on an F-test. Statistical significance was determined based on α = 0.05.Repeated measures analysis of variance was used to compare mean AUC between rotation and no-rotation conditions. The mean AUC for each condition was based on the average of the 15 frames across the 10 subjects. Analysis of variance was also used to compare rotation and no-rotation conditions with respect to the mean absolute difference in AUC between successive frames. This outcome measure examines structural changes while accounting for the temporal ordering of the frames. Variance component analysis  was used the obtain estimates of the inter-frame variance in AUC (VHML designed the study, participated in subject testing and drafted the manuscript.DMR provided the expertise for the variogram analyses and participated in writing the manuscript.JRF wrote software for data analysis, participated in the human subjects testing and writing the manuscriptJW acted as a consultant for the interpretation of ultrasound findings and contributed to writing the manuscriptEEK acted as a consultant for the analysis and interpretation of elastography findings and contributed to writing the manuscriptDST recruited and coordinated human subjects testing and participated in manuscript preparationNAB participated in the human subjects testing and performed ultrasound data analysesGJB participated in the design of the study, performed the statistical analysis and participated in writing the manuscript.MHK participated in the study design, performed the biopsy excisions and participated in writing the manuscriptAll authors read and approved the final manuscript.3-d animated rendition of collagenous sheets corresponding to ultrasound image shown in Figure 2CClick here for file


The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold.The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily – providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex.The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like) domains bind DNA in highly diverse manners. The largest evolutionary superfamilies of proteins cover such a large range of sequence space that the relationships shared by members may not be apparent by standard means of sequence comparison, and hence are often only recognized after structural determinations. Such has frequently been the case for the PD-(D/E)xK superfamily of nucleases. Within the superfamily, structures were first obtained for four restriction enzymes, of such diverse sequences that they were initially assumed not to share homology (reviewed in ). Since S. cerevisiae RPB5, an RNA polymerase subunit, where evidence suggests that it functions in protein-protein interactions [The unifying catalytic site characteristic of the superfamily is the presence of one or more catalytically essential divalent cations ,7. The cractions .Although extremely diverse in structure and sequence, modern sequence comparison methods have played their part in elucidating the full range of PD-(D/E)xK superfamily members ,13-15. NHere we show that the C-terminal region ('domain 3') of RecC can actually be dissected into two domains, the first of which is clearly related to PD-(D/E)xK superfamily nuclease domains  and particularly to the corresponding domain of RecB. The nuclease-like domain of RecC is inactivated and therefore possesses not even the metal-ligating residues of the PD-(D/E)xK motif. Instead, it carries out a function not hitherto observed in the superfamily, providing an aperture through which one strand of newly split DNA duplex is fed. Comparisons show that nuclease(-like) domains are extraordinarily versatile in their mode of interaction with duplex DNA. Characteristics of the RecC nuclease-like domain show that RecB and RecC share a common ancestor and reveal how evolution has progressed by sequential addition of domains to the C-terminus, as well as by altering function of, and repositioning of, existing domains.Domain 3 of RecC has been described as being of novel fold . StructuThe nuclease fold common to λ-exonuclease, RecB and now RecC is found in a wide variety of exo- and endonucleases, from restriction enzymes to Holliday junction resolvases, and enzymes of DNA repair . Within Interestingly, comparison of the nuclease domains of RecB, RecC and λ-exonuclease shows that the Rec subunits clearly share a more recent ancestor than the common ancestor of all three structures. As Figures As mentioned, nuclease domains as represented in the present PDB are extremely diverse in sequence but share conserved residues that bind essential metal ions and are almost invariably catalytically active. The recognition of the third domain of RecC as an inactivated nuclease domain highlights a wholly unexpected new function for a non-catalytic but clearly nuclease-like domain. As shown in Fig E. coli RecC the connection is a minimal β-turn and connections in other RecC sequences are also very short  in the results of the first iteration. In contrast, using an e-value cut-off of 0.0001 four iterations are required before RecC sequences, including that of E. coli RecC, appear among the significant hits. While the BLAST alignments centred on the helicase(-like) domains the C-terminal nuclease(-like) domains were sometimes matched, although PSI-BLAST runs of the nuclease domain of RecB failed to hit the nuclease-like domain of RecC, and vice versa. Similarly, structural comparisons show that both helicase domains and both α-helical insert domains of RecB are more similar to their counterparts in PcrA than to the corresponding RecC domains (not shown). Nevertheless, the clear homology of the RecB and RecC nuclease(-like) domains, evident in their common three α-helical bundle (see above) strongly suggests that RecB and RecC share a more recent common ancestor than they have in common with other extant helicases. How then to explain the apparently closer relationship of RecB with PcrA than with RecC? As was proposed for the nuclease(-like) domains (see above) it seems like the dramatic functional differences between corresponding RecB and RecC domains are responsible. As discussed above, the RecC nuclease-like domain is significantly shorter than the RecB nuclease domain in two key regions, each associated with its new role as provider of an ssDNA hoop. Thus, it seems plausible that the maintenance of helicase activity by the helicase domains of PcrA and RecB is responsible for their apparently closer relationship, the structural changes accompanying evolution of the helicase-like domains in RecC for new roles having obscured their more recent shared ancestry with RecB.The observation of inactivated helicase-like domains in RecC was not considered reason enough to propose the existence of homology between RecB and RecC extending over their whole length . Indeed,e domain , appearsThe recognition of homology between RecB and RecC, and the dissection of their domains leads to an interesting comparison with PcrA. In PcrA, a duplication of an ancestral RecA-like domain, already containing an all α-helical insert domain, is evident . In RecBThere is an interesting parallel to be drawn between RecBCD and AddAB , a different DNA repair system found in Gram positive bacteria where RecBCD is lacking . Protei


Protein sequence alignments have become indispensable for virtually any evolutionary, structural or functional study involving proteins. Modern sequence search and comparison methods combined with rapidly increasing sequence data often can reliably match even distantly related proteins that share little sequence similarity. However, even highly significant matches generally may have incorrectly aligned regions. Therefore when exact residue correspondence is used to transfer biological information from one aligned sequence to another, it is critical to know which alignment regions are reliable and which may contain alignment errors..PSI-BLAST-ISS is a standalone Unix-based tool designed to delineate reliable regions of sequence alignments as well as to suggest potential variants in unreliable regions. The region-specific reliability is assessed by producing multiple sequence alignments in different sequence contexts followed by the analysis of the consistency of alignment variants. The PSI-BLAST-ISS output enables the user to simultaneously analyze alignment reliability between query and multiple homologous sequences. In addition, PSI-BLAST-ISS can be used to detect distantly related homologous proteins. The software is freely available at: PSI-BLAST-ISS is an effective reliability assessment tool that can be useful in applications such as comparative modelling or analysis of individual sequence regions. It favorably compares with the existing similar software both in the performance and functional features. Protein sequence alignments are at the heart of many biological applications such as sequence database searches, annotation of new sequences, inference of functional regions, comparative protein modeling. Modern sequence comparison methods (e.g. PSI-BLAST ) often cThe importance of delineating reliable alignment regions has been recognized more than a decade ago, however, earlier studies focused on pairwise alignments -5. CurreHere, we present the PSI-BLAST Intermediate Sequence Search tool (PSI-BLAST-ISS) that is primarily designed to help identify reliable regions of the alignment as well as suggest potential alignment variants in unreliable regions. In comparative modeling PSI-BLAST-ISS can also help identify best matching structural templates. In addition, PSI-BLAST-ISS can be used to detect remote homologs that cannot be identified by a straightforward single PSI-BLAST search. However, it should be noted that the detection of remote homologs, unlike in the original and subsequent implementations of the Intermediate Sequence Search (ISS) strategy -17, is nSince PSI-BLAST-ISS might be most useful in comparative modeling we are going to refer to the sequence pair of interest as the target (query) and the template (reference) sequences throughout the article. However, it should be emphasized that the tool can be applied for any protein sequences that could be linked through common homologs, independently whether the three-dimensional structure for any of them is available or not.The main idea of PSI-BLAST-ISS is to obtain a number of alignment variants for the sequence pair of interest (target and template) and analyze their consistency. This idea has stemmed from previous manual analysis of multiple PSI-BLAST alignment variants suggesting that regions where variants do agree are likely to be aligned correctly and display close structural similarity .The whole PSI-BLAST-ISS procedure may be described as the following steps: 1) identification of multiple sequences related both to the target and template sequences, (2) formation of a representative set from these sequences by filtering out close homologs, (3) generation of sequence profiles for each sequence from this representative set by searching a sequence database with PSI-BLAST, (4) using each of the generated profiles to search a second sequence database that includes sequences of both the target and the template, (5) retention of all the instances of significant matches between the target and the template, (6) merging all significant target-template alignments by taking the target sequence as a frame of reference and (7) reducing the multiple variants of aligned template into the consensus sequence. The latter option enables contrasting of the region-specific reliability for multiple target-template alignments simultaneously. All the seven main steps are illustrated in a sketch of the data flow . By default, the target is searched against the non-redundant sequence database. Intermediate sequences are collected from the user-specified PSI-BLAST iteration in the resulting output file using the expectation value  threshold provided as a parameter. The reduced representative sequence set is constructed by filtering the initial set to a user-defined percentage of sequence similarity with CD-HIT , the sequence clusterization program (step 2). Optionally, a user may introduce a strict limit to the number of sequences to be included in the representative set or even supply independently pre-selected set of sequences. A PSI-BLAST-ISS user can also choose whether to collect intermediate sequences as complete protein sequences or just as sequence fragments matching the target sequence. In the case when the target sequence represents a domain that is also found in multidomain proteins the ability to select only homologous fragments of matching sequences may help to keep the ISS procedure from straying into the realm of unrelated sequences. Each of the intermediate sequences is used to generate a sequence profile in the form of the PSI-BLAST checkpoint file by running a user-defined number of PSI-BLAST iterations (step 3). The resulting checkpoint files are then used to restart PSI-BLAST searches in a second sequence database specified by the user (step 4). This database is expected to include sequences of both proteins of interest (target and template). In a common situation, when the template represents a structural template intended for use in comparative modeling, such a database may be derived by simply appending the target sequence to the PDB sequence database. In this case there is no need to define template(s) in advance since they are identified automatically. Searches against the second database generate corresponding multiple sequence alignments that contain a number of target-template alignment variants. The significance of the target-template alignment is then determined by counting the number of alignment variants that satisfy the expectation value threshold (step 5). Both parameters can be specified by the user. The significant target-template alignment variants are extracted and merged into a single multiple sequence alignment, where the target sequence is aligned with multiple instances of the template sequence according to different alignment variants (step 6). Such an alignment immediately reveals the regions where most  alignment variants are identical and thus might be considered reliable as well as those regions where there is little agreement between alignment variants and therefore unreliable. Often it is useful to analyze position-specific reliability for target alignments with multiple templates. However, it may be inconvenient to contrast/compare at once many multiple sequence alignments obtained by PSI-BLAST-ISS. To make this task easier we introduced a step (step 7) that reduces template alignment variants into a consensus template sequence for each of the target-template alignments. The consensus sequence is generated by analyzing each column of the alignment. A residue is considered conserved in the consensus template sequence if its repetition count in the corresponding position exceeds the user-defined conservation threshold.PSI-BLAST-ISS currently is implemented as a standalone UNIX-based tool meant to be installed and run locally. It consists of fairly independent modules linked together using Perl. Some of the sequence data processing tasks in PSI-BLAST-ISS are handled by a few modified SEALS scripts .PSI-BLAST-ISS produces several types of results. Perhaps the most informative output file is the FASTA-formatted sequence alignment between the target and automatically detected multiple template sequences, each represented as a consensus sequence derived from multiple alignment variants. The definition line for each consensus template sequence indicates the strength of the consensus in the interval from 0 to 1  and the number of significant target-template alignment variants that were used to produce the consensus. This output provides a possibility to simultaneously assess the alignment reliability between the target and multiple templates in a region-specific manner. In addition, the consensus strength and the number of significant target-template alignments may help in selecting templates that are structurally most consistent with the target. PSI-BLAST-ISS also produces individual FASTA-formatted multiple sequence alignment files for each target-template pair, where the target is aligned with multiple copies of the same template according to obtained multiple alignment variants. These alignment files provide a visual assessment of the region-specific alignment reliability as well as candidate alignment variants if further analysis of unreliably aligned stretches is needed. Finally, all the template sequences represented in the consensus alignment are collected together in a separate output file.Like for any method it is important to know how PSI-BLAST-ISS performs relative to other available methods. At the time of this study we have been aware of only two publicly available servers that estimate the position-specific reliability of sequence alignment using information from multiple sequences: the Consensus server  and SQUA. These proteins represent a variety of different structural folds and different degree of similarity to known structures. We ran PSI-BLAST-ISS for all the target sequences assessed in CASP6, but only those, for which PSI-BLAST-ISS with default parameters generated at least ten significant alignment variants with a structural template, were further analysed. The "gold standard" in evaluating sequence alignments is to compare them with the alignments derived from protein structure superposition. For most targets PSI-BLAST-ISS detected multiple templates but for evaluating its performance we only considered a single template for each target. The DaliLite structure comparison program [To compare PSI-BLAST-ISS and Consensus we chose protein sequences provided as prediction targets in the last round  of the c program  was usedThe data in Table A useful feature of PSI-BLAST-ISS is that it provides multiple alignment variants between the target and each template. Results in Table Often there is a need to choose a single or just a few best templates from a large number of distantly related target homologs. This becomes a challenge at low sequence similarity when the sequence signal is no longer a good indicator of structural relatedness . These targets do have evolutionary related structures in the PDB database but these relationships could not be detected by PSI-BLAST searches initiated with the target sequence. For this test we required at least one significant match to a PDB structure (template) from all intermediate sequence searches. To make the comparison compatible with the CASP6 setting we only considered structural templates that were available from PDB at the time of the CASP6 experiment. We also excluded from consideration those FR/H CASP6 targets, for which at least one domain could be matched to a PDB structure using a straightforward PSI-BLAST search. As a result, out of fourteen considered FR/H targets, PSI-BLAST-ISS was able to identify related structures for four of them . An interesting case is T0228. While direct PSI-BLAST search initiated with the T0228 sequence failed to find any related structure, PSI-BLAST-ISS identified several structures producing over ten significant matches each (a default parameter). The latter result stresses the fact that sometimes the space of homologous sequences might be skewed in such a manner that a single sequence search may not be very effective in identifying important relationships.We have described PSI-BLAST-ISS, a tool for delineating reliable alignment regions and suggesting possible alignment choices in unreliable yet structurally conserved regions. PSI-BLAST-ISS might be most useful in assessing target-template alignments in comparative modeling or judging whether the interpolation of biological information directly form alignments is feasible for individual sequence regions. Unlike two other recently published methods for predicting reliable alignment regions (SQUARE and the Consensus server) PSI-BLAST-ISS is not confined to reference (template) sequences with known three-dimensional structure. The performance of PSI-BLAST-ISS in alignment reliability estimation was directly compared with the Consensus server. We find that on a set of CASP6 targets PSI-BLAST-ISS on average is able to produce more extensive coverage of confident alignment or fewer errors, depending on the selected consensus stringency. The functionality of PSI-BLAST-ISS also extends into detection of non-apparent distant homologous relationships.Project name: The PSI-BLAST intermediate sequence search tool (PSI-BLAST-ISS)Project home page: Operating systems: Unix-based platformsProgramming language: PerlOther requirements: locally installed PSI-BLAST and CD-HIT License: NoneAny restriction to use by non-academics: NoneMM carried out the software development, programming work and participated in manuscript preparation. ČV conceived of the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


Diffusion-weighted imaging (DWI) is becoming an increasingly important tool for studying brain development. DWI analyses relying on manually-drawn regions of interest and tractography using manually-placed waypoints are considered to provide the most accurate characterisation of the underlying brain structure. However, these methods are labour-intensive and become impractical for studies with large cohorts and numerous white matter (WM) tracts. Tract-specific analysis (TSA) is an alternative WM analysis method applicable to large-scale studies that offers potential benefits. TSA produces a skeleton representation of WM tracts and projects the group's diffusion data onto the skeleton for statistical analysis. In this work we evaluate the performance of TSA in analysing preterm infant data against results obtained from native space tractography and tract-based spatial statistics. We evaluate TSA's registration accuracy of WM tracts and assess the agreement between native space data and template space data projected onto WM skeletons, in 12 tracts across 48 preterm neonates. We show that TSA registration provides better WM tract alignment than a previous protocol optimised for neonatal spatial normalisation, and that TSA projects FA values that match well with values derived from native space tractography. We apply TSA for the first time to a preterm neonatal population to study the effects of age at scan on WM tracts around term equivalent age. We demonstrate the effects of age at scan on DTI metrics in commissural, projection and association fibres. We demonstrate the potential of TSA for WM analysis and its suitability for infant studies involving multiple tracts.  •Evaluation of tract-specific analysis (TSA) for white matter studies in infants.•TSA improves white matter tract alignment over scalar-based registration.•TSA closely approximates native space tractography DTI values.•The first application of TSA to a neonatal population. Diffusion-weighted magnetic resonance imaging (dMRI) is increasingly being used to study brain development and injury in infants. Using metrics derived from diffusion tensor imaging (DTI)  we have A number of approaches have been used to analyse DTI data during development. Manually-drawn regions of interest (ROI)  or tractExploiting the sheet-like structure of many WM tracts, tract-based spatial statistics (TBSS) was introduced  and initTract-specific analysis (TSA)  is an alTSA offers potential advantages as an analysis tool. It is automated therefore reducing the time cost and inter-rater variability which affect manual-input methods. It characterises WM tracts as surfaces rather than aggregating tracts into a single core line thereby capturing the overall tract morphology. Theoretically TSA also offers improvements over TBSS by (i) employing a tensor-based rather than scalar-based registration; (ii) defining tracts individually and so making it possible to distinguish between adjacent tracts; and (iii) having a data projection search stopping criteria intended to limit crossing over into neighbouring tracts. TSA has been successfully applied to study pathologies such as paediatric chromosome 22q11.2 deletion syndrome  and amyoThe aim of this study is to evaluate the performance of TSA within the context of preterm infant data. We compare TSA with native space tractography as a gold standard, and with TBSS, a similar and widely-used method. Despite some known limitations, TBSS remains a widely-used tool, having been cited over 3000 times . Our evaluation of TSA involves (i) an assessment of TSA's ability to align WM tracts from different subjects and the accuracy of its data projection step in comparison to TBSS; and (ii) an application of TSA for the first time to a cohort of preterm infants at term equivalent age to determine whether TSA is able to detect developmental changes in diffusion properties of WM tracts.Permission for this study was granted by Queen Charlotte's and Hammersmith Hospitals Research Ethics Committee (07/H0704/99) and written parental consent was acquired prior to imaging. MR data were collected from 53 preterm subjects who were imaged between February and July 2013. All images were reviewed by an experienced perinatal neuroradiologist and cases with major focal lesions were excluded. Five data-sets were excluded; 2 unilateral haemorrhagic infarction, 1 cerebellar infarct, 1 cerebellar haemorrhage and 1 infant had temporal and cerebellar haemorrhages with cerebellar hypertrophy. 48 subjects  born at a median (range) gestational age (GA) of 30.6 (24.0–32.9) weeks and imaged at a median age of 41.9 (38.6–47.1 weeks) weeks post-menstrual age (PMA) were analysed in this study. The perinatal characteristics of the study group are summarised in 2, SENSE factor of 2.MR imaging was performed on a 3-T MR system sited on the neonatal intensive care unit. T1- and T2-weighted MR imaging and single shot echo planar dMRI data were acquired using an 8-channel phased array head coil. The pulse sequence parameters were as follows. 3D MPRAGE: repetition time (TR) = 17 ms, echo time (TE) = 4.6 ms, flip angle All examinations were supervised by a paediatrician experienced in MR imaging procedures. Infants were sedated with oral chloral hydrate (25–50 mg/kg) prior to scanning and pulse oximetry, temperature, and electrocardiography data were monitored throughout. Ear protection was used, comprising earplugs moulded from a silicone-based putty  placed in the external auditory meatus and neonatal earmuffs .eddy . Signal-to-noise-ratio (SNR) was calculated for each subject from the raw DW data. A 5×5 voxel ROI was manually placed in the central corona radiata of the b=0 volume and SNR was calculated as the mean signal divided by the standard deviation. For each subject deterministic tractography based on the FACT approach (http://dti-tk.sf.net) was used to delineate WM tracts in native space. Each subject's FA map was thresholded at 0.1 and whole brain tractography was seeded from each voxel with tracking parameters: maximum angle threshold of 45 and minimum FA threshold of 0.1. ROIs used to delineate tracts in native space were drawn manually for each subject, according to the protocol outlined in Diffusion-weighted images were visually inspected in 3 orthogonal planes for the presence of motion artefact and corrupt diffusion weighted volumes were excluded before tensor fitting. 33 subjects had no volumes excluded, eight subjects had one volume excluded, three subjects had two volumes excluded, three subjects had three volumes excluded and one subject had four volumes excluded. Non-brain tissue was removed using BET , images eddy  and the approach   registration used in TBSS  dimensionality reduction in TBSS and TSA from volumetric tracts to voxel-wise skeleton and surface skeleton, respectively; (ii) the projection of the maximum FA value; and (iii) misregistrations between the template and subject. The closer agreement between the values projected by TSA and native space tractography than those projected by TBSS may be due to better-defined stopping criteria for the data projection search in TSA. The search for the maximum FA value in TBSS can cross over into neighbouring tracts  and the Our results show a maturation-dependent increase in FA and decrease in diffusivity that concurs with previous studies in preterm infants . Age-relAssociation fibres such as the SLF, ILF and IFOF can be difficult to identify at birth in term infants as they mature more slowly . We wereFA values in the UNC demonstrated a strong positive correlation with PMA at scan and we observed a negative correlation between MD and RD in the frontal lobe projections with increasing PMA. The UNC has been shown to be affected by preterm birth  and simiOur results show limited correlation between AD and PMA. Previous studies have reported differing results for changes in AD associated with age at scan. Studies with similarly-sized cohorts found age-related changes only in the anterior limb of the internal capsule and the thalamus  while laWe found no correlation between GA at birth or post-natal day at scan and DTI metrics. Prematurity at birth has been shown to affect DTI metrics in larger cohorts , while aTSA has several advantages as an analysis tool. It is automated; its surface representations of tracts approximate closely native space diffusion data and TSA offers improved alignment between WM tracts.A major benefit of TSA is that it is anatomically specific, like other tracts-of-interest methods. Tract-of-interest approaches using tractography have been previously used to study development, averaging DTI values over the whole tract . The axoA possible application of TSA could be to study subjects where tractography is unsuccessful due to brain injury. As the tract skeletons are defined in the template it would be possible to project such subjects’ diffusion data onto the skeleton, as long as the registration was successful.2).A limitation of TSA, as assessed here, is the geometry it is able to describe. The choice made here, the FACT algorithm for tractography, is motivated by its suitability to the diffusion data at hand (32 gradient directions and a b-value of 750 s/mmWhile most of the TSA pipeline is automated, we have chosen to identify tracts in the study-specific template with manually-drawn ROIs to maximize the accuracy of the tract delineation. This step may be further automated with atlas-based techniques which have proven to be successful in recent studies . Such anWe have also omitted tracts that have a tubular structure, such as the fornix and cingulum. This is because TSA is ill-suited to such structures. The tract skeleton is determined by thinning the tract-boundary down to a medial surface. This strategy is poorly defined when there are multiple directions in which it possible to thin, as would be the case for cylindrical structures. For such tracts, it would be more appropriate to use methods such as those developed by .As part of our evaluation of TSA we have analysed only DTI-derived measures, however the framework allows data from other diffusion models, such as NODDI  or g-ratWe evaluate the performance of TSA against native space tractography, which serves as the gold standard, using TBSS as a benchmark, for the preterm population. This work demonstrates that TSA is a suitable method for infant studies using dMRI when particular tracts are to be targeted. The framework allows numerous WM tracts to be analysed and, by design, can easily be applied to large cohort studies. Here we have demonstrated the effects of age at scan on DTI metrics in nine tracts showing that TSA is indeed sensitive to the developmental changes.


Radical prostatectomy is the most common treatment for localised prostate cancer in New Zealand. Active surveillance was introduced to prevent overtreatment and reduce costs while preserving the option of radical prostatectomy. This study aims to evaluate the cost-effectiveness of active surveillance compared to watchful waiting and radical prostatectomy.Markov models were constructed to estimate the life-time cost-effectiveness of active surveillance compared to watchful waiting and radical prostatectomy for low risk localised prostate cancer patients aged 45–70 years, using national datasets in New Zealand and published studies including the SPCG-4 study. This study was from the perspective of the Ministry of Health in New Zealand.Radical prostatectomy is less costly than active surveillance in men aged 45–55 years with low risk localised prostate cancer, but more costly for men aged 60–70 years. Scenario analyses demonstrated significant uncertainty as to the most cost-effective option in all age groups because of the unavailability of good quality of life data for men under active surveillance. Uncertainties around the likelihood of having radical prostatectomy when managed with active surveillance also affect the cost-effectiveness of active surveillance against radical prostatectomy.Active surveillance is less likely to be cost-effective compared to radical prostatectomy for younger men diagnosed with low risk localised prostate cancer. The cost-effectiveness of active surveillance compared to radical prostatectomy is critically dependent on the ‘trigger’ for radical prostatectomy and the quality of life in men on active surveillance. Research on the latter would be beneficial.The online version of this article (doi:10.1186/s12885-017-3522-z) contains supplementary material, which is available to authorized users. Radical prostatectomy is the most common treatment for patients diagnosed with localised prostate cancer in New Zealand,  though iWatchful waiting is mainly used in patients with a life expectancy less than 10 years, but it was included in two randomised clinical trials to compare with radical prostatectomy , 7. The No randomised clinical trial with a follow-up over 10 years has been conducted comparing active surveillance and radical prostatectomy. Two published cost-effectiveness studies  comparinThis study was approved by Northern Y (Ref. No. NTY/11/02/019) and Multi-Region Ethics Committees (Ref. No. MEC/11/EXP/044). No inform consent is required for this study.An economic model was constructed, consisting of three Markov models with microsimulation  and were valued in 2012/13 New Zealand dollars (NZ$). A 3.5% discount rate was applied to future costs and utilities.The treatment costs Table  were basn = 1000) to capture variation in parameter values, with an inner loop microsimulation considering outcomes for a simulated population . The costs and utilities for each simulated man were calculated after each Markov cycle by summing the costs and utilities attached to the related health states and transitions in that cycle. The life-time costs and QALYs  per simulated man in each treatment arm were estimated by averaging the total costs and utilities of all cycles and applying a half cycle correction to all costs  and utilities.The model construction and data analysis were performed using TreeAge Pro 2015. The model used an outer loop  and for all cost distributions.Incremental analysis was performed in terms of incremental cost-effectiveness ratio (ICER) by dividing the incremental life-time costs with the incremental life-time utilities. Cost-effectiveness acceptability curves (CEAC) and frontier plots were also constructed to indicate the likelihood of each treatment being cost-effective under a range of willingness-to-pay values (the amount of money willing to pay for a QALY gained) .Five scenario analyses were conducted. The first scenario analysis used an annual conversion rate of 5% from active surveillance to radical prostatectomy. The 5% conversion rate was used in the cost model built by Corcoran et al. . Hayes eAcross all five age groups, men in the watchful waiting arm had the lowest life-time costs but also the poorest health outcomes in terms of both life years and QALYs Table . ExpecteFor younger men , radical prostatectomy appeared cost-effective compared to watchful waiting with ICERs of NZ$6432 to NZ$10,358 per QALY gained. Active surveillance was dominated (less effective and more costly) by radical prostatectomy for men aged 40–50 and was extended dominated by watchful waiting and radical prostatectomy for men aged 55.For men aged 60, active surveillance was cost-effective between willingness-to-pay values of around NZ$12,155–21,485 per QALY. At an indicative figure of NZ$30,000 per QALY, radical prostatectomy appeared cost-effective. However, for men aged 65 and 70, the ranges over which active surveillance was cost-effective included this indicative NZ$30,000 per QALY value . At much lower willingness-to-pay values , radical prostatectomy appeared cost-effective for the youngest patients (aged 45 and 50).The CEACs  caused by radical prostatectomy but not the costs of long-term complications. The long-term complications are mainly managed by GPs, and most of the costs are borne by patients and thus were not considered from the perspective of the Ministry of Health. In the active surveillance arm, radical prostatectomy was assumed to be used when cancer progression was detected. Not taking radiation treatment as definitive treatment into account is a limitation of this study.Active surveillance is less likely to be cost-effective compared to radical prostatectomy for younger men diagnosed with low risk localised prostate cancer. The cost-effectiveness of active surveillance compared to radical prostatectomy is critically dependent on the ‘trigger’ for radical prostatectomy and the quality of life in men on active surveillance.Early or unnecessary trigger of active treatment reduces the cost-effectiveness of active surveillance. If the quality of life for men under observational management was better than that for men having radical prostatectomy, active surveillance was cost-effective compared to radical prostatectomy, but was not cost-effective compared to watchful waiting for older men with a high annual probability of having radical prostatectomy in the active surveillance arm. More research on these specific points may allow a greater certainty when identifying the optimal management for men with low risk prostate cancer.


III–V semiconductors nanowires (NW) have recently attracted a significant interest for their potential application in the development of high efficiency, highly-integrated photonic devices and in particular for the possibility to integrate direct bandgap materials with silicon-based devices. Here we report the absorbance properties of GaAs-AlGaAs-GaAs core-shell-supershell NWs using photo-acoustic spectroscopy (PAS) measurements in the spectral range from 300 nm to 1100 nm wavelengths. The NWs were fabricated by self-catalyzed growth on Si substrates and their dimensions  allow for the coupling of the incident light to the guided modes in near-infrared (IR) part of the spectrum. This coupling results in resonant absorption peaks in the visible and near IR clearly evidenced by PAS. The analysis reveal broadening of the resonant absorption peaks arising from the NW size distribution and the interaction with other NWs. The results show that the PAS technique, directly providing scattering independent absorption spectra, is a very useful tool for the characterization and investigation of vertical NWs as well as for the design of NW ensembles for photonic applications, such as Si-integrated light sources, solar cells, and wavelength dependent photodetectors. The coupling of light to guided modes is essential for light harvesting and lasing applications; for example for the demonstration of NW lasing up to room temperature2, and monolithic integration of NW lasers on Si3. The resonant absorption in NWs has been studied numerically: for single NWs and NW arrays4, and for both parallel and normal excitation with respect to the long NW axis5. Recently, wavelength selective photodetectors6 and spin angular momentum generation7 based on the absorption enhancement have been proposed. Recent developments in fabrication of semiconductor NWs have already lead to a vast range of applications: photo voltaics11, light sources13, waveguides14, photodetectors15 and water reduction16, all of which exploit the confining properties of the NWs. In particular, NW solar cells reaching efficiencies up to 15.3% have been recently demonstrated owing to resonant absorption enabling bulk-like photocurrent generation with greatly reduced material consumption17. Furthermore, NWs can exhibit nonlinear optics effects as second harmonic generation18 and finally, the hybridization of dielectric NWs with metallic layers and/or nanostructures offers a wide range of new application possibilities such as novel yagi-uda antennas19, and plasmonic lasers22.The growing need for fast, integrated, nanoscale, low-cost photonic devices has triggered intensive developments of semiconductor nanowires (NWs). Main efforts have been directed to realize high quality nano-structures that are able to confine optical fields, and enhance the electrical and optical response in nano-scale dimensions. A single III-V semiconductor vertical NW has good waveguiding properties for energies above the bandgap owing to high refractive indices, and it can support a number of discrete photonic modes. These confined modes can lead to resonant absorption at specific wavelengths mainly defined by the combination of materials properties and their geometric dimensions23, photocurrents24 or photoluminescence excitation27. Recently, a scattering free photo-acoustic technique has been applied to determine the absorption edge of GaAsBi/GaAs NWs28. However, the absorption measurements that would provide the detection of the resonant NW behaviour and therefore direct detection of discrete waveguide modes are still lacking. In this work we used the photo-acoustic spectroscopy (PAS) technique to study absorbance properties of GaAs-AlGaAs heterostructure NWs grown by self-catalyzed technique on lithography-free Si/SiOx patterns29.Although the optical and electrical properties have been widely investigated and well understood, the characterization techniques are usually based on the indirect measurements of the resonant absorption by means of transmission/reflectionAlGaAs, and GaAs supershell thickness tGaAs. We investigate four samples with different NW dimensions, the parameters of which are given in Table The structures under examination are coaxial core-shell-supershell NWs with hexagonal cross section; schematic of a single NW is shown in Fig. A,C,D ~ 1 × 108 cm−2, while ρB~ 8 × 107 cm−2.The NWs were grown by molecular beam epitaxy on p-Si111) wafers using lithography-free Si/SiOx patterns for defining the nucleation sites. This technique enables fabrication of highly uniform NW ensembles with tailorable NW density (see Methods). In Fig. 11 wafersmn - magnetoelectric (TMmn-like), or EHmn - electromagnetic (TEmn-like), respectively. Here m is the m-th order of the corresponding Bessel and Hankel functions that are the solutions of a leaky guided wave in the infinitively long dielectric cylinder, and n corresponds to the n-th zero of these functions. In the case of finite NWs, and the illumination parallel to the NW axis, light scattering at the end facets can provide for the additional coupling where the leaky modes become guided5. Such modes result in resonant enhanced absorption that has HEmn or EHmn symmetry along NW, and we first numerically investigate their excitation in our samples by simulating the absorption cross section (see Methods and Supporting Information).High aspect ratio and high refractive indices of the materials used result in good waveguiding properties along the axes of our NWs. Since GaAs has bandgap wavelength at around 870 nm, material absorption at shorter wavelengths will lead to losses along the propagation, resulting in real component of the transverse wavevector inside NW. This means that the light coupled to these modes will transversely leak out, and they are correspondingly called leaky waveguide modes. Their electric field is mainly confined outside NW, contrary to the guided modes. Depending on the symmetry of the incoming electric field, for the TM or TE polarization, these modes can be HE11 mode for the sample A is excited at around 730 nm, while for the sample B HE11 resonance moves to around 790 nm  so that the light cannot impinge the microphone directly, thus largely reducing the effects of the scattered light .We then experimentally characterize our samples by measuring their absorption by means of the photo-acoustic (PA) technique. PA is a type of photothermal techniques, where absorption of incoming light beam leads to a non-radiative de-excitation process which generates heat, thus changing the thermal state of the sampleght Fig. . By this40. This means that with lower frequencies thermal signal takes much higher non-resonant contribution of Si substrate and parasitic (Al)GaAs growth. With the frequency increasing the contribution of the NW surface is higher, so we can see the resonant response which corresponds to the excitation of the discrete modes, as it is depicted in Fig. 11 peak which corresponds to the highest contribution of the NW surface that supports this resonance. Similar influence of frequency on the NW PA signal was recently reported for GaAsBi/GaAs core-shell NWs grown on Si28. However, no resonant features were observed most probably due to size non-uniformity of the investigated NW ensembles.In photothermal techniques the thermal diffusion length is inversely proportional to the square of the modulation frequencymod = 225 Hz.The contributions of the Si substrate and parasitic growth and NWs on the PA signal were investigated in detail by analyzing reference samples , which includes also the description of the shell layer growth and characterization of the coaxial heterostructure .The GaAs core was first grown by self-catalyzed growth mode. Then the Ga catalyst droplet was consumed in As2-flux in order to terminate axial growth. The AlWe have used Lumerical Finite Difference Time Domain (FDTD) solver to simulate the absorption cross section spectra of a single GaAs-AlGaAs-GaAs NW, by applying perfectly matched layers (PML) in all directions. A single NW is excited by total-field scattered-field source (TFSF) that is suitable for simulations of the absorption cross section in a box surrounding the NW  is connected with the input of the lock-in amplifier and analyses the signal with respect to the modulated frequency of the light source. The main element of the cell is a movable transparent quartz cylinder, with two optically polished faces, which offers some advantages: its movement along the cylinder axis direction changes the distance between the sample surface and the window, so that we can choose the optimum volume of the cell. All the samples are shined under a normal incidence, but our set-up is adaptable also for incidence angle and polarization scan.Photo-acoustic spectroscopy revealing resonant absorption of self-assembled GaAs-based nanowires – SUPPORTING INFORMATION


Anatomical variations of hepatic arteries may be problematic in pancreaticoduodenectomy (PD). We experienced pancreatic head cancer in a patient with rare variation of hepatic artery and performed PD successfully with the resection of this artery. A 75-year-old woman showed pancreatic head tumor on CT. Preoperative CT detected rare variation of hepatic artery; posterior segmental branch of right hepatic artery (RHA-PB) originating from posterior inferior pancreaticoduodenal artery. The image also demonstrated that there was a junction between RHA-PB and anterior branch of right hepatic artery (RHA-AB). We performed PD for suspected pancreatic head cancer. We divided RHA-PB for complete resection of cancer because we preoperatively knew that there was the junction between RHA-PB and RHA-AB. She was discharged uneventfully, and there was no evidence of local recurrence throughout the whole course. Careful preoperative assessment of hepatic blood supply is the key to perform successful PD even in this troublesome situation. Anatomical variations of hepatic arteries are of great importance for abdominal surgery and interventional radiology. Especially, aberrant right hepatic artery (RHA) may be problematic in pancreaticoduodenectomy (PD) . ResectiWe experienced locally advanced pancreatic head cancer in a patient with an unusual aberrant RHA and performed PD successfully by sacrificing this vessel without any complications.A 75-year-old woman with a history of surgical intervention for aortic aneurysm showed dilatation of her main pancreatic duct and pancreatic head tumor on CT scan at an annual medical check-up. Endoscopic ultrasound-guided fine needle aspiration of the pancreatic head tumor was performed, and the result was group 4. She was referred to our department for the surgical treatment of suspected pancreatic head cancer.Preoperative enhanced high-resolution CT detected pancreatic head tumor and major posterior segmental branch of RHA (RHA-PB) which originated from posterior inferior pancreaticoduodenal artery (PIPDA) and run through the tumor Fig. . The imaShe received PD combined with regional lymph node dissection. At laparotomy, RHA-PB was isolated from the hepatoduodenal ligament and confirmed that it originated from PIPDA in accordance with the preoperative findings, which revealed to run through the tumor. To preserve RHA-PB, we had to preserve posterior pancreaticoduodenal arcade and the common trunk of PIPDA, which decreased the curability of the tumor. We ensured that the color of the liver did not change after clamping of RHA-PB. Finally, we ligated and divided RHA-PB for complete resection of the pancreatic head tumor because we knew from the preoperative images that there was the junction between RHA-PB and RHA-AB at the hilar portion. We performed the operation without any complication and achieved R0 resection macroscopically .Her postoperative course was uneventful, and she was discharged on POD15 without hepatic or biliary ischemia. The pathological examination revealed that the tumor was diagnosed as an adenocarcinoma of the pancreas. The tumor invasion was seen near the intraparenchymal vessels Fig. , but R0 Although she died from chronic heart failure 13 months after the operation, local recurrence had not be seen throughout the whole course.We experienced pancreatic head cancer in a patient with rare variation of hepatic artery; RHA-PB originating from PIPDA. We preoperatively identified this unusual aberrant artery and the junction at the hilar portion, which led to successful PD with the resection of this unusual aberrant artery.We could not find a report on RHA-PB originating from PIPDA. In 1966, Michels et al. described 10 types of anatomic variations of hepatic artery  and manyWe performed PD successfully with the resection of this aberrant artery. The optimal surgical strategy in patients with aberrant RHA is under discussion. El Amrani et al. reported that aberrant RHA was preserved in most cases (87%) . PostopeIn these chaotic situations, we suggest the following strategy for aberrant RHA in PD. When there is aberrant RHA in preoperative image, you should assess the route with respect to the head of the pancreas and the position of tumor. When aberrant RHA is type I or type III, or the tumor is situated more than 10 mm from aberrant RHA , aberranWe experienced rare variation of hepatic artery during the resection of the pancreatic head tumor; RHA-PB originating from PIPDA. Careful preoperative assessment of hepatic artery system and its relationship with tumor and the head of pancreas is the key to perform successful PD even in this troublesome case.


The Average Information Content Maximization algorithm (AIC-MAX) based on mutual information maximization was recently introduced to select the most discriminatory features. Here, this methodology was applied to select the most significant bits from the Klekota-Roth fingerprint for serotonin receptors ligands as well as to select the most important features for distinguishing ligands with activity for one receptor versus another. The interpretation of selected bits and machine-learning experiments performed using the reduced interpretations outperformed the raw fingerprints and indicated the most important structural features of the analyzed ligands in terms of activity and selectivity. Moreover, the AIC-MAX methodology applied here for serotonin receptor ligands can also be applied to other target classes.The online version of this article (doi:10.1007/s11030-017-9729-8) contains supplementary material, which is available to authorized users. Fingerprints, which are a representation of a chemical compound structure in the form of a bit string, have been widely used in chemoinformatics for many years –9. They N) and To resolve the aforementioned difficulties with application of high resolution fingerprints, the AIC-MAX algorithm  was receThe algorithm extends the application of existing techniques –18, 20 aAmong the popular fingerprints –25, the software , 26. Commentclass2pt{minimThe AIC-MAX algorithm selected one hundred bits for each target (number optimized in a previous study) . In tota ligands . Moreovementclass2pt{minimIn the second experiment, AIC-MAX was applied to select the most important features for distinguishing ligands with activity specific to one receptor versus another. The procedure was repeated for all pairs of receptors (66 times). The set of “selective features” could be applied to search for selective ligands, which is an essential goal of 5-HTR ligand research. Analysis of the 5-entclass1pt{minimarvations . The algTo evaluate the potential of selective bits, machine-learning experiments  aimed at the separation of compounds that act on individual target compared with other targets were conducted . ClassifThe results Fig.  indicateompounds .Experimental studies confirmed that since AIC-MAX algorithm maximizes, a discriminatory power of a group of bits  and the resulted representation contains enough information to characterize active compounds as original KRFP fingerprint. Therefore, it can be applied in the wide spectrum of screening applications aimed for particular target as well as for searching the compounds selectivity potential, which is a one of the most important challenges in computer-aided drug design.Reduced fingerprints especially should be utilized in machine-learning experiments where application of previous conclusions should ensure outstanding results , 33.In this paper, we presented the application of the AIC-MAX algorithm to identify the most significant chemical patterns for fingerprint representation of serotonin receptor ligands. Moreover, we demonstrated the performance of the AIC-MAX algorithm for selecting the most important substructures to distinguish ligands between two closely related receptors, which is one of the most demanding challenges in computer-aided drug design. The experimental studies confirmed that AIC-MAX is able to produce a reduced representation that preserves almost all meaningful information contained in original KRFP fingerprint and provides efficient numerical computations as well as outperforms the original fingerprint.Supplementary material 1 (docx 1023 KB)Below is the link to the electronic supplementary material.


Self-care practice in type two diabetes is a critical factor to keep the disease under control. Despite the important role of self-care practices in management of diabetes were recognized to be useful and effective in achieving diabetes control and preventing its complication, findings of previous studies in Ethiopia were confirmed that, aspects of self-care practices were more problematic. So that this study focus on magnitude of self-care practice and associated factors among diabetic patients.Among the total 284 respondents, their mean age was 52.19 years and about 178 (62.7%) had poor diabetic self-care practice. Having glucometer at home , knowing fasting glucose level , attending diabetic education , perceived benefit , perceived barrier , and self-employment  were significantly associated with good self-care practice. Self-care practice in diabetes patient is a critical factor to keep the disease under control and about 95% of the disease management is usually carried out by the affected individual or their families .Diabetes mellitus and its complications bring extensive economic loss in diabetic patients with their family, to health systems and to the national economies through direct medical costs and loss of work and incomes .In developing countries, where the resources are limited, and treatment costs of diabetes are constantly increasing, the practice of self-care component among patients with diabetes may result in better economic and therapeutic outcomes .Consistent implementation of recommended self-care behaviors in individuals with type two diabetes requires collaboration between the patient and the provider in an enabling health care system with adequate facilities and resources . This isDespite the important role of self-care practices in management of diabetes were recognized to be useful and effective in achieving diabetes control and preventing its complication, findings of previous studies in Ethiopia were confirmed that, aspects of self-care practices were more problematic –9.There is no study conducted in Ethiopia specifically in Tigray Region regarding self-care practice on diabetic patients and the few studies conducted in developing countries have also discrepancy on the self-care practice among diabetes patients. Therefore, this study focus on magnitude of self-care practice and associated factors among diabetic patients.The study was conducted in public Hospitals of central zone, Tigray region, Ethiopia. The research was conducted from April 1 to March 1 2017.A Hospital based cross-sectional study design was conducted.Were all type two diabetic patients who visits general public Hospitals of the central zone of Tigray, Ethiopia.Were all type two adult DM patients who visit general public Hospitals of the central zone of Tigray at the time of the data collection period.We were excluded critical ill patients from the study participants.Total sample size was calculated using an EPI Info software version 7.1.1 with the parameters for the single proportion formula. Confidence level, 95%, marginal error (d) 5% and 56.6% prevalence of self-care practice was taken from a study conducted in TASH, Addis Ababa in 2012  andHaving glucometer at home showed significant association with good self-care practice. The odds of having glucometer at home were 3.7 times more associated with good self-care practice than compared to those who were not having glucometer at home. This study was similar to a study conducted in TASH in (2012) [Knowing glucose level was significantly associated with good self-care practice. This study showed that those who knew fasting glucose level were 2.7 times more associated with good self-care practice than their counterpart. This might be due to a patient encourages to perform essential self-care behaviors such as healthy eating, being physically active, risk reduction behavior and also it encourages to apply the recommended action to self-care practice.Attending diabetic education was significantly associated with good self-care practice. Patients who were attending diabetic education were 2.5 times more likely associated with the outcome variable than to their counterpart. This study is consistent with studies conducted in Addis Ababa Public Hospitals in (2016)  in thoseThose who had favorable attitudes to perceived benefit had significant association with the outcome variable of good self-care practice. Those favorable attitudes to perceived benefits were 2.4 times more associated with good self-care practice than to their counterpart. This study was consistent with a study conducted in Nigeria in (2014)  12.4 timThose who had favorable attitudes to the perceived barrier had significant association with the outcome variable of good self-care practice. Those favorable attitudes to the perceived barrier were less likely associated with good self-care practice than to the opposite side by 52.9%. This study is consistent with studies conducted in Harare Town in (2012) [Those who, recruited as self-employed were 5.9 times associated with good self-care practice than those unemployed to good self-care practice. This study is consistent with study conducted in Nekemte referral Hospital in 2014  around fThis study established that, more than half of respondents had poor self-care practice. So that, Health care personnel and Ethiopian diabetic association must increase the patient’s awareness to the importance of self-care practices domains and strongly promote the practice among diabetes patients by strengthening IEC program.There may have been recalled bias and social desirability bias since the self-care practices of the study participants were based on self-reports and performance of these behaviors were not observed and could not be confirmed.The study design was cross-sectional, the direction of causal relationships between variables can’t always determine.