Genetic recombination is a major contributor to the ongoing diversification of HIV. It is clearly apparent that across the HIV-genome there are defined recombination hot and cold spots which tend to co-localise both with genomic secondary structures and with either inter-gene boundaries or intra-gene domain boundaries. There is also good evidence that most recombination breakpoints that are detectable within the genes of natural HIV recombinants are likely to be minimally disruptive of intra-protein amino acid contacts and that these breakpoints should therefore have little impact on protein folding. Here we further investigate the impact on patterns of genetic recombination in HIV of selection favouring the maintenance of functional RNA and protein structures. We confirm that chimaeric Gag p24, reverse transcriptase, integrase, gp120 and Nef proteins that are expressed by natural HIV-1 recombinants have significantly lower degrees of predicted folding disruption than randomly generated recombinants. Similarly, we use a novel single-stranded RNA folding disruption test to show that there is significant, albeit weak, evidence that natural HIV recombinants tend to have genomic secondary structures that more closely resemble parental structures than do randomly generated recombinants. These results are consistent with the hypothesis that natural selection has acted both in the short term to purge recombinants with disrupted RNA and protein folds, and in the longer term to modify the genome architecture of HIV to ensure that recombination prone sites correspond with those where recombination will be minimally deleterious. Recombination is a process involving the movement of genetic information within or between DNA/RNA molecules. Homologous recombination, where the fragment of transferred genetic information replaces a homologous fragment within its destination DNA/RNA molecule, is important from an evolutionary perspective because within individual genomes it can both remove harmful mutations and facilitate the accumulation of beneficial mutations. By creating novel combinations of nucleotide polymorphisms homologous recombination can also enable far wider exploration of a sequence space than is achievable by mutation alone Homologous recombination, hereafter referred to simply as recombination, features prominently in the evolution of many viruses. In these organisms recombination does not necessarily involve the breakage and re-ligation of DNA/RNA molecules. In retroviruses such as HIV, for example, it predominantly occurs when RNA copies of the viral genome are being reverse transcribed into DNA by the viral enzyme, reverse transcriptase Although the capacity to recombine can provide viral species with a number of evolutionary benefits, many of the individual recombination events that occur between any particular pair of viruses will be deleterious; especially if they occur between distantly related genomes in vitro protein evolution experiments that the most viable of the chimaeric proteins that are expressed from recombinant genes tend to have lower degrees of predicted folding disruption relative to wild-type proteins than do randomly generated chimaeras It has been demonstrated in in vitro recombination experiments Besides potentially disrupting intra-protein amino acid interactions, it is similarly possible that whenever biologically functional nucleic acid secondary structures are present within virus genomes, recombination could disrupt nucleotide-nucleotide interactions within these. When in their single-stranded RNA configuration, HIV genomes have a high degree of secondary structure, much of which is potentially biologically functional Here we test whether the distinctive recombination patterns evident within naturally occurring HIV genomes http://hiv-web.lanl.gov/) and contained a maximum of three reference sequences for each HIV-1M subtype, up to two sequences for each of 53 recognised HIV-1M circulating recombinant forms and 197 apparently unique recombinants. The alignment was trimmed to include only the protein coding sequences of the 274 genomes. The 434 recombination events detected within the sequences of this dataset were all manually checked with a range of breakpoint localisation and recombinant sequence identification tools available within the program RDP4 http://computingforbiology.org/patterns-of-recombination-in-hiv both as an alignment in FASTA format, and a RDP4 readable file containing information on all of the analysed recombination events). A recombination event is considered here to be a recombinant sequence, a set of two breakpoints within the recombinant sequence and the identities of sequences in the dataset that most closely resemble the parental sequences that recombined in order to form the recombinant sequence. For any particular recombination event we differentiate between the parental viruses by referring to the sequence in the dataset that most closely resembles the sequence that provided the biggest fraction of the recombinant's genome as the “major” parent and the sequence resembling that which provided the smaller fraction as the “minor” parent.A set of aligned patient derived HIV-1 group M sequences was obtained that had been previously analysed in great detail to characterise inter- and intra- subtype recombination events , were simply those identified in our dataset as most closely resembling the actual parents. For each detected recombination event we refer hereafter to the 5′ breakpoint in its corresponding M-recombinant as the “start position”, the 3′ breakpoint as the “end position”, and the number of sites differing between the major and minor parents between these two positions as the “event-length”. For each of the M-recombinants, multiple simulated recombinant sequences, called S-recombinants, were then generated from the same major and minor parental sequences and with the same event-length but with randomly selected start positions (such that the end positions were simply determined by the event-length). Start positions that resulted in end positions falling beyond either the end of the gene of interest (for the protein folding disruption tests) or the end of the genome alignment (for the RNA folding disruption tests) were excluded. Keeping the event-length constant between the M- and S-recombinants ensured that these all had either exactly the same number of polymorphic translated amino acid sites (for the protein folding test) or exactly the same genetic distance (for the RNA folding tests) from both the major and minor parental sequences – something that was crucial for our permutation-based tests of recombination-induced protein and nucleic acid structural disruption. See The protein folding and secondary structure disruption tests performed here both relied on a permutation test involving the generation of sets of simulated recombinants with precisely the same genetic distances to the parental viruses but with breakpoints in random genome locations. Genetic distances were computed as the number of nucleotide differences between a pair of sequences . Given the breakpoint positions and parental sequences associated with a particular recombination event, an et. al. 2007 This test is based on that presented by Lefeuvre In order to test whether recombinant protein sequences avoid protein fold disruption more than can be accounted for by chance, random recombination events were simulated in the manner described above – each of the real recombination events was shifted up or down along the sequence by a random amount, whilst maintaining the same number of polymorphic non-synonymous codon sites (i.e. sites within homologous codons that encode different amino acids) as in the real recombinant. E-scores were calculated for each of the M-recombinants and their corresponding sets of S-recombinant proteins (1000 for each M-recombinant) and a permutation test was performed which counted the fraction of times that the sum of E-scores for the set of real M-recombinant proteins were less  than the sum of E-scores for each of 100,000 sets of S-recombinant proteins. This fraction corresponds to the p-value that the real recombination events that are collectively represented amongst all the M-recombinants are not collectively less disruptive of protein folding than are those represented amongst their corresponding sets of S-recombinants: For a particular gene a p-value <0.05 therefore suggests >95% confidence that recombination events detectable under natural conditions within that gene are less disruptive of protein folding than would be expected in the absence of either (1) selection disfavoring the survival of viruses that express chimaeric proteins with disrupted folds or (2) mechanistic factors that cause recombination events to occur most frequently at genomic sites where they will have minimal impact on protein folding.Tests for recombination-induced RNA secondary structure disruption that we performed were similar to those used to test for protein tertiary structure disruption. For each detected recombination event, one M- and ten S-recombinant sequences were generated and each of these sequences was computationally folded along with the major and minor parental sequences using an optimised version of the UNAfold program The aberrant base-pair test: the number of predicted base-paired nucleotides that were present in a M/S-recombinant, but were not present in either of the parental sequences.The base-pair disruption test: the number of predicted base-paired nucleotides that were present in both parental sequences, but were not present in the M/S recombinant.The minor parent base-pair disruption test: the number of predicted base-paired nucleotides that were present in the minor parent, but were not present in the M/S recombinant.The major parent base-pair disruption test: the number of predicted base-paired nucleotides that were present in the major parent, but were not present in the M/S recombinant.For each of these metrics we obtained (1) a list of disruption scores for the M-recombinants and (2) a ten times longer list of disruption scores for the S-recombinants. These lists were compared using a one-tailed Wilcoxon-rank sum test to determine whether the disruption scores of the M-recombinants were significantly lower than those of the S-recombinants. In these tests low disruption scores for the M-recombinants coupled with an associated p-value <0.05 would indicate with >95% confidence that recombination events detectable within HIV genomes are less disruptive of RNA folding than would be expected in the absence of either (1) selection disfavoring the survival of viruses in which recombination has disrupted RNA folding or (2) mechanistic factors that cause recombination events to occur most frequently at genomic sites where they will have a reduced impact on RNA folding.Recombination that occurs between divergent genome fragments having largely independent evolutionary histories can potentially disrupt coevolved intra-genome interactions such as those occurring between amino acids within three-dimensional protein folds (i.e. intra-protein interactions). Here chimaeric proteins resembling those expressed by actual viruses were computationally tested to determine whether they displayed lower degrees of predicted folding disruption than those of randomly generated protein chimaeras. Actual recombinants sampled from nature would be expected to display less disruption of intra-protein interactions than simulated recombinants either if natural selection disfavoured the survival of recombinants expressing misfolded proteins, or recombination breakpoints tended to coincidentally occur most frequently at sites where they would have minimal impact on protein folding.In order to more rigorously test whether the chimaeric proteins that are expressed by HIV-1M recombinants tend to display lower degrees of protein folding disruption than can be accounted for under random recombination in the absence of selection against misfolded protein chimaeras, individual HIV-1M proteins were analysed using a previously described permutation-based “avoidance of protein folding disruption” test Similar to the findings of a recent study using an alternative approach to that described here Although three of the eight proteins analysed here displayed no detectable signal of lower than expected recombination-induced fold disruption , in at least one case (gp41), this may simply be due to low numbers of recombination breakpoints having been detected within the gene encoding this protein: a fact which reduces our ability to detect a signal in this protein. In this regard it is noteworthy that for all three of these proteins, the mean estimated fold-disruption in the M-recombinants was consistently lower than that of the S-recombinants , tests 2 to 4 examined whether individual base-pairs within the minor and major parent secondary structures were maintained in the secondary structures of M-/S- recombinants.The results of test 1 indicate that M-recombinants tend to have significantly fewer aberrant base-pairs than S-recombinants . The results of test 2 suggest that the number of disrupted base-pairings  in the M-recombinants was significantly lower than those in the S-recombinants . These two tests collectively imply that, relative to parental sequences, the M-recombinants have significantly better preserved base-pairing configurations than do the S-recombinants.Tests 3 and 4 relaxed the criterion in test 2 that considered only base-pairs that were present in both the minor and major parental sequence secondary structures. Specifically, test 3 counted the number of disrupted base-pairings between the minor parental and M-/S-recombinant sequence secondary structures, whereas test 4 counted the number of disrupted base-pairings between the major parental and the M-/S-recombinant sequence secondary structures. For test 3 there was no significant evidence that fewer base-pairs present in the minor parental sequence secondary structures were disrupted in the M-recombinants than in the S-recombinants . Test 4, however, yielded marginal evidence  that fewer major parental sequence base-pairings were disrupted in the M-recombinants than were disrupted in the S-recombinants.In these two tests it is entirely understandable that, relative to minor parental base-pairing disruptions, there were far fewer major parental base-pairing disruptions in both the M- and S-recombinants since by definition the M/S-recombinants are genetically far more similar to their major parents than their minor parents. It is therefore expected that, of the four tests, Test 3 would be the least likely to produce any evidence of secondary structures being less disrupted in the M-recombinants than in the S-recombinants.It is noteworthy that these tests only indicated significant evidence of lower degrees of folding disruption in the M-recombinants than would be expected under random recombination when we considered the structure of the complete HIV-1 coding region. When we applied these and related tests to individual genome regions corresponding to known biologically functional structural elements (such as the Rev response element), they yielded no evidence that that M-recombinants had less disrupted structural elements than S-recombinants (data not shown). While it is possible that, relative to selection favouring maintenance of proper protein folding, selection favouring the maintenance of biologically functional RNA secondary structures has a much smaller influence on patterns of recombination in HIV, it is also possible that our RNA folding disruption tests were simply less powerful than the protein folding disruption tests. In this regard, the RNA folding disruption tests had four potentially important shortcomings: (1) the actual parental sequences of naturally occurring recombinants were not used in these tests and it is entirely possible that with these in hand subtle structural differences between the actual and simulated recombinant genomes would have been clearer; (2) the individual recombination events that were analysed involved single pairs of 5′ and 3′ recombination breakpoints and were probably not representative of natural recombinants which frequently have more than two detectable breakpoints; (3) the accuracy of computational secondary structure prediction is not perfect and it is likely therefore that incorrectly inferred base-pairing interactions decayed (at least slightly) the power with which disruptions of actual base-pairing interactions could be estimated; (4) the possible inclusion of non-viable viruses within the set of analysed sequences could have decreased the power of our tests because it would have violated the implicit assumption that all of the analysed HIV genomes were reasonably fit and were therefore likely free of recombination-induced RNA structure disruption. However, focusing our analyses on recombination events in CRFs (addressing shortcoming 4) and on only the most plausible biologically well characterised secondary structure elements (addressing shortcoming 3) failed to yield any stronger evidence of selection disfavouring the survival of recombinants with disrupted genomic secondary structures (data not shown).et. al. have proposed that the distribution of secondary structural elements within the HIV-1M genome may maximise the chances that recombinant genomes will express properly folded chimaeric proteins by “directing” recombination breakpoints to protein domain boundaries. It remains unclear, however, how any analogous mechanism might maximise the probability of recombinant genomes having properly folded RNA secondary structures; especially since it is specifically the recombination breakpoints that occur within RNA structures that are expected to be the most disruptive of these structures. It is nevertheless possible that sequence determinants of recombination frequency besides secondary structure – such as sequence conservation We have confirmed here that recombination events detectable in the coding regions of a number of HIV-1 proteins tend to be less disruptive of both intra-protein amino-acid – amino-acid interactions and intra-genomic nucleotide – nucleotide secondary structural interactions than would be expected if recombination were random and all recombinants were equally viable. Although this result is entirely consistent with the hypothesis that natural selection has strongly impacted the distribution of recombination events that are detectable within HIV genomes that have been sampled from the global epidemic, it does not indicate the time-scale of this selection. Specifically, while it is likely that selection over the short-term acts against newly generated recombinant genomes that have either misfolded RNA structures or express misfolded chimaeric proteins, it is similarly plausible that selection acting over the longer-term has configured the underlying structure of HIV-1M genomes so as to minimise the deleterious effects of recombination


Solid organ transplantation is the only treatment for end-stage organ failure but this life-saving procedure is limited by immune-mediated rejection of most grafts. Blood vessels within transplanted organs are targeted by the immune system and the resultant vascular damage is a main contributor to acute and chronic graft failure. The vasculature is a unique tissue with specific immunological properties. This review discusses the interactions of the immune system with blood vessels in transplanted organs and how these interactions lead to the development of transplant arteriosclerosis, a leading cause of heart transplant failure. The success of organ transplantation as a curative therapy is hindered by the eventual failure of almost all grafts due largely to immune-mediated rejection . Also, tBy its nature, organ transplantation results in the exposure of the immune system to an abundance of foreign antigens associated with inflammation, the former being a result of genetic differences between organ donors and recipients and the latter being caused by ischemic and mechanical damage during the transplantation procedure. The result is that T and B cells of the adaptive immune system specifically recognize graft-derived antigens  and become activated to elicit effector responses that reject transplanted organs. T cells recognize graft-derived peptides bound to major histocompatibility complexes  expressed by graft cells and recipient antigen-presenting cells . This leads to the induction of cell- and antibody-mediated alloimmune responses , 3. AlloIn response to T cell recognition of pMHC molecules, T cells undergo rapid activation, proliferation, and differentiation into effector cells. Effector CD8 T cells are equipped to specifically induce cell death of target cells by expressing cytotoxic molecules, such as granzymes and perforin, which are contained within cytotoxic granules. Death ligands, such as FasL and TRAIL, are also expressed on CD8 T cell membranes –8. In adIn addition to providing a conduit for tissue oxygenation and delivery of nutrients, blood vessels also interact intimately with the immune system to control the outcome of immune responses. Endothelial changes in the microvasculature are essential for leukocyte migration into sites of inflammation. Human endothelial cells (ECs) also basally express MHC class I and II molecules and up-regulate both molecules in response to inflammatory cytokines such as IFNγ and TNF, thereby enabling them to directly present alloantigens to T cells and be targeted by alloreactive effector T cells –16. Furtin vitro and granzyme B alone is capable of inducing EC death in a delayed fashion by proteolyzing extracellular proteins required for adhesion-mediated cell survival , to activate antigen-presenting cells and vascular cells . They alHMGB1 is a chromatin binding protein that is universally expressed in cells. It is released into the extracellular space after necrotic cell death but can also be secreted in a regulated manner by macrophages. It binds to TLR4 and receptor of advanced glycation end-products (RAGE) to activate the immune stimulating properties of antigen-presenting cells . HMGB1 iThe above examples highlight the role of alarmins in the activation of allogeneic immune responses and development of TA. Recent evidence has established a novel role for the alarmin IL-33 in preventing allogeneic immune responses, cardiac transplant failure, and TA. IL-33 is expressed in non-hematopoeitic cells including ECs, is released after cellular damage, and has been established to promote the activation of protective immune responses following virus infection . In contDuring immune responses, the endothelium is “activated” by cytokines to express cell adhesion molecules and chemokines that facilitate the recruitment of leukocytes from the blood into tissues. During this process, the endothelium also undergoes morphological changes that increase vascular permeability to plasma proteins . These vProfiling of the immune response in clinical samples of TA has revealed a predominance of Th1 cytokines and associated chemokines . These fThe inflammatory cytokines IL-1β, IL-1α, and TNF are also abundant in TA lesions and all are induced early after transplantation –136. TheDue to their polymorphic nature and cell surface abundance, donor HLA molecules are a major class of antigens recognized by antibodies in graft recipients , 147. ThSome graft recipients also develop graft-reactive antibodies toward non-HLA molecules. This likely arises from an aversion of tolerance stemming from broad-scale inflammation that triggers activation of autoreactive lymphocytes and/or that creates neo-antigens through the proteolytic cleavage of self-proteins. Non-HLA antibodies that target vascular cells bind to vimentin, a novel fragment of perlecan termed LG3, and angiotensin II type 1 receptor –53. IncrBesides intimal thickening, another change in allograft arteries that ultimately drives ischemic graft failure is vasomotor dysfunction. The vascular endothelium is essential for regulating arterial vasomotor function, acting on vascular smooth muscle cells to control the dilation and constriction of blood vessels –160. TheNitric oxide is an important endothelial-derived factor that induces arterial dilation . In blooTh17 cells have been implicated in vascular changes that occur in TA , prompti4) levels through the induction of GTP cyclohydrolase I, the rate limiting enzyme in the BH4 synthesis pathway  is also a key regulatory component of arterial vasomotor function. A discussion of EDCFs tends to be focused on endothelin (ET-1) since it is one of the most potent endogenous vasoconstrictors produced in humans and there are clear clinical indications for pathophysiological roles of ET-1, including in TA –186. TNFSmall arteries in humans and other mammals exist in a state of partial constriction. This allows a basal level of endothelial-independent arterial tone against vasodilators and vasoconstrictors that can act to influence arterial diameter and resultant blood flow. Allogeneic immune responses induce smooth muscle cell death in the media of allograft arteries and this likely reduces the functional properties of arteries . In a raThe expression of cytoprotective and immunoregulatory proteins by tissue cells is essential for preventing pathological tissue damage and resultant immunopathology that can occur during immune responses toward pathogens. Similar processes may also be operational in a transplant setting and, as such, are pertinent to any discussion of alloimmune-mediated vascular injury. Profiling gene expression in non-rejecting or tolerized grafts has identified the increased expression of “cytoprotective” genes A20, hemeoxygenase-1 (HO-1), Bcl-xL, and Bcl-2 in non-rejecting grafts, suggesting a role for them in accommodating graft survival , 192.c release from the mitochondria , and immunoglobulin-like transcript-3 and -4 (ILT3/4). IDO is an enzyme that degrades tryptophan, resulting in the release of kynurenines. The depletion of local levels of tryptophan can inhibit the proliferation of immune cells and kynurenines can actively inhibit immune cell activation . The index vivo-expanded regulatory T cells, which suppress effector T cell responses, can prevent arterial remodeling reflective of TA (Therapeutic prevention of TA requires a combination of strategies to specifically inhibit immune responses and to directly prevent hyperplastic responses of vessel wall cells. Non-specific immunosupression with cyclosporine is a mainstay in heart transplantation but the eventual failure of most grafts and the association of this drug with side effects necessitate the need for improved approaches. The “holy grail” of transplantation therapy is the induction of tolerance to specifically prevent immune activation toward transplanted grafts while maintaining protective immunity. Many preclinical studies demonstrate the induction of tolerance and prevention of TA using co-stimulatory blockade –219. In ve of TA , 225.ex vivo modification of allograft artery cells using viral transduction for the prevention of TA since it is likely that inhibition of vascular cell death by forced expression of protective genes, such as A20, can reduce the development of TA in preclinical models (ex vivo modification of graft cells prior to transplantation, such therapeutic approaches yet to be translated into a clinical setting.In addition to preventing immune activation, anti-proliferative drugs such as mTOR inhibitors inhibit smooth muscle proliferation and resultant intimal thickening in TA. These inhibitors first found use in the prevention of intimal hyperplasia after restenosis , 227. Stl models . AlthougBlood vessels possess unique immunological features that define the outcome of immune responses. Cytotoxic damage of vessel wall cells and the alteration of vascular cell phenotypes by different components of the allogeneic immune response drives the remodeling of arteries in transplanted organs and this culminates in the development of TA. Further understanding these pathogenic mechanisms will be essential for future advancements that are able to specifically prevent immune activation toward allograft blood vessels and the hyperplastic responses of vessel wall cells. Also, similar immunopathological mechanisms that contribute to the development of TA are also involved in other immune-mediated arteriosclerotic conditions, such as giant cell arteritis and atherosclerosis, so insights obtained from studies on TA could also be informative for these diseases , 230.Anna Von Rossum, Ismail Laher, and Jonathan C. Choy wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Systemic Lupus Erythematosus (SLE) is a multisystem autoimmune connective tissue disease that has a strong female predominance 9:1) seen especially in women of child-bearing years  seen esp. ClinicaRNA was extracted from monocytes from SLE patients and healthy controls previously collected in the laboratory. Bioinformatics analysis was undertaken to identify potential genes targeted by miRNAs of interest. The primers specific to the miRNAs were designed and optimised following which gene induction of miRNAs was determined by PCR. In order to investigate the expression of miRNAs, densitometric analysis of the gel electrophoresis was determined.In the study, the expression of miRNA-107 and miRNA-132 were investigated. The expression of miR-107 was significantly greater in SLE patients than in healthy controls (p<0.00362), whilst there were no appreciable differences in miRNA-132 expression in SLE patients and healthy controls. SMURF1 and SOCS5 were identified as potential genes targeted by miRNA-107, however no significant change in expression in either genes were observed in SLE patients versus controls.There was a significant increase in the expression levels of miRNA-107 in SLE patients, while no significant changes in miRNA-132 was demonstrated. Further investigation into potential gene targets for miR-107 is required in order to understand the pathological importance of dysregulation of this miRNA in SLE and its therapeutic implications


Sites are defined from complexes between a protein and a pharmacological ligand. The database provides the all-atom description of the protein, its ligand, their binding site and their binding mode. Currently, the sc-PDB archive registers 9283 binding sites from 3678 unique proteins and 5608 unique ligands. The sc-PDB database was publicly launched in 2004 with the aim of providing structure files suitable for computational approaches to drug design, such as docking. During the last 10 years we have improved and standardized the processes for (i) identifying binding sites, (ii) correcting structures, (iii) annotating protein function and ligand properties and (iv) characterizing their binding mode. This paper presents the latest enhancements in the database, specifically pertaining to the representation of molecular interaction and to the similarity between ligand/protein binding patterns. The new website puts emphasis in pictorial analysis of data.The sc-PDB database (available at  Recenterature –9.The sc-PDB is a specialized structure database focused on ligand binding site in ligandable proteins . We haveThe first publicly available version of sc-PDB has been released in 2004. The database has been annually updated with regular additions of new features (See Supplementary Table S1 for a summary of changes since the database creation). Not only the quality and the precision of data improved over the 10 years, but new tools have allowed global analysis of data. A major example is the clustering of sites for proteins present in multiple copies in the database . The newThe sc-PDB data are directly compatible with computational methods, such as docking, molecular mechanics and electrostatic calculations. Unlike the PDB, which generally does not represent hydrogen atoms nor defines ionization state of titratable groups, the sc-PDB provides an all-atom model of molecules: (i) hydrogen atoms are added to amino acids considering that arginine and lysine are positively charged and aspartic and glutamic acids are negatively charged, (ii) hydrogen atoms are added to other residues according to ionized templates built from HET group dictionary , (iii) thttp://cheminfo.u-strasbg.fr/scPDB/ABOUT.The current sc-PDB release contains 9283 entries, representing 3678 different UniProt  proteinsThe total size of compressed database is 1.5 GB. Its downloadable content is summarized in Table The latest sc-PDB release enables the user to depict protein–ligand complexes according to different needs and complexity levels. For example, a medicinal chemist may be primarily interested in the PoseView 2D sketch highlighting the ligand structure, binding site boundaries and main interactions . A cheminformatician may focus on the nearby tabulated list of protein–ligand interactions including involved atoms and a full topological description  of each interaction . Last, a structural biologist can access a 3D picture of the complex embedded in the OpenAstex viewer  . The intWater is by essence the biological fluid. The role of water in molecular recognition events is not yet fully understood although it has been extensively studied experimentally and theoretically . These water molecules are expected to be hardly displaceable by a ligand because of tight binding to the protein . Other high quality databases derived from the PDB also facilitate the comparison of the binding sites across a protein family  shares significant 3D similarity with a single site in sc-PDB, that of GTP in a murine homologous protein .Local structural similarity between non-homologous proteins can account for the promiscuity of a ligand, and thus can help explaining the side effects of a drug or suggest its repositioning toward a novel target and therapeutical indication ,25. The The non-bonded interactions between a ligand and its protein define a 3D pattern that characterizes the binding mode. We have recently developed a new geometrical method to encode and compare protein–ligand interaction patterns . BrieflyE. coli adenylosuccinate synthetase  shares significant similarity with 25 complexes in sc-PDB, representing 19 different proteins bound to GDP, GTP or close analogs. The two top scorers are respectively a homologous protein in wheat (PDB ID: 1DJ3) and the functionally unrelated signal recognition particle protein , interaction pattern, tabulated results  and charts  can be downloaded in the relevant file format . In case of binding site/binding mode similarity searches, aligned molecules are also downloadable Table .http://cheminfo.u-strasbg.fr/scPDB/ABOUT.The website specifications are detailed at Supplementary Data are available at NAR Online.


Here we demonstrate that the crack growth in the upper region of the branch-stem interface of conifer trees proceeds along a narrow predefined region of transversally loaded tracheids, denoted as sacrificial tissue, which fail upon critical bending moments on the branch. The specific arrangement of the tracheids allows disconnecting the overloaded branch from the stem in a controlled way by maintaining the stem integrity. The interface microstructure based on the sharply adjusted cell orientation and cell helical angle secures a zig-zag crack propagation path, mechanical interlock closing after the bending moment is removed, crack gap bridging and self-repairing by resin deposition. The multi-scale synergetic concepts allows for a controllable crack growth between stiff stem and flexible branch, as well as mechanical tree integrity, intact physiological functions and recovery after the cracking.Biological materials possess a variety of artful interfaces whose size and properties are adapted to their hierarchical levels and functional requirements. Bone, nacre, and wood exhibit an impressive fracture resistance based mainly on small crystallite size, interface organic adhesives and hierarchical microstructure. Currently, little is known about mechanical concepts in macroscopic biological interfaces like the branch-stem junction with estimated 10 Internal interfaces in structured biological materials appear at all hierarchical levels and contribute decisively to the overall tissue integrity and function2456810131519Picea abies [L.] Karst). The interface has to support not only basic physiological functions like water and nutrient transport but has to be morphologically and mechanically adapted to static and dynamic loads in order to secure the mechanical safety of the tree and a certain level of damage tolerance2123In this work, we have analysed microstructural, mechanical and self-repairing mechanisms at a branch-stem interface of a Norway spruce (cf.26In A scanning X-ray diffraction (XRD) analysis of a branch-stem radial-section  was used23In order to analyse the morphology of the interface during crack propagation, a statically loaded branch was examined using X-ray computed tomography. Axial and tangential views document the presence of the zig-zag mechanism at early crack growth stages  as well expected crack path, however, the orientation of the tracheids  with a diameter of 10 to 15 cm and age of 20 years were used in the experiments. Annual ring width of the juvenile wood material ranged from 0.5 to 4 mm.Sections of freshly cut spruce trees . The stem body was mounted onto a carrier and the branch was continuously loaded by the movement of the cross head of the machine. The force transmission point was 100 mm from the stem surface. A force displacement curve was recorded during the experiments.X-ray diffraction analysis of MFA  was perfGE phoenix nanotom system equipped with a tungsten X-ray tube providing a spatial resolution of about 10 μm.Tomography characterization was performed using a commercial How to cite this article: Müller, U. et al. Synergy of multi-scale toughening and protective mechanisms at hierarchical branch-stem interfaces. Sci. Rep.5, 14522; doi: 10.1038/srep14522 (2015).


The mammalian brain is a complex organ composed of many specialized cells, harboring sets of both common, widely distributed, as well as specialized and discretely localized proteins. Here we focus on the human brain, utilizing transcriptomics and public available Human Protein Atlas (HPA) data to analyze brain-enriched  polyadenylated messenger RNA and long non-coding RNA and generate a genome-wide draft of global and cellular expression patterns of the brain. Based on transcriptomics analysis of altogether 27 tissues, we have estimated that approximately 3% (n=571) of all protein coding genes and 13% (n=87) of the long non-coding genes expressed in the human brain are enriched, having at least five times higher expression levels in brain as compared to any of the other analyzed peripheral tissues. Based on gene ontology analysis and detailed annotation using antibody-based tissue micro array analysis of the corresponding proteins, we found the majority of brain-enriched protein coding genes to be expressed in astrocytes, oligodendrocytes or in neurons with molecular properties linked to synaptic transmission and brain development. Detailed analysis of the transcripts and the genetic landscape of brain-enriched coding and non-coding genes revealed brain-enriched splice variants. Several clusters of neighboring brain-enriched genes were also identified, suggesting regulation of gene expression on the chromatin level. This multi-angle approach uncovered the brain-enriched transcriptome and linked genes to cell types and functions, providing novel insights into the molecular foundation of this highly specialized organ. The brain is a complex organ that controls a variety of bodily functions, including maintenance of homeostasis, processing of sensory information, cognition and generation of behaviors. These functions are executed by circuitries composed of specialized neurons supported by glial cells  that each express sets of genes that determine their phenotype and physiological properties.The human genome projects ,2 revealIt has recently become evident that subsets of long non-coding RNAs (lncRNAs) regulate transcription and translation as precursor of microRNAs, by binding to microRNAs or interacting with microRNA binding sites , by chroHere we analyzed genes expressed in a functionally important area of the human brain, the frontal cortex (FC). By comparing 27 tissue types representing all major organs and tissues in the human body, brain-enriched protein coding  and non-http://www.brainspan.org). This analysis revealed a correlation coefficient >0.9, indicating reproducibility of sequencing experiments in general.The transcriptomes of 26 peripheral human organs  and three frontal cortex samples  were anaGenes expressed in the frontal cortex were classified according the categories determined to describe expression profiles in a large set of related and unrelated tissues ,19–24. I–6) were found  results for >300 proteins, including 190 brain-enriched proteins , were anIn cortical regions the neuronal population is roughly divided into two main classes, excitatory, glutamatergic pyramidal projection neurons (≈75%) and inhibitory, mostly GABAergic interneurons (≈25%). Among the list of brain-enriched genes we found several proteins expressed in pyramidal-like neurons including widely expressed neurogranin  brain-enriched genes in the analyzed samples  are geneWe also investigated the global expression of molecular components of myelin expressed in oligodendrocytes , includiThe third class of glial cells ‘populating’ the brain is microglia. These cells derived from hematopoietic stem cells that invaded the brain during embryonic development or macrophages that entered the brain from the bloodstream later in life. All well known microglia genes, including integrin alpha M chain  provides the brain with sufficient oxygen and glucose. Several genes generally expressed by endothelial cells such as the membrane protein caveolin 1 (CAV1) are expressed in blood vessels  in all oDuring the initial analysis of the deep sequence data several genes enriched in a limited number (2–7) of tissue types were found. These are clearly non-housekeeping proteins that can have either similar or more diverse functions in different tissues. Several group-enriched genes are expressed in peripheral organs that contain cell types that originate from the neural crest, a developmental structure closely related to the neuro-ectoderm that forms the brain. For example, ELAV -like 4 is expressed in peripheral ganglion cells  and endoRNA-sequencing reveals the full mappable sequence of available transcripts and can be utilized to identify splice variants coded from each gene. For example, different transcripts for N-terminal EF-hand calcium binding proteins (NECABs) 1 and 2 are expressed in different organs. NECAB1 is brain-enriched (FPKM 49) but also expressed in heart muscle (FPKM 8). Based on the sequence data it is evident that the full-length transcript (ENST00000417640) is expressed in the brain, whereas a truncated transcript (ENST00000521366) is predominantly expressed in heart muscle . This waBased on Ensembl annotations, we analyzed the expression levels of 6,969 long non-coding RNAs (biotype lncRNA in Ensembl 73). We identified 2,959 lncRNA transcripts in the 27 tissue and organ types analyzed and found 661 lncRNA in frontal cortex. Out of these, we identified 49 brain-enriched and 38 group-enriched (including brain) lncRNAs . DetaileDue to its heterogeneity in function and cellular composition, multiple brain regions need to be analyzed to capture all transcripts expressed in the brain. Hawrylycz, Lein and colleagues were the first to carry out a comprehensive study on gene expression in 900 anatomically defined regions and sub-regions of the human brain . Here weFirst focus of this study was to generate a comprehensive list of genes with elevated expression in the human brain compared to analyzed peripheral tissues and to further characterize these brain-enriched protein-coding genes, by determining enriched biological functions and cellular expression. GO analysis of brain-enriched genes (including group-enriched), using all genes expressed in the brain as background, revealed many enriched GO-terms directly associated with development of the nervous system or synaptic signaling . The strDo all proteins have a single molecular function and is adaptation of tissue and organs regulated by expression of genes and transcripts with specialized properties? The fact that we identified hundreds brain-enriched genes supports the hypothesis that organ function and properties are determined by the expression of proteins with specific molecular characteristics and cellular functions. However, the fact that we did not find many brain-specific genes, suggests a more context-dependent functional adaptation of proteins resulting in specific tissue and organ functions. This is supported by the many group-enriched genes expressed in brain and non-related tissue types (e.g testis and liver) or the expression of BBB proteins ABCB1 (P-gp) and ABCG2 (BCRP) in glandular cells in colon. We also explored NECAB1 splice variants expressed in brain and heart muscle. The fact that NECAB1 expressed in heart lacks the N-terminal calcium binding EF-hand motif suggests a different cellular function of this protein in heart muscle and brain.An additional benefit of our approach is the possibility to identify and quantify the expression of lncRNAs. Here we analyzed the genomic organization and expression patterns of brain-enriched non-coding genes in relation to protein-coding neighboring genes. The results presented here suggest a consistent pattern of neighboring genes with similar enriched expression characteristics, and the existence of chromosomal ‘hot spots’ containing several brain-enriched coding and non-coding genes. For example a 3,185 kb region at position q16.1 of chromosome 6  containsIn summary, we report for the first time the integration of deep sequencing transcriptomics of brain tissue homogenates with precise localization data on the single cell level using IHC results linking genes to proteins, cells and brain functions. This information will not only help to better understand brain evolution and physiology, but could also be utilized to direct the search for possibilities to intervene with disease processes.http://www.uppsalabiobank.uu.se/en/). All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board  and # 2011–473 (RNA)), and consequently the need for informed consent was waived by the ethics committee. The use and analyses based these human tissues has previously been described [Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained form the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank , using a phred quality threshold of 20. All reads shorter than 54 bp after trimming were discarded. The processed reads were mapped to the GRCh37 version of the human genome with Tophat v2.0.3 [http://picard.sourceforge.net/). To obtain quantification scores for all human genes, FPKM (fragments per kilobase of exon model per million mapped reads) values were calculated using Cufflinks v2.0.2 [http://www.R-project.org/) with the addition of package ‘gplots’ (http://CRAN.R-project.org/package=gplots). A network analysis  containing triplicate 1-mm cores of 46 different types of normal tissue were generated as previously described . TMA secThe immunohistochemical staining pattern was visually evaluated for antibodies targeting the genes of interest, and staining profiles were compared with RNA values in 27 analyzed tissues. Each antibody was scored for reliability, taking into account the effect of antibody titration on intensity of immunoreactivity. We considered an antibody to be reliable, if the staining pattern was (i) consistent with the expression profile, (ii) partly consistent with the RNA profile together with supporting western blot or (iii) was consistent when obtained with multiple antibodies recognizing different epitopes of the target protein. Only such antibodies were included in this study . In totawww.proteinatlas.org/about/download). The primary data (reads) are available through the Array Express Archive (www.ebi.ac.uk/arrayexpress/) under the accession number: E-MTAB-1733. The transcript profiling data  for each gene in each cell and tissue is available in the most recent version (v13) of the Human Protein Atlas (www.proteinatlas.org).All the IHC and expression  data are available for downloads without any restrictions  were analyzed to determine grey matter: white matter ratios (B-C). Grey matter areas are characterized by the presence of neurons with larger nuclei , whereas white matter structures do not contain neurons but do contain large numbers glial cells with smaller nuclei . Sample information is summarized in table. HE stainings of sample #1 could not be analyzed due to severe tissue damage during cryosectioning.(EPS)Click here for additional data file.S2 Fighttp://web.stanford.edu/group/barres_lab/brain_rnaseq.html). The expression data was scaled and subjected to unsupervised hierarchical clustering using Euclidean distance to visualize clusters of genes more abundantly expressed in the different cell types. Similar to results for our IHC analysis we find the majority of brain-enriched genes to be expressed in a single cell-type. Note the overrepresentation of neurons, astrocytes and oligodendrocytes and underrepresentation of microglia and endothelial cells.Cellular expression of human brain-enriched and group-enriched genes was extracted from the RNA-Seq transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex ((EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLS)Click here for additional data file.


Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10 This situation has changed dramatically over the past two decades as new effective treatment strategies have emerged, leading to achievement of complete response (CR) in a considerable number of patients. It is now established that achievement of CR according to the CLL International Workshop (IW-CLL) guidelines2Chronic lymphocytic leukemia (CLL), a CD51–1010 neoplastic cells. If indeed achievement of CR forecasts freedom from disease, it is conceivable that the deeper the CR, the greater the effect on disease suppression and perhaps even on overall survival and cure. New combination treatments which incorporate biologic agents and monoclonal antibodies into the treatment regimens induce deeper responses, leading to a higher incidence of CR; however, more sensitive techniques for residual disease detection would probably show that the CR group actually encompasses a vast variety of responses. Therefore, the incorporation of sensitive techniques into disease status evaluation in histological negative malignancies allows a more precise segregation of treatment responses, which is vital for clinical decision-making and prognostic purposes.Nevertheless, CR defined by clinical findings, blood count, and bone marrow cellularity is not equivalent to disease eradication, and the classic definition of CR actually includes patients harboring 10Assessment of minimal residual disease (MRD) as a measurement of tumor load in hematologic malignancy is becoming a tool for monitoring the depth of disease response, predicting early relapse, and tailoring treatment. Monitoring MRD as a tool for detecting disease presence in patients without histological evidence of the tumor has been incorporated into studies in acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), as well as mantle cell lymphoma (MCL).4Minimal residual disease in B-cell malignancies has been monitored in clinical studies using two major methods. The first is fluorescence-activated cell sorting (FACS), also referred to as “flow cytometry” (FLC), which detects cells harboring a specific phenotype and depicts their number among the normal cell population, and the second is allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) which sequences and amplifies patient-specific DNA in the immunoglobulin heavy-chain variable region (IgHV). Of note, FLC is usually used for detecting CLL cells at diagnosis and following treatment, but routine laboratory methods cannot detect a low tumor burden and are thus not valid for MRD analysis. Hence, a distinction must be made between MRD evaluation and routine disease detection by FLC.Implementation of MRD assessment into clinical practice lags behind due to the lack of standardized detection methods and absence of adequate laboratory facilities in some centers. Moreover, evidence-based guidelines for decision-making based on MRD are still unavailable. Another issue related to the applicability of MRD assessment in routine practice is that certain malignancies show vast heterogeneity of markers best defining them; hence, designing a standard routine method that will fit everyone is difficult.+CD5+ light-chain restricted cells as CLL. Back in 1992, Robertson et al.Minimal residual disease assessment in CLL began two decades ago in studies utilizing poorly specific and insensitive two-color FLC methods, referring to all CD19Later studies began using more complex multi-color multiparameter FLC as well as PCR to assess MRD as a secondary end-point, and examined a variety of treatment regimens, coming up with results in line with those presented above. These findings added solid evidence for the role of MRD surveillance in CLL during first-line as well as advanced-line treatments.In a study by Bosch et al.,Hillmen et al.The most substantial evidence for the benefit of achieving MRD negativity in first-line therapies comes from the German CLL Study Group (GCLLSG) trial designed, among other things, to compare MRD status in two different arms using four-color FLC 2 months after treatment.A retrospective analysis of over 200 patients treated with a variety of first-line therapies conducted by Santacruz et al.The issue of MRD negativity has also been addressed in advanced-line treatments. In the study by Moreton et al.,In a similar vein, among 37 patients with resistant or relapsed CLL treated with FCM in the study by the Spanish GELCAB ,Minimal residual disease has also been evaluated in the post-allogeneic stem cell transplant  setting. Post-alloSCT follow-up presents challenges in decision-making due to the fact that while physicians have a potential therapeutic tool that can be constantly manipulated for disease control, studies have not determined the exact scenarios requiring intervention, or the best intervention method. A study by the German CLL Group examined the MRD status at several time points after alloSCT and demonstrated enhanced event-free survival in MRD-negative patients at 12 months.Importantly, these two studies report an association between MRD surveillance per se and improved event-free survival, suggesting that once the treating physician was equipped with the knowledge of MRD status, treatment modifications were made leading to improved survival. Optional treatment modifications applied in MRD-positive patients may include the use of donor lymphocyte infusions or reduction of immunosuppressive drug dose. This observation emphasizes the need for trials specifically addressing the question of treatment tailoring according to MRD dynamics after transplantation.The available data imply that MRD negativity is a potent landmark on the way to improved survival, raising questions about its potential applicability for treatment de-escalation and toxicity reduction in patients having reached MRD negativity early in the course of therapy. Two prospective studies addressed this issue suggesting that MRD negativity can guide decisions regarding therapy duration and intensity. In the study by Strati et al.,11These two studies show that MRD negativity is an independent prognostic factor at any time point, suggesting that once reached it may provide sufficient relapse risk “protection” which is not enhanced with treatment continuation.If so, strict adherence to the full-length treatment protocols might prove to be unwarranted, and shortening or de-escalation of therapy could reduce toxicity in patients achieving early and deep responses. This hypothesis needs to be proven in a randomized trial, but currently available data are nevertheless thought-provoking.−4 ,6 cells per test tube. Conceptually, cells are distinguished by their differential co-expression of CD19 and CD5 together with the absence or low expression of CD22, CD79b, and CD81, and over-expression of CD43. Specific gating strategies have been established for sample analysis according to this standardization. The following test tubes are required: (1) CD20 CD38 CD19 CD5; (2) CD81 CD22 CD19 CD5; (3) CD43 CD79b CD19 CD5.One of the limitations in the interpretation of presented data is related to the fact that the studies have employed different methods of MRD measurement, which complicates the comparison. However, at least three studies−2 cells with a 100% positive predictive value, thus obviating the need to proceed with more extensive tests if clearly demonstrating residual CLL cells, and (2) an antibody combination that is becoming increasingly important, and includes CD45 CD14 CD19 CD3, for the removal of contaminating double-stained non-B-cells and for setting the detection threshold.Two additional tubes are recommended: (1) a screening tube containing the sIgλ SIgk CD19 CD5 combination, which can detect above 10−4, then a comment should be made indicating that the sample is inadequate, and an explanation is to be provided.For MRD-positive samples, the consensus for reporting the level of MRD in CLL is the percentage of CLL cells among all leukocytes after indicating the limit of detection. For MRD-negative samples, the report should specify that CLL percentage is under the indicated level of detection. If the limit of detection is above 10Flow cytometry is an accessible and inter-laboratory reproducible method, and, as such, it yields results that can be incorporated into real-time decisions.Following the ISA publication, other groups have proposed alternative combinations with the intent to optimize cost and complexity of the ISA panel, or even improve its sensitivity.+CD19+ that had otherwise been defined as CLL and lowered the positive predictive value of MRD positivity. This method also reduced time and complexity of the analysis and allowed for analysis of cases with very hypocellular marrow or lymphopenia due to reduction in the number of events required for analysis. Very good concordance with the four-color assay was observed for detection in the range of 10−4–10−5.The same group that issued the 2007 ISA approach conducted further studies under the auspices of the European Research Initiative in CLL, and proposed an alternative six-color FLC assayRaponi et al.−5, but the 10-color assay could eliminate contaminating CD3+CD19+ cells more efficiently, improving the positive predictive value of the test, and lowering the number of leukocytes required for analysis from 1×107 in the ISA to 2×106.A study by Sartor et al.−4–10−6 disease cells, it requires patient-specific reagents and is laborious and expensive. Its results can also be obscured by low quality and quantity of DNA. According to this method, DNA is amplified with primers specific to the immunoglobulin gene rearrangement present in the neoplastic cell following individual patient sequencing of the variable domain region of the immunoglobulin. This is indeed a very sensitive technique; however, only real-time PCR can provide a quantitative answer regarding the number of malignant cells, a prerequisite for precise MRD evaluation. Therefore, a combination of real-time PCR with ASO-immunoglobulin heavy-chain variable region (IgHV) primers is required for sensitive quantitative measurement of PCR copy numbers.As previously mentioned, FLC is not the only method employed in MRD studies in CLL. While ASO-PCR is generally considered a more sensitive method,Naturally, efforts have been made to compare both methods of MRD surveillance in an attempt to select the best sensitive but yet applicable method. The study by Raponi et al.Bottcher et al.−4 disease level. Notably, in this study, as opposed to the investigation by Bottcher et al.,Finally, Rawstron et al.Another question encountered in practice is whether peripheral blood (PB) examination suffices for MRD analysis. The study conducted by Rawstron et al.Raponi et al.The available data provide strong evidence for the incorporation of MRD assessment into two aspects of CLL patient care. It may serve as an independent individualized prognostic indicator predicting PFS and OS in patients with CLL, regardless of the type of treatment, and it may guide treatment-related decision-making.Available data support the determination of MRD levels using the ERIC consensus four-color FLC in PB, since its high sensitivity and negative predictive value compared to RQ-ASO-PCR, as well as good concordance with bone marrow findings, reassure that positive cases are rarely missed. Flow cytometry assays utilizing more than four colors in each study tube may replace the ISA in the near future. Certain scenarios, such as MRD assessment earlier than 3 months after treatment, or analysis of extremely low numbers of cells, may warrant bone marrow examination and the use of RQ-ASO-PCR; however, these scenarios are limited.Further studies are needed to resolve issues regarding the optimal time points for MRD evaluation following different treatment protocols and the value of MRD surveillance in the era of new biological agents. The issue of appropriate clinical decisions based on MRD levels is unresolved. The assumption that MRD level could guide chemoimmunotherapy tailoring or pre-emptive immune manipulations in alloSCT patients remains to be investigated. Notably, in the alloSCT setting, interventions based on MRD data are already a working theory, although not yet evidence-based.While the information about prospective studies intended to guide real-time treatment decisions according to MRD levels is extremely limited, at least one such trial is underway, examining lenalidomide maintenance therapy for MRD-positive patients concluding chemo-immunotherapy . The CLL International Workshop has recently recommended investigating the significance of MRD in clinical studies as a priority; so, the proportion of such studies among the long list of clinical trials in CLL is expected to rise in the upcoming years. Studies of MRD-tailored therapy could establish the role of MRD monitoring as a tool for extending survival by treatment escalation in MRD-positive patients, on the one hand, and as a tool for minimizing toxicity by treatment de-escalation in MRD-negative patients, on the other hand.


Positron annihilation spectroscopy (PAS) was used to examine the effect of defined Cd-rich and Te-rich annealing on point defects in Cl-doped CdTe and Ge-doped CdZnTe semi-insulating single crystals. The as-grown crystals contain open-volume defects connected with Cd vacancies  Cadmium telluride (CdTe) and Cadmium zinc telluride (CdZnTe) are important materials for multiple applications in solar cells124In spite of the high interest and expectation on CdTe/CdZnTe-detectors, till nowadays they have been hardly commercialized due to high expenses for fabrication high quality crystals. A CdTe crystal with a high resistivity The electric properties are affected by lattice defects forming energy levels in the band gapPositron annihilation spectroscopy (PAS) including Positron lifetime (LT) and Coincidence Doppler broadening (CDB) spectroscopy was usually used for the study of the structure of point defects in CdTe/CdZnTe in the past1314151617This work reports on an investigation of the influence of defined annealing in Cd or Te vapour on the defect structure of Cl-doped CdTe and Ge-doped The paper is organized as follows. In Section Experimental we introduce samples, show the impurity content and summarize technical details of experiments. In Section Theory, we introduce methods for lifetime calculations and discuss their limits. Section Results and Discussion presents galvanomagnetic, positron back-diffusion measurements and theoretical calculations of lifetimes of positrons captured in different point defects. The subsection Lifetime spectroscopy reports the results of temperature dependent positron lifetime spectroscopy and their analysis based on three-state simple trapping model. This subsection is divided to the three parts describing for the undoped CdTe, Cl-doped CdTe and Ge-doped CdZnTe. The next subsection contains the results of CDB spectroscopy and their interpretation.−1. Nominally undoped CdTe (u-CdTe) single crystal grown by the vertical gradient freeze method at fairly increased Cd overpressure 1.6 atm was used as a reference sample.The Cl-doped CdTe (CdTe:Cl) and Ge-doped 22Na activity of 1 MBq deposited on a 2 μm thick Ti foil was used as a positron source. The LT measurements were performed at 295 K (room temperature - RT) and at 123 K (low temperature), respectively. In both temperatures typically, 107 positron annihilation events were collected in LT spectra, which were subsequently decomposed into exponential components. The contribution of positron annihilations in the source spot and in the covering foils were always subtracted. In addition, temperature-dependent LT measurements were carried out in the temperature range of 123–295 K at a heating rate of ~1 K hour−1. Here the statistics of LT measurements were carried out using a digital spectrometerCDB spectroscopyS (sharpness) parameterS parameter on positron energy was fitted using the VEPFIT codePositron back diffusion measurement was performed on a continuous magnetically guided slow positron beam with energy of incident positrons adjustable in the range from 0.05 to 30 keV. Doppler broadening of the annihilation photopeak was measured by a HPGe detector with the relative efficiency of 35% and the energy resolution of 15 cm−3. A 30% error of GDMS analysis was declared.Glow Discharge Mass Spectrometry (GDMS) was used for the chemical analysis. The concentrations of elements exceeding Positron properties were calculated using density functional theory (DFT) within so-called standard schemewhere Positron wave functions i-th positron state. In the present calculations we considered the positron ground state only.where The positron annihilation rate (i.e. the inverse of positron lifetime) is determined using the expressionc is the speed of light, and γ denotes the electron enhancement factor describing the pileup of electrons at the positron sitewhere γ, were treated by two approaches:local density approximation (LDA) utilizing the parametrization by Boroński and Nieminenet al.generalized gradient approach (GGA) within the approach introduced by Barbiellini The electron-positrons correlation, i.e. the correlation potential The electron density The ATSUP-LDA and ATSUP-GGA calculations of positron parameters were performed on d electrons3236k-point grids generated using the Monkhorst-Pack schemea = 6.60 Å is in a reasonable agreement with the experimental value of 6.48 Å. Ionic relaxations were not considered at the present stage of calculations. In construction of the positron potential the frozen core electron orbitals were added to the self-consistent valence electron density calculated by VASP. In the following text this approach is denoted VASP-GGA.It is well known that LDA overestimates positron annihilation rates especially with i–th atom and a shell characterized by principal (n) and orbital (l) quantum numbers is calculated by the formulaThe momentum distribution of annihilating pairs was calculated employing the ATSUP-based approach described in refs where In order to mimic the effect of the finite resolution of the experimental setup, the theoretical momentum distribution curves were convoluted with a Gaussian with FWHM of VCd-related defects are suppressed and the electron density is afforded by residual noncompensated donors. The as-grown CdTe:Cl and CdZnTe:Ge samples exhibit rather high resistivity, which is typical for the growth of Te-rich materials, where excess donors dominating above shallow acceptors are compensated by VCd8The galvanomagnetic properties of as-grown u-CdTe, CdTe:Cl and CdZnTe:Ge and annealed CdTe:Cl and CdZnTe:Ge samples are summarized in n-type conductivity due to the suppressed VCd and dominating shallow donors ClTe. The free carrier concentration of ~1017 cm−3 therefore gives a lower limit for the concentration of Cl dopants in the samplep-type conductivity contrary to CdTe:Cl. Such a behaviour is well known in annealed undoped or weakly doped CdTe/CdZnTep-type conductivity in both samples. This is in agreement with expected VCd formation.Annealing in Cd vapour leads to a decrease in resistivity for both samples. CdTe:Cl exhibited Positron lifetimes, calculated using ATSUP-LDA, ATSUP-GGA and VASP-GGA approaches for various positron states in CdTe are listed in In order to compare positron lifetimes for various defects with the experimental values we use ratios While Positron lifetime was calculated also for C defect. One can see in the figure that positron density in AC becomes asymmetric since it expanded towards the Cl nearest neighbour.In Cl doped CdTe The results of LT measurements, i.e. lifetimes The u-CdTe crystal exhibits a single component LT spectrum (except of the source contribution) with lifetime of ~290 ps, which fits in the CdTe bulk lifetimes reported in literature, see S parameter on the energy E of incident positrons in plotted in S parameter approaches a plateau. One can see in Results of positron back-diffusion measurement, i.e. dependence of the 2s−1 which is in very good agreement with the value 2s−1 reported for CdTe by Neretina et al.The positron diffusion length determined in the CdTe region is 9 m−2 as determined by etch pitting technique. We performed an additional independent test whether positrons in u-CdTe crystal are annihilated in the free state and the lifetime of ~290 ps measured in this sample can be indeed considered as the bulk lifetime. The u-CdTe sample was mechanically grinded by F600 SiC paste with grain size 9 μm. Plastic deformation caused by grinding introduced dislocations into the sample. The grinded u-CdTe sample exhibits two-component LT spectrum with lifetimes The u-CdTe crystal exhibits very low density of dislocations which does not exceeds 10equals to the positron bulk lifetime, i.e. τ1), (ii) positrons trapped in shallow traps  corresponding to the three positron states: (i) free positrons  was used for analysis of the LT spectra measured at RT. The solution of 2-STM with the initial conditions whereThe LT spectrum Decomposition of LT spectrum of the as-grown CdTe:Cl measured at RT revealed two exponential components, see The concentration of vacancy clusters is directly proportional to the trapping rate et al.Extended investigations of CdTe:Cl with chlorine content from 100 to 3000 ppm performed by Krause-Rehberg A-centres −1. This value falls into the expected range for The analysis of the LT spectrum of the as-grown CdTe:Cl measured at 123 K revealed a new component with lifetime Note that Rydberg states are characterized by a lifetime which is close to the bulk lifetime54n-type conductivity, caused by Annealing the CdTe:Cl sample in Cd vapour led to a significant reduction of the intensity of the long-lived defect component and shortening of its lifetime down to Subsequent annealing of a CdTe:Cl sample in Te vapour leads to the appearance of a defect component with lifetime Referring to the concentration of defects , we can Similar estimation can be done also for the as-grown CdTe:Cl sample where the concentration of S-parameter on the energy E of incident positrons for the as-grown CdTe:Cl crystal is plotted in c] in the as-grown CdTe:Cl sample can be estimated from shortening of the positron diffusion length using the expression57The as-grown CdTe:Cl crystal was characterized also by positron back-diffusion measurement on the slow positron beam. The dependence of the v is the specific positron trapping rate to defects. From LT studies we know that the as-grown CdTe:Cl sample contains where At RT the CdZnTe:Ge as-grown crystal exhibits a defect component with lifetime Taking into account electrical neutrality of defects and positron lifetime comparable to Annealing of CdZnTe:Ge crystal in Cd vapour led to the disappearance of the defect component with lifetime The subsequent annealing of CdZnTe:Ge in Te vapour resulted in the restoration of the defect component with lifetime The CDB spectroscopy was employed in order to obtain direct information about chemical environment of The CDB results for CdZnTe:Ge samples are presented in In spite of extensive research, the basic properties of native point defects in CdTe/CdZnTe are still far from agreed understanding. Both formation and ionization energies are not settled and analyses based on various defect models or experimental data produce incompatible defect characteristics58Detailed characterization of defect structure of CdTe:Cl and CdZnTe:Ge samples summarized in Similarly, the intentional doping by Cl or Ge donors supplied the only extrinsic elements with the large enough concentration to arrange the detected density of revealed A-centres and complexes. Nevertheless, suggested interpretation of the defect structure of complexes does not represent the only possibility to create neutral or acceptor-type complex in CdTe and CdZnTe. An analogous defect might be created also without participation of extrinsic atoms taking only native point defects into account. That is the complex of Cd vacancy and Te anti-site defect A-centres. These shallow traps are able to confine positrons at low temperatures only when thermal de-trapping becomes sufficiently small. The lifetime of positrons localized in the shallow traps is shorter than the CdTe bulk lifetime. The as-grown CdZnTe:Ge crystal contains Point defects in CdTe:Cl and CdZnTe:Ge crystals have been characterized by LT and CDB spectroscopies combined with galvanomagnetic measurements. The as-grown CdTe:Cl crystal exhibits larger point defects with open volume comparable with several vacancies. The low temperature LT studies in CdTe:Cl have proved the existence of negatively-charged shallow traps interpreted as Rydberg states associated with Cl-related How to cite this article: Šedivý, L. et al. Positron annihilation spectroscopy of vacancy-related defects in CdTe:Cl and CdZnTe:Ge at different stoichiometry deviations. Sci. Rep.6, 20641; doi: 10.1038/srep20641 (2016).


How very long-term (over many years) motor skill training shapes internal motor representation remains poorly understood. We provide valuable evidence that the football brain of Neymar da Silva Santos Júnior  recruits very limited neural resources in the motor-cortical foot regions during foot movements. We scanned his brain activity with a 3-tesla functional magnetic resonance imaging (fMRI) while he rotated his right ankle at 1 Hz. We also scanned brain activity when three other age-controlled professional footballers, two top-athlete swimmers and one amateur footballer performed the identical task. A comparison was made between Neymar’s brain activity with that obtained from the others. We found activations in the left medial-wall foot motor regions during the foot movements consistently across all participants. However, the size and intensity of medial-wall activity was smaller in the four professional footballers than in the three other participants, despite no difference in amount of foot movement. Surprisingly, the reduced recruitment of medial-wall foot motor regions became apparent in Neymar. His medial-wall activity was smallest among all participants with absolutely no difference in amount of foot movement. Neymar may efficiently control given foot movements probably by largely conserving motor-cortical neural resources. We discuss this possibility in terms of over-years motor skill training effect, use-dependent plasticity, and efficient motor control. It is well-established that motor practice induces plastic changes in the human central motor system even in adults. For example, when people practice a novel hand motor skill, the central motor representation in the hand section of the primary motor cortex (executive locus of voluntary motor control: M1) expands when they repeat the practice for days or weeks  uptake widely in the forelimb section of M1 even when the monkeys perform the acquired motor skill  substantially reduces the recruitment of foot motor regions during foot movements. We scanned his brain activity with a 3-tesla fMRI while he rotated his right foot (ankle) at 1 Hz and compared his brain activity with that obtained from three age-controlled professional footballers, two top-athlete swimmers, and one amateur footballer. We assumed that professional footballers have trained to perform various types of foot movements, e.g., “manipulating” and kicking a ball in many different ways by controlling ankle joint, through their intensive over-years daily training. Even though top-athlete swimmers have also trained their foot movements, these are usually highly-patterned movements and less variety is required. Thus, it is very likely that professional footballers have been exposed to richer sensory-motor experiences of foot movements and their brains must have stored a variety of repertoire of foot motor skills as compared to swimmers and amateur footballers. We hypothesized that neuronal resource in motor-cortical foot regions allocated to control a given simple foot movement is smaller in professional footballers than in swimmers and amateur footballer, because previous studies have shown that musicians (pianists and keyboard players) who should have richer sensory-motor experiences and a variety of repertoire of hand/finger motor skills reduce recruitment of motor areas during finger movements compared with musically naïve control , two Spanish national-training-center level swimmers  and one amateur footballer . The following are the years of their football experience : 16 for Neymar, 12 for JS, 13 for SC, 19 for XB, and 9 for SH. No such football experience was reported by the swimmers. Except XB and YL, all the participants reported that they prefer to kick and dribble with their right foot. The local ethics committee of the ALOMAR hospital in Barcelona approved this study. All participants gave written informed consent, and the experiment was carried out based on the principles and guidelines of the Declaration of Helsinki (1975). The public disclosure of Neymar’s brain activity and the use of his name solely for academic purposes were approved by Neymar’s representative.2; flip angle, 90°; echo time, 35 ms). We collected functional volumes every 3 s (TR = 3000 ms) that comprised 48, 3.0-mm thickness slices without interslice gaps. The whole brain was within the field of view .A 3.0-T MRI scanner with a head-coil  provided T1-weighted anatomical images (MP-RAGE) and functional T2*-weighted echo-planar images . Thus, the participants completed four rightward runs and four leftward runs in each session. We collected 40 functional volumes (TR = 3 s) during the foot movements in each session. For Neymar, we conducted one extra (third) session where he generated much larger movements  by a digital video camera  that was located away from the end of the scanner bed (just outside the five gauss line) at approximately the same height as the bed. Thus, the movements were recorded from the view of the bottom of the foot. The bed’s width was also recorded for the calibration to quantify the foot movements (see below).We analyzed the fMRI data using Statistical Parametric Mapping software  that was implemented in Matlab 2007a . We excluded the first five functional images of each session from our analysis to allow for magnetization equilibrium. The remaining functional images were realigned to correct for head movements and co-registered to each participant’s anatomical image. Both the functional and anatomical images were normalized to the Montreal Neurological Institute (MNI) template brain using the standard SPM5 defaults. The functional images were smoothed with an isotropic 8-mm full-width at half-maximum Gaussian kernel. Finally, the value in each voxel was normalized by converting it into a percentage increase from the mean of the voxel in each session.After the preprocessing, we fitted a linear regression  model to the data obtained from each participant (three sessions for Neymar and two sessions for the others). We prepared a separate regressor for each session. In each regressor, 8 runs (4 rightward and 4 leftward) were modeled with boxcar functions convolved with the canonical hemodynamic response function in SPM5. The regressor specified each run period with a hemodynamic delay in each session. Since Neymar had three sessions, we prepared three regressors for him and two for the others. We also included the head motion parameters estimated in the realignment procedure as regressors in each session to minimize the effects of the head motion artifacts. Finally, in the analysis, we removed the slow signal drifts (frequency > 128 s).p < 0.001 (uncorrected). Since our main interest was the motor-cortical foot regions, we set the region-of-interest (ROI) in the medial-wall foot motor regions . ROI was defined based on our previous study where we identified the foot sections in the human medial-wall motor regions  when we evaluated its significance in terms of the spatial extent of activation within the ROI  across the participants and counted the number of active voxels . We also analyzed the data obtained from the third session for Neymar. We again consistently used the same voxel-wise threshold . After calibrating the sizes of the foot movements using the reference (bed width), we analyzed them in a two-dimensional plane perpendicular to the camera view. The location of the right big toe was plotted frame-by-frame (30 frames per second). Then we calculated the toe’s total movement distance in each run and separately averaged the movement distance for each session of each participant. This piece of data was used in the correlation analysis described above  across participants by calculating Spearman’s rank correlation coefficient.For the following statistical evaluation, we used statistical software . In the 2-session analysis, we performed a nonparametric correlation analysis between the year of football experience and the data  was eight for the pro footballers and six for the other participants, i.e., two values for each participant.In the single-session analysis, we performed a nonparametric Mann-Whitney test .Next, we examined whether the data obtained from a particular participant were significantly different from those obtained from the remaining participants . Here, All the participants successfully performed their foot movements in synchronization with the 1-Hz metronome sounds. The mean movement distances across two sessions for the professional footballers ranged from 3.1 to 5.3 m and those obtained from the other participants  ranged from 3.3 to 8.5 m. As we describe the details of statistics below, no significant difference was observed between the two groups in terms of movement distance .T-value in the medial-wall activity was also smallest in his brain . Negative correlation (r = −0.3) was also observed between the year and the maximum T-value, but this did not reach significant level.In this analysis, we depicted the general features of the individual brain activity during the foot movements and found that the movements activated the medial-wall motor regions in all the participants. The brain activity consistently formed the largest cluster in the entire brain across participants Figure . The numT-values had no clear relationship with the movement distance across participants , the number of active voxels was significantly smaller in the pro footballers than in the other participants . Neymar’s maximum T-values also showed a significantly smaller trend . This strongly indicates the consistency of our finding: smaller activity in Neymar’s medial-wall foot motor regions during foot movements. The additional individual analysis when we included his third session data showed that the number of active voxels and the maximum T-values were significantly smaller than those in the remaining six participants , with absolutely no difference in movement distance (p = 0.45). Thus, among all participants, the size of the medial-wall activity and its intensity were smallest in Neymar’s brain even though he generated comparable foot movements in size.Even when Neymar made larger movements in the extra third session  and that the number of active voxels and the maximum T-values in amateur SH were greater than those in the other participants . This means that the amateur footballer recruited the greatest medial-wall activity among all the participants . The relatively smaller number of participants and of experimental sessions and the lacking of complete non-athlete novice were the limitation of the current study. However, we found that, among all the present participants, the size of the medial-wall activity and its intensity were smallest in Neymar’s brain even though he generated comparable foot movements in size, which was consistently observed across experimental sessions Figures , 2. ThusThe relatively small size of the medial-wall activations during foot movements in professional footballers Figures , 2 seemsAmong the present professional footballers, Neymar’s medial-wall activation was the smallest both in size and strength Figures , 2. Togeper se.Anyhow, we speculate how Neymar’s brain controls his foot movements based on recent findings in non-human primates (Picard et al., Neymar may efficiently control foot movements by largely conserving motor-cortical neural resources probably with higher reproducibility and less effort. Ideally, a greater range of foot movements should be assessed to further substantiate this conclusion. It is intriguing to speculate that conserving motor-cortical resources when performing simple foot movement may in turn expand the possible control capacity for a wide range of football skills since the remaining resources can be assigned to control a variety of movements of lower extremities. We may also assume that this fundamental capability of his football brain could allow him to spend neural resources to focus more on cognitive aspects during a football game, such as anticipating/predicting and detecting the actions of other players (cf. Wright et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allergy to Apium graveolens is one of the more severe food allergies. Six celery allergens have been characterised, Api g 1-6, while additional allergens have characterised from other members of the Magnoliospida class, including kiwi, peanut and ragweed, suggesting homologous allergens may also be present in celery. The participants of the Omics Allergy School 2013 were provided with celery bulb and leaf extracts for two dimensional separation and immunoblotting.Two dimensional electrophoresis was used to separate the bulb and stalk protein extracts of two strains of celery. Allergenic proteins were detected using sera from a celery allergic patient.Hundreds of celery bulb and stalk proteins were separated using 2DE, using both silver stain and fluorescent labelling for increased sensitivity. The protein profile of two celery strains were distinctly different. A similar IgE binding pattern was observed to the bulb of the two strains of celery with 20-30 spots detected, while the binding pattern of the leaves of the two strains differed. Proteins have been identified by MALDI-ToF-MS/MS, with a de novo-sequencing approach used for protein identification due to limited protein sequences for celery. Post translational modifications of protein isoforms have been quantitatively labelled by H216O/ H218O-trypsin and subsequently analysed by nanoLC-ESI-MS/MS. PNGase treatment has been used for addressing protein glycosylation.Allergy School participants were able to contribute to the separation of celery extracts and to the identification of celery allergens.