FRB), length (FRL), and root area index (RAI) were significantly lower for spruce than for beech in mixed stands. Vertical profiles of fine root attributes adjusted to one unit of basal area (BA) exhibited partial root system stratification when central beech is growing with spruce competitors. In this constellation, beech was able to raise its specific root length (SRL) and therefore soil exploration efficiency in the subsoil, while increasing root biomass partitioning into deeper soil layers. According to relative values of fine root attributes (rFRA), asymmetric below-ground competition was observed favoring beech over spruce, in particular when central beech trees are admixed with spruce competitors. We conclude that beech fine rooting is facilitated in the presence of spruce by lowering competitive pressure compared to intraspecific competition whereas the competitive pressure for spruce is increased by beech admixture. Our findings underline the need of spatially differentiated approaches to assess interspecific competition below ground. Single-tree approaches and simulations of below-ground competition are required to focus rather on microsites populated by tree specimens as the basic spatial study area.Scientists are currently debating the effects of mixing tree species for the complementary resource acquisition in forest ecosystems. In four unmanaged old-growth spruce-beech forests in strict nature reserves in southern Sweden and northern Germany we assessed forest structure and fine rooting profiles and traits (≤2 mm) by fine root sampling and the analysis of fine root morphology and biomass. These studies were conducted in selected tree groups with four different interspecific competition perspectives: (1) spruce as a central tree, (2) spruce as competitor, (3) beech as a central tree, and (4) beech as competitor. Mean values of life fine root attributes like biomass ( Fagus sylvatica L.) and Norway spruce [Picea abies (L.) Karst], Pretzsch and Schütze  show evidence of species interaction and its effects on tree performance and stability. Several studies in managed mature beech-spruce mixtures in Germany and Austria found indications for vertical root system stratification  Karst.] or oak  expand its distribution range northwards  finding evidence for vertical root system stratification in the unmanaged spruce-beech forests, (2) quantifying effects of spruce-beech competition in different mixture constellations on fine root structural traits, and (3) evaluating the competitive status of both species below ground considering different levels of interspecific competitive pressure.Our present study includes a temperate near-natural forest within the high montane zone of the Harz Nationalpark (Northern Germany) with climate and site conditions comparable to the hemi-boreal forests of our other two study sites in Southern Sweden (see Table Picea abies (L.) Karst.] and European beech (Fagus sylvatica L.) are located in the boreo-nemoral zone  covered with moraine sediments whereas at the Harz site both metamorphic “Hornfels” and sedimentary “Grauwacke” sandstones prevail, partly covered by loess (eolic silt sediment). All sites have comparably low amounts of fine-textured soil and are riddled with boulders. Soil traits of all three sites are comparable with a moderate to thick accumulation of organic material 5–12 cm; the humus type is moder to raw humus  or raw humus (Siggaboda) with a high C/N ratio. Fine soil material is dominated by silt (40–65%) and sand (18–52%); the clay proportion is relatively low (8–17%) and the texture can be classified as sandy silt or silty sand, respectively. The soil type is a Haplic Podzol BGR,  with a hdbh), tree heights, and basal area (BA) .All three mixed stands are dominated by spruce with higher mean values of stem density, diameter at breast height (A) Table . The SweForest stand structures were recorded in a 1 ha square core plot (100 × 100 m) in the center of the semi-natural forest representing a typical section of the old-growth stand. We subdivided the plot using a 20 m-grid  of 7 cm and larger in winter 2004 at Siggaboda and in spring 2005 at the other sites. For each tree, species, cardinal location coordinates of the stem using above mentioned equipment and dbh with a girth tape were recorded. Subsequently, a tagging system  was used for temporarily (between 2004 and 2007) marking and numbering each measured tree.Within the entire 1 ha core plot, we assessed each tree with a A) spruce as the central tree, (B) spruce as competitors, (C) beech as the central tree, and (D) beech as competitors. This design was completed by monospecies groups with comparable intraspecific competitive status  that was only used as a reference for analyses of mixed species representation .We studied fine root structure of mature trees focusing on tree groups with one central tree with its mostly four–five competitors . Therefore, beech-spruce tree groups representing four different types of interspecific competitive situations were selected:  until the solid bedrock layer was reached. The maximum mineral soil depth sampled varied from 20 to about 40 cm. Various studies revealed that the maximum of fine roots is found in the top soil layers  and vitality . We applied morphological criteria for the identification of dead root material: dead root parts exhibits a dark discoloration of the central cylinder and a decreased flexibility of root segments . The reported measurements allowed the calculation of specific root length  and specific root surface area (SRA)  from the ratio of fine root length (FRL) and root biomass, and fine root surface area and rooting biomass, respectively.All recorded root fragments were then dried for 48 h at 40°C and weighed in order to measure living fine root biomass , and thus variable species abundance above-ground :The data was included in a relational database running on the open source database system PostgreSQL and analyzed with the software package Statistica 9  and SAS JMP 9 (SAS Institute Inc. 2010). Differences between mean root traits were assessed either by the Kolmogorov-Smirnov-two-sample test, or by the Kruskal-Wallis H test when more than two samples or groups were compared. We used the respective tree BA per hectare of either spruce or beech as a reference unit for the comparison of fine root attributes (FRA are the life fine root attributes in terms of FRB (g m−2), FRL (m m−2), and RAI (cm2 m−2) of a central tree (CT) or n competitors (COMP), respectively. BA (m2 ha−1) is the BA of either spruce or beech, and FRAad are fine root attributes adjusted to the same BA (1 m2 ha−1) of either one central tree or of n competitors .where CT) or competitor (COMP) constellations of spruce and beech were further assessed by calculating relative fine root attributes  in mixed groups with either CT or COMP constellations to those of pure stand groups (Equation 3). This enables the assessment of under- (<1) or overrepresentation (>1) of beech and spruce below ground growing in different mixed stand constellations under interspecific competition.The fine root representation of either central tree  attained higher adjusted fine root abundance  along the vertical rooting profile than the competitors  exhibited significantly higher fine root abundance in both the humus layer and deeper soil layers compared to beech competitors (COMP). This was not the case for spruce with a quite equal allocation of fine root proportion along the profile for both CT and COMP constellations. A spatial separation of the fine roots of spruce and beech was visible for the deeper soil horizons (10–40 cm) when growing with beech as the central tree  and spruce as competitors  resulting in high beech and low spruce fine root quantities. This partially vertical stratification was mainly due to high plasticity of beech fine rooting as a central tree. No stratification was visible in the organic layer and top soil where both species reached their maximum abundances.For both spruce and beech, central trees (SRL and specific fine root surface area (SRA) attained values for mixed beech (CT and COMP) of SRL 12.7 ± 5.5 m g−1 and of SRA 367.3 ± 125.9 cm2 g−1 compared to mixed spruce of SRL 6.6 ± 3.8 m g−1 and of SRA 253.6 ± 108.1 cm2 g-1 . However, total SRL and SRA differences between CT and COMP constellations within both species were not significant.Total means of specific SRL and SRA support the idea of higher plasticity of beech fine rooting . For spruce in contrast, we did not find such changes between central spruce trees (CT) and its competitors (COMP) rooting behavior. However, significant higher SRL and SRA values of CT constellations in the humus layer  and the top soil  points to higher competitive investments for rooting space sequestration in the upper soil horizons of single central spruce trees (CT), whereas single beech trees (CT) invested more in deeper soil horizons with less competitive abundance of spruce roots. Comparing beech and spruce as interspecific competitors (COMP), one can observe quite similar vertical rooting behavior , beside the above mentioned fact that the overall level of beech is much higher than that of spruce.The vertical profiles of g Figure . WhereasrFRA, cf. Equation 3) in mixed CT and COMP constellations  and COMP  constellations on interspecific tree species representation compared to the intraspecific one (Table rFRA). Two major findings were derived: (1) spruce is underrepresented in fine root abundance in mixed stands (mean rFRA < 1) whereas beech is overrepresented (mean rFRA > 1); (2) both beech overrepresentation and spruce underrepresentation address CT constellations with beech as the central tree (B). This indicates that beech rooting was favoured by an asymmetric interspecific competition of both species when several spruce competitors grew together with a single beech tree. But this was not the case for CT constellations with central spruce and beech competitors where we found a quite symmetric interspecific competition. Combining results from the existing mixtures (means of A + D) of central spruce (CT) and competing beech (COMP) or vice versa (means of B + C) one can assess fine root representation effects of the mixtures compared to either beech or spruce pure groups. It turned out that due to the low fine root representation of spruce no existing mixture reaches the high fine root representation of pure beech. However, both mixture constellations  lead to higher fine root representation compared to the pure spruce plots.For the analyses of fine root representation  of the mixed stand plots  in Sweden  is related to the remarkably low tree abundance above ground  compared to the Swedish plots. The positive relationship between tree dimension (BA) and soil exploration (FRB) on microsite and tree level has been demonstrated by Bolte and Villanueva  of central beech and competing spruce in deeper soil horizons  and mixed beech (12.7 m g−1) lie within the lower part of the range for spruce and beech (d < 2 mm) of 4.5–26 and 5.7–31.5 m g−1, respectively, reported by the extensive meta-analyses of Ostonen et al.  are similar to those reported for mixed spruce by Bolte and Villanueva  of beech does not mean that resource exploitation is more efficient compared to spruce  increased SRL in deeper soil layers. This indicates additional soil exploration activities at low carbon costs in soil depths from 20 cm downwards fitting well to the findings of changed root system partitioning favouring deeper soil horizons for increasing complementary resource exploitation and “underground niche separation”  adjusted to the same unit of above ground performance (1 m2 BA per hectare) with adjusted values for monospecies plots  is in line with other studies reporting an over-proportional reduction of fine root abundance in beech mixtures with spruce  surrounding a single beech tree (CT) have a low fine root representation possibly reflecting intensive above ground competition between central beech and spruce competitors leading to increased biomass partitioning toward aboveground tree compartments. Beech fine root representation on the other hand is favored by interspecific competition with spruce. The found morphological belowground plasticity (variation of rooting depth in CT constellation and of SRL/SRA) reflects the high crown plasticity (Dieler and Pretzsch, The relative fine root representation is a measure to compare mixed species abundance (SRL and with this soil exploration efficiency in the subsoils while increasing root biomass partitioning toward deeper soil layers. Moreover, asymmetric below-ground competition was observed favoring beech toward spruce in a mixed constellation with central beech. We conclude that beech fine rooting is facilitated in the presence of spruce by lowering competitive pressure compared to intraspecific competition whereas the competitive pressure for spruce is increased by beech admixture. This is most obvious when central beech trees are admixed with spruce competitors. Our findings underline the need of spatially differentiated approaches to assess interspecific competition below ground. Since tree competition is a process that affects tree at an individual scale, stand scale analyses should be complemented by single tree approaches, above and below ground. In line with the recent development of crown competition assessments and stand simulation methods (e.g., Pretzsch et al., The results of the presented study exhibited an only partially vertical root system stratification in the subsoil in the three spruce-beech old-growth stands and depended on specific mixture constellation: beech central tree with spruce competitors. In this mixture constellation, beech was able to raise 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.


Headache diagnostic software (HDS), developed by Headache center, Neurology Clinic in Belgrade integrates a diagnostic expert system able to suggest the correct ICHD-II diagnosis once all clinical characteristics of a patient’s headache have been collected.Purpose The aim of this pilot study was to test its diagnostic accuracy, the usefulness and applicability in the diagnosis of primary headache disorders and medication overuse headache (MOH).Forty patients with clinical diagnosis of migraine, tension type headache (TTH), cluster headache and MOH with an Internet access agreed to fill the web-based headache diary 4-6 weeks after their first consultation in Headache center. This diagnostic diary is developed exclusively based on the ICHD-II criteria, and represents the core of HDS. We analyzed the level of agreement between diagnosis given after clinical interview and obtained by HDS as well as patients’ level of compliance.Patients’ understanding of the diary proved highly satisfactory which was followed by good compliance (77.5%). Number of patients with one headache diagnosis was 22; 9 patients received 2 or 3 different diagnosis. All patients with a clinical diagnosis of migraine (21) had at least one migraine attack recognized by HDS; episodic TTH was less frequent diagnosed using standard clinical interview than by DHS. In all MOH (4) and cluster headache patients (3) there was complete concordance between clinical and software generated diagnosis.Web-based HDS can be considered as advanced, user-friendly, useful and reliable diagnostic tool in headache center.


Mycobacterium tuberculosis (MTB) strains is a worrisome trend seen during recent years. Rapid detection of MDR strains is very important to restrict their spread in the population. Gold standard methods for drug susceptibility testing (DST) of MTB are either costly or very slow. Nitrate reductase assay (NRA) is one of the methods for rapid detection of resistance. This technique is based on the capacity of M. tuberculosis to reduce nitrate to nitrite. The WHO recommends that the NRA be used as direct test on smear-positive sputum specimen or as an indirect test on Mycobacterium tuberculosis isolates grown from conventional solid cultures. We evaluated the performance of NRA as rapid, reliable & inexpensive method for drug-susceptibility testing of Mycobacterium tuberculosis against first line antitubercular drugs, Rifampicin (RIF) and Isoniazid (INH).Increase in multi-drug resistant (MDR) M. tuberculosis isolated from sputum samples of pulmonary tuberculosis patients were subjected to NRA and absolute concentration method for comparison.80 strains of Out of 80 isolates, 12 strains were resistant to INH & 11 strains were resistant to RIF and 9 strains are resistant to both INH & RIF constituting MDR strains. Sensitivities and specificities were 99%, 98% for RIF and 99%, and 100% for INH by NRA as compared to Absolute Concentration. However median time of obtaining results was shorter using NRA (9-10 days) compared to Absolute Concentration (30-40 days).Mycobacterium tuberculosis.We conclude that NRA has the potential to be a useful tool for rapid DST of


The complete funding information is: "This study was supported by Clinical Translational Science Institute (CTSI)(1UL1RR029893) seed fund to PL, Northwestern Memorial Hospital Dixon Translational Research Fund and Marsha Rivkin Center For Ovarian Cancer Research Award to JJW, and predoctoral fellowships from NYU CTSI TL1 (1UL1RR029893) and NYU Molecular Oncology and Immunology Training grant (T32 CA009161) to GD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."


Selecting the treatment of choice for multilevel cervical myelopathy remains a controversial issue. The most important factor in choosing a treatment method for patients with cervical myelopathy is the sagittal curvature and the site of compressive pathology.A total of 20 cases were available for the last 4 years with acceptable follow-up (f/u) of at least one year after surgery. Clinical examinations were obtained before surgery, 3 months post-surgery and at f/u. Evaluating the operative results performed by analyzing the recovery rate using the Japanese Orthopaedic Association (JOA) scoring system.Analysis were conducted on a total 20 patients undergoing surgery via anterior approach (9 patients), posterior approach with fixation (3 patients), and anterior. Posterior approach (8 patients). Two patients underwent surgery with hybrid decompression fixation technique. There were two patients who had decreased level of the JOA score which need posterior decompression. One patient had early onset post of infection in posterior approach.Our strategy base on naderi algorithm were effective and the follow up showed stable clinical results which depended on the completely removal of the possible causes of the pathology.Cervical spine, Multilevel, Myelopathy


To the Editor: Mediterranean spotted fever (MSF) is endemic in Italy, where it is a reportable disease. From 1992 to 1998, the Italian Ministry of Health was notified of approximately 8,500 cases of human rickettsioses presumed to be MSF. MSF occurs more commonly in some central (Lazio) and southern  regions  for ompA gene and restriction analysis under conditions previously described by Roux et al.  and aligned sequence data with homologous sequences of reference strains of the spotted fever group rickettsiae retrieved from the GenBank database. Sequence analysis showed 100% similarity with the homologous sequence of Israeli spotted fever Rickettsia reference strain ISTT CDC1 (GenBank accession no. U43797). The Israeli spotted fever Rickettsia belongs to the R. conorii complex .Molecular analysis of spotted fever group


Global evaluation of influenza vaccination in children indicates that current recommendations are not followed. Most children at high risk for influenza-related complications do not receive the vaccine, and increased efforts are needed to protect them. Furthermore, immunizing healthy infants 6–23 months of age and their close contacts should be strongly encouraged. Vaccinations are recommended for children with recurrent acute otitis media or recurrent respiratory tract infections and possibly for healthy daycare and school-age children because of the potential socioeconomic implications of influenza. Issues that need to be addressed include educating physicians and parents concerning influenza-related illness and complications, cost-effectiveness and safety of licensed vaccines, adequate vaccine supply, and availability of intranasal products. The most important of these obstacles are lack of understanding of the risks for complications and lack of knowledge of efficacy (Seven main obstacles to complying with recommendations for vaccination in children exist: 1) lack of understanding of the risk for influenza complications in children; 2) lack of knowledge of annual immunization’s efficacy in primary prevention; 3) parents’ negative reaction to parenteral vaccine administration (“Not another shot!”); 4) need for two priming doses in children <9 years old followed by annual administration; 5) fear of limited protection in younger and high-risk children; A number of studies of adult (particularly elderly) populations have shown that knowing risk factors for influenza complications, favorable perceptions of the vaccine, and clinician recommendations are the main variables predicting the administration of influenza vaccination  and require annual administration to maintain protection  were randomized to receive the vaccine (n = 67) or no vaccination (n = 66). During a 6-month period, 24 vaccine recipients (35.8%) experienced 32 episodes of AOM, and 42 control participants (63.6%) experienced 64 episodes. The overall efficacy of vaccination in preventing AOM was 43.7%  . Recurrent AOM is common in infants and children, and its possible sequelae make prevention desirable (= 0.002)  (13). Mo>6 episodes per year if >3 years; >8 episodes per year if <3 years) were randomized to receive the intranasal virosomal influenza vaccine (n = 64 with 176 household contacts) or a control placebo (n = 63 with 173 household contacts). During the influenza season, vaccinated children had fewer respiratory infections or febrile respiratory illnesses, received fewer prescribed antimicrobial and antipyretic drugs, and missed fewer school days than the controls (A second group of children who could be considered for influenza vaccine are those with recurrent episodes of respiratory tract infections (RRTIs). A large number of children without any immunologic problems experience multiple episodes of RRTIs during the first years of life; although these generally have a benign prognosis, they can cause substantial medical and socioeconomic problems  in 555 school-aged children in Russia demonstrated that both were efficacious in preventing school absenteeism by reducing the number of missed school days by 47% to 56% compared to missed school days in unvaccinated children . AlThe socioeconomic importance of influenza in childhood is confirmed by economic analyses showing that vaccinating healthy preschool and school-age children can lead to health and economic benefits during epidemic and pandemic periods , and the central role of children in disseminating the infection in households and communities. The issues that need to be addressed include educating physicians and parents about the illness caused by influenza, the cost-effectiveness and safety of licensed vaccines, adequate vaccine supplies, and the availability of intranasal products. Improved compliance associated with nasal administration should increase the use of influenza vaccination. Only a heightened and regular demand for influenza vaccine will result in sufficient vaccine supplies at all times (not just on a year-to-year basis) and place us in a better position to detect a novel pandemic influenza virus strain.


The recent article by Prud'homme et al  suggestsThe above examples clearly illustrate the role of neuropilin-1 in tumor progression in systemic malignancies and the urgent need to identify further inhibitors of neuropilin-1 expression.


Critically ill patients with inadequate lid closure are susceptible to developing exposure keratopathy. Protective measures can prevent this and reduce the risk of subsequent microbial keratitis and irreversible visual loss. Of two published surveys ,2, one wA team of researchers telephoned all general ICUs and other specialty critical care units in England caring for sedated, ventilated patients and asked a supervising nurse questions from a questionnaire piloted earlier in London.Two hundred and seventeen out of 267 ICUs (81%) participated. One hundred and thirty out of 217 (60%) ICUs had an eye care protocol. Sixty-six per cent of units with protocols assessed lid closure compared with 65% of those without. Geliperm application was the most common protective therapy , followed by Lacrilube . Most ICUs used a combination of methods. The total estimated incidence of ocular complications in the last year was 502. The most recent complications witnessed included corneal ulceration (23 cases), microbial keratitis (11) and chemosis (23). Cases of severe visual loss were caused by anterior ischaemic optic neuropathy following prone positioning (two cases) and microbial keratitis in a patient's only functioning eye.There is a need for protocols that encourage proper eyelid position assessment, effective protection of the ocular surface and referral to ophthalmologists in the event of any complications or any loss of corneal clarity.


Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients. The molecular chaperone Hsp90 (90 kDa heat-shock protein) is required for managing conformational development, stability and function of proteins in the cellular environment E coli homologue, HtpG Structural and biochemical studies have established Hsp90 as an ATP-dependent system that operates as a homodimer in a functional cycle associated with the ATP binding and hydrolysis Modern biophysical approaches including X-ray crystallography, electron microscopy and small-angle X-ray scattering (SAXS) have provided a more detailed characterization of the Hsp90 conformational states during progression through the ATPase cycle Computational approaches have been instrumental in revealing the atomic details of the inter-domain communication pathways which may regulate the conformational equilibrium of the molecular chaperone. We have recently reported the first computational study of the Hsp90 allosteric binding with an atomic level analysis of the conformational motions and the inter-domain communication pathways in the full-length yeast Hsp90 dimer in the apo form and complexes with ATP and ADP Mechanistic and thermodynamic models of allosteric communication have become widely accepted and broadly applied to analyze mechanisms of allosteric regulation in proteins and biological networks Structure-functional studies of Hsp90 regulation have proposed a number of functional motifs that may contribute to the regulation of Hsp90 activity, including ATP hydrolysis and allosteric interactions. Nevertheless, molecular mechanisms that control the relationship between conformational diversity and biological functions of Hsp90 lack a detailed quantitative characterization. In this work, we integrated molecular dynamics (MD) simulations and modeling of the principal collective motions with the energy landscape models and structure-functional analyses of regulatory interactions to characterize functional dynamics of the Hsp90 molecular chaperones and formulate a unifying theoretical framework that may adequately describe and rationalize molecular principles of Hsp90 dynamics and function.We begin by providing a road-map through the manuscript and formulating main questions, computational experiments conducted and hypotheses tested. In the present work, we integrated all-atom MD simulations with modeling of principal correlated motions, the energy landscape analysis and structure-functional characterization of regulatory interactions to investigate functional dynamics and mechanisms of the Hsp90 molecular chaperones. The following specific objectives were pursued in this study: (a) to quantify molecular mechanisms by which functional dynamics and allosteric motions of Hsp90 may be connected with the probabilistic nature of the underlying energy landscape; (b) to identify and characterize functional hotspots shared by the Hsp90 chaperones that could coordinate functional dynamics and “conformational selection” of the functional states. We proposed that conformational dynamics and functional motions of Hsp90 may be described using the energy landscape models and principles of “conformational selection” (or “population-shift”) between preexisting conformational ensembles In the first section, we discussed the results of MD simulations performed for all structural forms of Hsp90. A comparative analysis of the conformational ensembles highlighted fundamental aspects of Hsp90 dynamics that may be exploited by the chaperone to accommodate functional requirements during the ATPase cycle. In the second section we characterized functional dynamics and principal collective motions of the molecular chaperone. This analysis demonstrated that a central evolutionary conserved characteristic of Hsp90 dynamics is the presence of long-range inter-domain motions that enable allosteric signaling. We concluded that allosteric interactions may provide a mechanism for structural adaptation of Hsp90 by shifting the dynamic equilibrium between the ensembles of functional states. A switch-based conformational change is a hallmark of complex biomolecular systems that function via allosteric signaling and are often regulated by a relatively small number of functional hot spots , , The crystal structures of the full-length Hsp90 dimer from three different species revealed a similar domain architecture, yet significant differences in the global conformational flexibility and the relative domain orientation Figure 1Figure S1) that may serve as stabilizing “handlers” in assisting the central “strap” of hydrophobic residues (residues 755–765) to ensure structural integrity of the M-domain and CTD  of MD trajectories. The structural distribution of the protein mobility and the cross-correlation maps of protein residue fluctuations were computed along the dominant principal component (PC) modes. This analysis identified the shapes of the low frequency modes and the protein regions subjected to correlated and/or anti-correlated motions along the selected low frequency modes.Sstructural mapping of the HtpG mobility projected along the low frequency modes revealed a heterogeneous and diffuse network of structurally stable regions distributed across all domains of the extended HtpG form , the catalytic loop in the M-domain (residues 332–342) and the adjacent H10 helix (residues 343–366) are among functional regions that become protected upon ATP binding The full-length mammalian Grp94 in complex with either ATP or ADP has a semi-open, V-shaped conformation of the dimer , , , The ATP-bound yeast Hsp90 is characterized by structurally constrained conformation, stabilized by a dense network of both intra-monomer and inter-monomer interactions , , , 8These findings are in accordance with insights by Wolynes and coworkers Within the energy landscape framework, long-range cooperativity of allosteric interactions during allosteric transitions may favor a combination of the population-shift and induced-fit mechanisms Allosteric signaling is often enabled by a relatively small number of evolutionary conserved and sparsely connected regulatory hotspots. In this section, we describe a computational approach to identify and characterize a network of conserved functional motifs responsible for regulation of Hsp90 dynamics. We propose that functional motifs coordinating functional dynamics and collective motions of the Hsp90 chaperone may serve as central regulators of Hsp90 activity. Structurally rigid and conformationally flexible residues may often occupy proximal positions to the hinge sites, since the regions of high and low structural stabilities could participate in binding sites We focus on the low-frequency functional motions represented by the normalized mean square fluctuation (NMSF) of the Hsp90 chaperone residues, along the two lowest frequency modes. These profiles can reflect salient features of the intrinsic protein dynamics , , According to this analysis, the NMSF profiles along the two slowest modes of HtpG motions confirmed the presence of anti-correlated motions between the NTD and CTD in the open apo-HtpG form , 10. TheInterestingly, this charged linker in Grp94 (residues 287–327) was deleted in the protein construct and replaced by four glycine residues in the crystal structures In the framework of the energy landscape model, this inter-domain region may facilitate cooperative interactions and efficient allosteric communication networks in HtpG. The alterations in this region observed in Grp94 may introduce a degree of inter-domain frustration that would detrimentally affect allosteric interaction networks. These findings could rationalize several important biochemical experiments REILQD-342 in HtpG . When Hsp90 dimer forms an initial complex with two Cdc37 molecules Figure S2). According to our mechanistic model, the binding sites of these motifs may recognize the “incoming” co-chaperone molecules, and the Cdc37 dimer would then bind the two linker regions. The regulatory elements of these motifs may then “pass” the binding signal information via allosteric communication pathways and trigger switching of the thermodynamic equilibrium towards a more open and flexible ADP-bound state of Hsp90 suitable for recognition of protein kinase clients.Hence, allosterically regulated conformational equilibrium of Hsp90 and co-chaperone binding are closely associated and may be modulated via the stochastic nature of the underlying energy landscape. Based on our findings and agreement with structure-functional experiments , IV-539 containing a pivotal switch point Ile-538 in HtpG  emerged as a central element of the CTD regulatory motif involved in coordinating the inter-domain communications. The evidence of an efficient molecular communication between NTD and CTD regions at the interface is of special importance given a rapidly growing interest in developing novel and specific Hsp90 inhibitors targeting allosteric C-terminal nucleotide binding site Hence, we identified universal functional motifs shared by the Hsp90 chaperones in different species that could play a universal role in coordinating functional dynamics and principal collective motions of Hsp90. These motifs may also serve as central functional regulators of Hsp90 activity, including control of ATP hydrolysis, the inter-domain communications and protein client binding. We observed that recognition sites may correspond to the conformationally mobile regions along slow modes of principal motions. Moreover, the recognition sites are often strategically positioned close to the regulatory anchor motifs containing catalytic and/or hinge residues. According to our results, functional coupling of structurally stable and conformationally mobile elements in the regulatory motifs is what may enable allosteric signaling and recognition of co-chaperones and protein clients. The central result of this study is that a small number of functional motifs may be utilized by the chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, co-chaperone binding and protein client recognition. Mutations, drug targeting, and probing protein binding in these regions would allow furthering pinpointing specific residues responsible for various functions including protein binding.It becomes more apparent now that functional dynamics of Hsp90 is closely associated with the ability of the molecular chaperone to bind and control activity of numerous protein clients. During client binding, the Hsp90 chaperone can adopt equilibrium between structurally different states that is maintained even after substrate loading In this work, we identified and characterized evolutionary and functionally conserved elements of the Hsp90 chaperone that may serve as key regulators of collective motions and hubs of long-range communication networks. We propose that functional clusters corresponding to stable regions in principal collective motions define minimally frustrated anchor sites (“hot spots”). These minimally frustrated residue clusters with optimized local interactions may be critical for regulation of allosteric signalling between distant protein regions. The proximal recognition sites (“soft spots”) may be functionally coupled to the anchoring regulatory residues and form networks of communicating interaction clusters. These “soft spots” may be formed by locally frustrated residues, whose interaction networks are energetically optimized and prone to dynamic modulation by mutations or protein client binding. Hence, allosteric signaling may involve a dynamic coupling between structurally rigid  and plastic  clusters of residues. A switch in state at a soft cluster upon protein client binding can shift the thermodynamic balance and activate a specific network of allosterically coupled residues. This model is consistent with the central role of regulatory nodes as “discrete breathers” or independent dynamic segments in allosteric signaling Overall, the proposed mechanistic picture of Hsp90 function is consistent with studies showing that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected groups of residues, suggesting that rapid transmission of allosteric signals through a small network of distantly connected residue clusters may be a universal requirement encoded in the molecular chaperones and possibly other macromolecular assemblies. Our analysis has also confirmed that Hsp90 homologues sharing the same fold architecture may exhibit similar dynamical characteristics, including conservation of principal collective motions. The results support a mechanistic hypothesis according to which that the interaction networks regulating collective motions of the Hsp90 chaperone may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of Hsp90 functional states is likely to be readily modulated by co-chaperones and protein binding partners.Targeting allosteric binding sites and interrupting protein client binding with Hsp90 can produce a broad-spectrum antitumor activity and a low risk of drug resistance due to a combinatorial blockade of multiple signaling pathways. While there has been a steady progress in structural studies of the Hsp90-NTD complexes with small molecules, much less is known about the molecular basis of allosteric Hsp90 binding with co-chaperones and protein clients. A second nucleotide-binding domain has been reported to exist in the C-terminus of Hsp90, and specific inhibitors to this domain are represented by novobiocin and the related coumarin antibiotics The results of our study could guide design of experiments for probing allosteric binding of celastrol by introducing mutations in the communicating residues from the regulatory and recognition motifs of Hsp90. Integration of computational and experimental approaches may thus facilitate experimentally-guided mapping of allosteric binding states and help to specify whether Hsp90 or its co-chaperone Cdc37 is the primary target for binding of celastrol. Recent evidence suggests that Hsp90 interacts and stabilizes a growing list of protein kinases, and Cdc37 serves as the key co-chaperone for a large portion of the yeast kinome +) and chloride (Cl−) counter-ions were added to achieve charge neutrality in MD simulations at physiological concentration of 0.15 mol/L. All Na+ and Cl− ions were placed at least 8 Å away from any protein atoms and from each other. The system was subjected to initial minimization for 20,000 steps (40 ps) keeping protein backbone fixed which was followed by 20,000 steps (40 ps) of minimization without any constraints. Equilibration was done in steps by gradually increasing the system temperature in steps of 20 K starting from 10 K until 310 K and at each step 15000 steps (30 ps) equilibration was run keeping a restraint of 10 Kcal mol−1 Å−2 on protein alpha carbons (Cα). Thereafter the system was equilibrated for 150,000 steps (300 ps) at 310 K (NVT) and then for further 150,000 steps (300 ps) at 310 K using Langevin piston (NPT) to achieve uniform pressure. Finally the restrains were removed and the system was equilibrated for 500,000 steps (1 ns) to prepare the system for simulation. An NPT simulation was run on the equilibrated structure for 20 ns keeping the temp at 310 K and pressure at 1 bar using Langevin piston coupling algorithm. The integration time step of the simulations was set to 2.0 fs, the SHAKE algorithm was used to constrain the lengths of all chemical bonds involving hydrogen atoms at their equilibrium values and the water geometry was restrained rigid by using the SETTLE algorithm. Nonbonded van der Waals interactions were treated by using a switching function at 10 Å and reaching zero at a distance of 12 Å. The particle-mesh Ewald algorithm (PME) as implied in NAMD was used to handle long range electrostatic forces.MD simulations of the Hsp90 structures (each of 20 ns duration) were performed for a closed ATP-bound conformation of yeast Hsp90  (PDB ID 2CG9) α atoms) i and j was calculated for each of the 20 ns MD simulation trajectories: snapshots from trajectories were taken every 200 ps, overall translation and rotation were removed, and only Cα was kept for analysis.Protein flexibility was analyzed by combining the results of MD simulations with the principal component analysis of conformational ensembles To obtain collective motion coordinates that represent the overall dynamics of each trajectory, PCA was performed, in which the covariance matrix Figure S1Time-Dependent History of High Occupancy Salt Bridges. Thermal fluctuations of the salt bridges in Grp94 (A) and yeast Hsp90 (B). (A) The depicted high occupancy hydrogen bond interactions in the M-domain and CTD of Grp94 are Glu564-Lys530 (in blue), Asp573-Lys493 (in red), and Glu548-Arg660 (in green). (B) The depicted high occupancy hydrogen bond interactions in yeast Hsp90 are Glu165-Arg174 , Glu355-Lys335 , and Asp506-Lys426 .(TIF)Click here for additional data file.Figure S2Allosteric Coupling of the Regulatory and Recognition Sites in the Hsp90 Chaperone. The coordinated involvement of the inter-domain charged linker, the catalytic functional loop from the M-domain and the projecting recognition loops are shown in blue spheres (only main chain is shown). The ADP-bound Hsp90 crystal structure (PDB ID 2IOP) was used for clarity of illustration. The protein structure is shown in ribbons colored according to secondary structures.(TIF)Click here for additional data file.


Elevated intracranial pressure (ICP) episodes are associated with poor outcome and should be prevented. We developed models to predict these episodes 30 minutes in advance, and to predict long-term neurological outcome by using dynamic characteristics of continuous ICP and mean arterial pressure (MAP) monitoring.The Brain-IT  dataset Predictions of elevated ICP episodes Figure  were extDynamic data in continuous MAP and ICP monitoring allows prediction of elevated ICP. Adding information of the first 24 hours of ICP and MAP to known risk factors allows accurate prediction of neurological outcome at 6 months.


Whereas the use of discrete pitch intervals is characteristic of most musical traditions, the size of the intervals and the way in which they are used is culturally specific. Here we examine the hypothesis that these differences arise because of a link between the tonal characteristics of a culture's music and its speech. We tested this idea by comparing pitch intervals in the traditional music of three tone language cultures  and three non-tone language cultures  with pitch intervals between voiced speech segments. Changes in pitch direction occur more frequently and pitch intervals are larger in the music of tone compared to non-tone language cultures. More frequent changes in pitch direction and larger pitch intervals are also apparent in the speech of tone compared to non-tone language cultures. These observations suggest that the different tonal preferences apparent in music across cultures are closely related to the differences in the tonal characteristics of voiced speech. Tonal differences between traditional Eastern and Western music are readily heard. Explanations often refer to the use of different scales The use of pitch in speech varies greatly among languages. The most obvious example is the broad division of languages into “tone” and “non-tone” groups Based on these differences, we asked how, if at all, differences in the use of pitch in the traditional music of tone and non-tone language speaking cultures compares with the use of pitch in speech. To address this question, we compiled databases of speech and music from several tone and non-tone language cultures. The analysis focused on two aspects of pitch dynamics: the frequency of changes in pitch direction , and the size of the pitch intervals used. These aspects were chosen because they differentiate tone and non-tone language speech and play a central role in the structure of musical melodies U = 9843.5, P<0.001; U = 2317, P<0.001; t = -11.4, P<0.001), whereas intervals equal to or larger than a major second occur more often . The only exception in this overall pattern is major thirds (400 cents), which are more frequent in the music of non-tone language cultures (7.7% vs. 2.3%). t = −5.6, P<0.001), whereas larger intervals occur more often .  and three that use non-tone languages . The patterns apparent in music parallel those in speech. Thus the music of tone language cultures changes pitch direction more frequently and employs larger melodic intervals. Similarly, the speech of tone language cultures changes pitch direction more frequently and employs larger prosodic intervals. Presumably tone language speech exhibits these characteristics because the lexical meaning of each syllable in a tone language is tonally determined The only exception to this pattern is the use of major thirds. Despite the greater frequency of larger prosodic intervals (≥200 cents) in the speech of tone compared to non-tone languages, there are fewer melodic major thirds (400 cents) in the music of tone language cultures see . A possiIn sum, co-variation of tonal characteristics in the music and speech of the tone and non-tone language speaking cultures we examined indicates an intimate relationship between these two modes of social communication, providing a way of explaining at least some aesthetic preferences in biological terms.Monophonic folk melodies from tone and non-tone language speaking cultures, all of which could be either played on an instrument or sung, were obtained from scores and MIDI format files obtained principally from National University of Singapore and the Singapore National Library Board. The tone language database comprised 50 traditional Mandarin, 20 traditional Thai, and 20 traditional Vietnamese melodies. The non-tone language database comprised 50 traditional American, 20 traditional French, and 20 traditional German melodies. To mitigate cross-cultural contamination by modern media, all compositions pre-dated 1900, often by hundreds of years.Tone and non-tone speech samples were acquired by recording monologues read by native speakers of each of the 6 relevant languages . Each speaker read 5 emotionally neutral monologues translated into the appropriate language . Prior tThe analysis of music focused on melodic slope reversals, and melodic interval size. Melodic slope reversals were defined as any change in the direction of the pitch contour of a melody. For each melody, the number of local minima and maxima was tabulated and divided by the total number of notes; this value was multiplied by 100 to give the incidence of melodic slope reversals per 100 notes. Melodic interval size was defined as the pitch difference (in cents) between adjacent notes see . The analysis of speech focused on two aspects of pitch dynamics analogous to those examined in music: prosodic slope reversals, and prosodic interval size. Prosodic slope reversals were defined as any change in the direction of the simplified pitch contour of a speech recording Click here for additional data file.Figure S2Music analysis. (A) Musical notation of the traditional American melody “Home on the range”. (B) The same melody reformatted for analysis. Notes are represented by black bars with open circles marking their beginnings and endings. The length of each bar is proportional to the duration of the note. Local maxima (max) and minima (min) indicate slope reversals in the melodic pitch contour; melodic interval size (i) is the vertical distance between successive notes. Dashed lines have been added to aid visualization of the melodic pitch contour.(TIF)Click here for additional data file.Figure S3Speech analysis. (A) Recording of the sentence “I applied for a job that would give me a good work experience” spoken in American English (B) Fundamental frequency (F0) of the recording in (A) over time . (C) F0s in Panel (B) segmented into syllables with simplified contours. Syllables are represented by black bars with open circles marking their beginnings and endings. (D) Output of the Prosogram analysis. Local maxima (max) and minima (min) indicate slope reversals in the prosodic pitch contour; prosodic interval size (i) is the vertical distance between successive syllables. Dashed lines have been added to aid visualization of the prosodic pitch contour.(TIF)Click here for additional data file.Figure S4Interval size in the music and speech of tone and non-tone language speaking cultures sorted into descending and ascending. (A) The distributions of descending (left panel) and ascending (right panel) melodic interval sizes per melody in the tone (red) and non-tone (blue) language music databases. Unisons (0 cents) are shown in both panels. (B) The distributions of descending (left panel) and ascending (right panel) prosodic interval sizes per speaker in the tone (red) and non-tone (blue) language speech databases. The left panel inset shows the percentages of large (≥200 cents) vs. small (<200 cents) descending prosodic intervals . The right panel inset shows the percentages of small (<200 cents) vs. large (≥200 cents) ascending prosodic intervals .(TIF)Click here for additional data file.Figure S5Slope reversals in the music and speech of tone and non-tone language speaking cultures sorted by the individual languages examined. (A) Box plot showing the distribution of the number of melodic slope reversals per melody  in the Mandarin, Thai, and Vietnamese melodies (red), and in the English, French, and German melodies (blue). Horizontal yellow lines indicate medians; colored boxes specify inter-quartile ranges and dashed lines the ranges without outliers. (B) Box plot of the number of prosodic slope reversals per speaker  in the Mandarin, Thai, and Vietnamese speech (red) and English, French, and German speech (blue). Format is the same as in (A). Crosses indicate outliers (defined as greater/lesser than 1.5x the inter-quartile range). See (TIF)Click here for additional data file.Figure S6Interval size in the music and speech of tone and non-tone language speaking cultures sorted by the individual languages examined. (A) The distribution of absolute melodic interval sizes per melody in Mandarin, Thai, and Vietnamese melodies (red), and in the English, French, and German melodies (blue). (B) The distributions of absolute prosodic interval sizes per speaker in the Mandarin, Thai, and Vietnamese speakers (red), and the English, French, and German speakers (blue). See (TIF)Click here for additional data file.Figure S7Comparison of melodic intervals arising from pentatonic vs. heptatonic scale structure. (A) Illustration of heptatonic and pentatonic scales on piano keyboards . The pattern within a single octave repeats in each octave. (B) Histogram of the percentages of all possible intervals ≤ 500 cents arising from analysis of the scales in (A). Red bars represent pentatonic scale percentages, and blue bars heptatonic scale percentages.(TIF)Click here for additional data file.Table S1Statistics comparing the number of slope reversals in tone and non-tone language music and speech databases for each possible pair of the cultures examined. (A) Statistics for melodic slope reversals in music. (B) Statistics for prosodic slope reversals in speech; n1 and n2 refer to the sample sizes of group 1 and group 2. (DOC)Click here for additional data file.Table S2Statistics for the comparisons of interval size distributions in the tone and non-tone language music and speech databases for each possible pair of the cultures examined. (A) Statistics for melodic interval size distributions. (B) Statistics for prosodic interval size distributions; n1 and n2 refer to the sample sizes of groups 1 and 2. (DOC)Click here for additional data file.Text S1Supporting Methods and Results. This file gives an overview of the methods and results used in this manuscript.(DOC)Click here for additional data file.


In patients with diabetes mellitus, coronary artery disease (CAD) is a major cause of mortality and results in substantial morbidity. Non-invasive detection of CAD in diabetic patients is challenged by silent ischemia and microvascular disease. Pharmacological stress CMR perfusion imaging (CMRPI) may identify evidence of flow limiting CAD at high resolution and tissue contrast. We therefore sought to test the hypothesis that stress CMRPI can provide robust prognostication to identify diabetic patients at high risk for major adverse cardiac events (MACE).Stress CMRPI was performed on 138 consecutive patients with diabetes  referred for assessment of ischemia. Rest and vasodilator stress CMRPI were performed each using a bolus of 0.1mmol/Kg of gadolinium, followed by late gadolinium enhancement (LGE) and cine imaging. Myocardial ischemia was defined by the presence of a stress induced perfusion defect, reversible at rest, in the absence of LGE in the same territory. At a median follow up of 21 months (IQR 30 months), 21 MACE (15%) had occurred (14 cardiac deaths and 7 AMIs).By univariable analysis, the presence of myocardial ischemia by CMRPI portended to 3-fold increase in MACE hazards . The absence of myocardial ischemia and LGE by CMRPI indicated a low annual rate of cardiac death (0.9%, Figure Stress CMRPI provides robust prognostication for major cardiac events in patients with diabetes. While a CMR study negative in both myocardial ischemia and LGE indentifies diabetic patients with lower event rates for MACE, the presence of myocardial ischemia by CMRPI indentifies a subgroup of diabetic patients at high risk for cardiac events.


Xenotropic murine leukemia virus-related virus (XMRV) infects different human cells in vitro. In this study the pathogenic effect of XMRV in lymphoid tissue was investigated by infection of human tonsil histocultures.Human tonsils were obtained from healthy donors, dissected into small blocks and cultured on top of gelfoams. Tissue blocks were left uninfected or infected with an XMRV stock obtained from a 22Rv1 cell culture supernatant in the presence or the absence of AZT or Raltegravir. Viral infection was evaluated by PCR at different times in the cells migrating out the tissue and at day 14 in tissue cells. In addition, tissue cells were immunophenotyped and the expression of XMRV envelope protein was analyzed by western blot.Cells migrating out the tissue and tissue cells were positive for XMRV and both AZT and Raltegravir blocked the detection of viral DNA. Despite the presence of XMRV DNA, tissue lysates exhibited undetectable expression of XMRV env proteins by WB. XMRV infection did not modify the percentage of CD3 , CD4 (47 vs 48), CD8 (20 vs 19), or CD19 cells (29 vs 27) neither the naive/memory cell ratio, nor immune activation markers, as evaluated by the expression of HLA-DR and CD38.XMRV could specifically infect human lymphoid tissue cells although this process does not culminate in an explicit productive infection. This infection did not result in changes of T or B cells nor in immune activation, suggesting that lymphoid tissue could latently support XMRV infection.


Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins.This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome.This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development. The antigen receptor loci of B and T lymphocytes exhibit a unique mechanism of control amongst the genes of multicellular organisms. The production of functional immunoglobulin (Ig) and T cell receptor (TCR) genes is accomplished through a tightly regulated process of recombination. Variable (V), diversity (D) and joining (J) gene segments of antigen receptor loci are assembled into a functional coding unit by a series of site-specific recombination events mediated by the products of recombination activating gene (RAG)1 and RAG2 [Escherichia coli [Assays of V(D)J recombination have relied extensively upon the transfection of extrachromosomal plasmid substrates into RAG-expressing cell lines and the recovery of these plasmids in hia coli -6. Many hia coli -5. This hia coli -9, or rehia coli -12. Furthia coli . Here, wDiscosoma and enhanced green fluorescent protein (EGFP). The system was designed such that unrearranged substrates would express the DsRed gene, whereas substrates that had undergone RAG-mediated recombination would replace DsRed expression with EGFP expression. In one construct, the EGFP open reading frame was placed on the opposite strand to DsRed and the RSS motifs arranged such that the segment containing DsRed and EGFP would be inverted by RAG-mediated recombination J recombination can occur either by deletion of the DNA between RSS motifs or by inversion of the intervening segment Figure . We geneThe inversion and deletion constructs were stably transfected into two cell lines representing different developmental stages of the B cell lineage. The cell line 300-19P is a pre-B cell line, expresses RAG1 and RAG2 and is known to catalyse V(D)J recombination, whereas WEHI231 is a B cell line in which RAG gene expression has been silenced and is therefore unable perform V(D)J recombination. The expression of RAG1 and RAG2 in these two cell lines was confirmed using reverse transcription polymerase chain reaction (RT-PCR) Figure . Pools oThese transfectants were then analysed for expression of the DsRed and EGFP reporter genes at the mRNA level. RT-PCR analysis established that expression of the EGFP gene was restricted to the RAG-expressing cell line and that EGFP transcripts could be detected in cells transfected with both the inversion and deletion construct Figure . A largeOur aim was to identify V(D)J recombination at the single cell level. We therefore analysed expression of the DsRed and EGFP molecules by fluorescence. Fluorescence microscopy of the stably transfected RAG negative WEHI231 and RAG positive 300-19P cells indicated that expression of EGFP was restricted to the 300-19P cells. Furthermore, EGFP expression was clearly detectable in both the deletion and inversion transfectants Figure . ExpressThe process of V(D)J recombination has evolved to generate a repertoire of antigen receptors. Large repertoires are generated by both combinatorial diversity and by the imprecise nature of the recombination process whereby the location of the double strand break is not fixed and nucleotides can be added or deleted from the free DNA end, generating extensive diversity in complementarity determining region (CDR)3 of the antigen receptor -16. Clonin situ in the absence of selection. Previously, assays of V(D)J activity have relied heavily upon extrachromosomal recombination substrates that are transiently transfected into cells before recovery and transformation into E. coli [In the system described here, the use of the twin fluorescent protein system distinguishes individual cells that have recombined a V(D)J recombination substrate from those which carry the substrate in a configuration representing germline gene segments. The ability to assay the absence of recombination is important when testing the effects of mutations or local chromatin environment on the recombination process. Importantly, the twin fluorescent protein system allows recombination to be detected  E. coli -6. Chrom E. coli ,18 and fcis-acting sequences (RSS12 + RSS23) need to be provided to ensure correct expression of the exogenous gene (unpublished results). The ability to switch gene expression from one gene to another by harnessing endogenous recombinase activity may have practical applications in the B and T cell lineage analogous to the use of the Cre/Lox system or other exogenous recombinases [Interestingly, the twin fluorescent protein system also indicates that V(D)J recombination could be used to control the expression of exogenous genes. Just 67 bp of mbinases .The inversion and deletion plasmids  for selection.Murine B cell lineage cell lines (300-19P and WEHI231) were maintained in RPMI medium supplemented with 10% foetal calf serum and 50 μM 2-mercaptoethanol. The recombination substrates were transfected into pre-B (300-19P) and B (WEHI231) cell lines by electroporation. Approximately 20 μg of linearised DNA were electroporated  into 10DNA and RNA were isolated from 10 ml aliquots of culture using Genelute kits  as per the manufacturers' protocols. The optical density was measured at 260 nm and the concentration of the nucleic acid calculated; 500 ng of genomic DNA was used as template in a PCR reaction. Recombination events were detected by PCR using appropriate primers  using the FL1 channel of a FACS Calibur flow cytometer . The mean fluorescence intensity of EGFP expression was compared between transfected and non-transfected cells and the data expressed as a fold increase in fluorescence with non-transfected cells assigned an arbitrary value of 1.The authors declare that they have no competing interests.GPC designed the overall strategy, GPC and GBS designed the constructs, GBS performed the experiments, EADW assisted with flow cytometry and GPC and GBS wrote the paper.


MyoG and Myf6) than the genes encoding myogenic determination factors (Myf5 and MyoD). Additionally, the functional divergence within the myogenic determination factors or differentiation factors was also under positive selection pressure. The positive selection sites were more frequently detected from MyoG and MyoD than Myf6 and Myf5, respectively. Amino acid residues under positive selection were identified mainly in their transcription activation domains and on the surface of protein three-dimensional structures. These data suggest that the functional gain and divergence of myogenic regulatory factors were driven by distinct positive selection of their transcription activation domains, whereas the function of the DNA binding domains was conserved in evolution. Our study evaluated the mechanism of functional divergence of the transcriptional regulation factors within a family, whereby the functions of their transcription activation domains diverged under positive selection during evolution.The functional divergence of transcriptional factors is critical in the evolution of transcriptional regulation. However, the mechanism of functional divergence among these factors remains unclear. Here, we performed an evolutionary analysis for positive selection in members of the myogenic regulatory factor (MRF) gene family of vertebrates. We selected 153 complete vertebrate MRF nucleotide sequences from our analyses, which revealed substantial evidence of positive selection. Here, we show that sites under positive selection were more frequently detected and identified from the genes encoding the myogenic differentiation factors ( Recent studies in evolutionary genetics have provided several lines of evidence supporting the role of positive selection in the evolution of many genes. These studies have suggested that positive genetic selection is also the major evolutionary force in addition to neutral mutations and random genetic drift MyoD), myogenic factor 5 (Myf5), myogenin (MyoG), and Myf6 (MRF4) genes. All MRFs share a conserved basic helix-loop-helix (bHLH) domain that is required for DNA binding and dimerization with other proteins, such as E protein. All four MRFs are characterized by their capacity to convert a variety of cell lines into myocytes and to activate muscle-specific gene expression Myf5 and MyoD are myogenic determination factors and contribute to myoblast determination, which is activated in proliferating myoblasts before overt differentiation. In contrast, MyoG and Myf6 are myogenic differentiation factors that contribute to the differentiation of myoblasts and act downstream of Myf5 and MyoD, though Myf6 partly acts at both the determination and differentiation levels Myf5 and MyoD have redundant functions in myoblast determination and can compensate for the functional loss of each other, Myf5 plays a more critical role during the early determination of epaxial muscle, whereas MyoD is more critical for hypaxial muscle determination Myogenesis involves two major temporally ordered steps. First, myogenic progenitor cells (myoblasts) originate from mesenchymal precursor cells, and second, these cells then terminally differentiate into mature muscle fibers Myf5, MyoD, MyoG, and Myf6 genes were duplicated from a single invertebrate gene Caenorhabditis elegansAnthocidaris crassispinaDrosophila melanogasterCiona intestinalis, it gives rise two different transcripts of MRFs (MDFa and MDFb) as a result of alternative splicing. Moreover, in cephalochordates, the amphioxus have two MRF genes: BMD1 and BMD2MyoD in ascidians suggest that the regulation pattern of multiple MyoD genes has evolved under selective pressure before the MRF genes were duplicated into multiple copies Myf5 and MyoD genes were then duplicated from one of these two lineages, whereas MyoG and Myf6 were duplicated from the other lineage during vertebrate evolution. Therefore, the functional redundancy between Myf5 and MyoD as well as between MyoG and Myf6 might be due to their common genetic origin Genome duplication is believed to be a major genetic event that occurs during the evolution of a gene family from a single gene to multiple gene copies The mechanisms underlying the evolution of the MRF gene family during their duplication remain unclear. In particular, the evolutionary forces affecting the functional divergence of the four MRFs genes have not been fully elucidated. In this study, we investigated the mechanisms underlying the evolution of the four MRF genes with particular emphasis on the selective pressures imposed on the branches and sites of MRFs during vertebrate evolution. Our study provides several lines of evidence for the role of positive selection in the functional divergence of transcription factors.Myf5 and MyoD), but not in the myogenic differentiation factors (Myf6 and MyoG)  . Two amiand MyoD .Nucleotide mutations in coding sequences are important for the evolution of gene functions. The likelihood ratio (LR) tests of site models in the CODEML program of phylogenetic inference by maximum likelihood (PAML4) Typically, the relatively short period of positive selection is usually followed by long periods of continuous negative selection MyoG lineage  and myogenic differentiation factors (MyoG/Myf6) play distinct roles in myogenesis. The functional divergence between these factors was estimated using the DIVERGE 2.0 program Myf5/MyoD and MyoG/Myf6 branches, which was significantly greater than 0 (p<0.01). Thus, the functional divergence between Myf5/MyoD and MyoG/Myf6 was significant. Twenty-nine residues have a stringent threshold of a posterior ratio higher than eight. Most of these sites were located in the BASIC, MYF5 domains and C-terminus, which might be critical for the functional divergence between the myogenic determination factors (Myf5 and MyoD) and myogenic differentiation factors (Myf6 and MyoG) (Myf5/MyoD lineage (branch a in MyoG/Myf6 lineage (branch b in The myogenic determination factors (nd MyoG) . The rolnd MyoG) . Using tMyoD coding sequences  tree of 53 vertebrate equences  was geneequences . No siteequences . Using aequences . These rMyoD and MyoG, no branch or site under positive selection was identified in the vertebrate Myf5 gene  analysis, a total of 55 positive selection sites during the divergence of MRFs were identified using the site and branch-site models of PAML4. We plotted the genetic location of positively selected sites onto the protein secondary structure and three-dimensional structure . PositivdN) and synonymous (dS) substitution rates  were transcribed by a single MRF gene, which was different than lower invertebrates, whereby a single MRF ortholog was transcribed Ciona intestinalisMyf5 and MyoD evolved from one of these lineages, whereas MyoG and Myf6 (MRF4) evolved from another lineage Myf6 and MyoG (Myf5 and MyoD) and the myogenic differentiation factors (MyoG and Myf6). In addition, two amino acid regions (SXXTSPXSNCSDGM and SSLDCLSXIVXRIT) might be critical in the functional gain of the myogenic determination role in Myf5 and MyoD  and differentiation factors (Myf6 and MyoG) was driven by positive selection. Positive selection sites responsible for this divergent process were identified from the BASIC, MYF5 domains and C-terminus  was also under positive selective pressure. The evolution processes of MyoD in all vertebrates are driven by positive selection on the BASIC and MYF5 domains  also diverged under positive selection. The positive selection on the BASIC and C-terminus were identified in the bird MyoG lineage and the teleost MyoG lineage . The MRF sequences were aligned by the program MUSCLE or ClustalW, and all gaps were eliminated by manual edition  and synonymous (dS) substitution rates were calculated by the Nei-Gojobrotri (Jues-Cantor) method as implemented in the MEGA5.0 program to measure the pairwise sequence distances of the three domains among different MRFs The CODEML program in the PAML4 Three-dimensional structures of the proteins were predicted using the worldwide web following the methods of a case study using the Phyre server Myf5/MyoD) and myogenic differentiation factors (MyoG/Myf6). The Type I functional divergence was measured as the coefficient of functional divergence, θ (ranging from 0–1), which was calculated by model-free estimation (MFE) and maximum-likelihood estimation (MLE) under a two-state model. The value of θ represents the functional divergence The DIVERGE 2.0 program Table S1Likelihood Ratio Tests for the positive selection on all MRF genes.(XLS)Click here for additional data file.Table S2Likelihood Ratio Tests for the positive selection on each of the MRFs.(XLS)Click here for additional data file.File S1Alignment results for the 153 vertebrate MRF coding sequences.(NEXUS)Click here for additional data file.File S2Alignment results for the 53 vertebrate MyoD coding sequences.(NEXUS)Click here for additional data file.File S3Alignment results for the 43 vertebrate MyoG coding sequences.(NEXUS)Click here for additional data file.File S4Alignment results for the 19 mammalian MyoG coding sequences.(NEXUS)Click here for additional data file.


Dear Editor,The increasing incidence of aspergillosis emphasizes to improve the diagnostic tools. AspergilA standard isolate of Aspergillus fumigatus PTCC5009 was grown in potato dextrose broth. DNA extraction of Aspergillus fumigatus, Fusarium oxysporum PTCC5115 and Aspergillus flavus PTCC5004 as control species were done according to established methods.[4] BloodThe alignment of the three DNA sequences was performed with the program Gene Works , Selection of the primer sequences was based on a close check for sequences with matching homologies in current DNA databases (Gen Bank). By using a nested PCR technique, two pairs of o primers  derived from sequences of the A. fumigatus 18s rRNA gene (GenBank No.) were chosen for PCR assays. For eachThe two mentioned primers with the highest sensitivity and species specificity of Aspergillus which amplified a fragment of 404 bp, were followed by AFU5S and AFU5AS which produced an internal fragment of 335 bp. These primer-binding sites are located in the 3' part of the 18S rRNA gene and in variable region V7-V9 (AFU7AS) or V8-V9 (AFU5AS), with no sequence overlap between the primers used in the first and second PCRs to reduce contamination problems. Comparison of clinical characteristics of the patients, and their PCR results showed that IA negative patients had an age>25, WBC count>3000 while positive patients had an age<30 and WBC count=1000-2000.Our results showed that out of 30 BMT patients, six patients had positive nested PCR results (20%). So detection of Aspergillus DNA from blood is possible and helpful in diagnosis of IA and can assist in distinguishing this disease from others . Most of


We surveyed intensivists to evaluate their stated use of systemic steroids in the ICU. The efficacy of steroids in septic shock and ARDS remains uncertain and clinicians' perceptions of competing indications and contraindications may jeopardize future randomized controlled trials (RCT). Knowledge of current practice will inform the design of future RCTs addressing the efficacy of systemic steroids in septic shock and ARDS.We designed and conducted a self-administered survey of intensivists practicing in academic settings with expertise in ARDS clinical research. We generated questionnaire items in focus group sessions with content experts and refined them through a standardized process of clinical sensibility, pilot and intra-rater reliability testing. Respondents used a four-point scale to grade how frequently they would administer systemic steroids in a 14 different clinical situations and reported their opinions of 16 near absolute indications or contraindications for systemic steroids. Local research staff distributed the survey to all intensivists practicing in the 11 centres (Canada and USA) with most patients enrolled in the OSCILLATE trial.In total, 103 of 125 potential respondents returned completed surveys (response rate 82%). A majority of respondents 'almost always' prescribe systemic steroids in the setting of recent systemic steroid use and low blood pressure (93%), significant bronchospasm in a mechanically ventilated patient (93%) and vasopressor refractory septic shock (52%). A majority of respondents would 'almost never' prescribe steroids in severe community-acquired pneumonia (81%), ALI (76%) and ARDS (65%). One-half (50%) would 'almost never' prescribe steroids for severe ARDS (50%). The near absolute indications selected by a majority of respondents were 'known adrenal insufficiency' (99%) and 'suspicion of cryptogenic organizing pneumonia' (89%). The only near absolute contraindication selected by a majority of respondents was 'systemic fungal infection' (52%).Certain clinical conditions may prompt intensivists to almost always prescribe systemic steroids and reduce equipoise for future placebo-controlled trials. Moreover, this survey shows that in selected academic centres a majority of intensivists do not prescribe corticosteroids for pneumonia, ALI and ARDS.


Objective. The aim of this study was to identify patients with Parkinson's disease who showed loss or decrease of nocturnal blood pressure fall (nondipper patients) as a marker of autonomic dysfunction. Presence or absence of orthostatic hypotension was considered to investigate whether alterations in circadian blood pressure pattern are associated with posture-related dysregulation of blood pressure. Methods. 40 patients with Parkinson's disease underwent 24-hour blood pressure monitoring. 21 patients were diagnosed with arterial hypertension and received anti-hypertensive drugs. Nondipper patients were defined as having nocturnal decrease of mean systolic and diastolic blood pressure less than 10%. Presence or absence of orthostatic hypotension was determined by Schellong's test. Results. We identified 35 nondipper patients (88%). Nondipping was detected in 20 patients with orthostatic hypotension (95%) and in 15 patients without orthostatic hypotension (79%). 18 patients with hypertensive and 22 patients with normal blood pressure values were detected. Conclusions. In conclusion 24-hour blood pressure monitoring showed a high prevalence of nondipping in 40 patients with Parkinson's disease with and without orthostatic hypotension independent of coexisting arterial hypertension and antihypertensive treatment. 24-hour blood pressure monitoring may be useful to identify non-dipping as a marker of autonomic dysfunction in patients with Parkinson's disease. Parkinson's disease is a multisystem degeneration . Beside Involvement of the autonomic nervous system (ANS) occurs in the early stages of the disease . Cardiovascular dysautonomia, especially orthostatic hypotension, is frequently reported in Parkinson's disease .Involvement of peripheral components of the ANS can be demonstrated by MIBG scintigraphy.MIBG scintigraphy shows reduced cardiac uptake of MIBG (meta-[123I]iodobenzylguanidine) representing loss of postganglionic myocardial sympathetic nerve fibers in patients with Parkinson's disease and autonomic failure . HoweverFurther effects of autonomic cardiovascular dysfunction are demonstrated in earlier studies such as baroreceptor reflex dysfunction, alterations in circadian blood pressure regulation, and supine hypertension –15. FindAmbulatory blood pressure monitoring is frequently used in the diagnosis and therapy monitoring of arterial hypertension and allows an evaluation of the circadian blood pressure evaluation , 18.The aim of this study was to identify patients with Parkinson's disease who showed loss or decrease of nocturnal blood pressure fall (non dipper patients) as a potential marker of autonomic dysfunction.Presence or absence of orthostatic hypotension was considered to investigate whether alterations in circadian blood pressure pattern are associated with posture-related dysregulation of blood pressure.40 patients with Parkinson's disease underwent ambulatory 24-hour blood pressure monitoring. Parkinson's Disease was diagnosed according to the criteria of the United Kingdom Parkinson's disease Society Brain Bank . None ha20 of the patients were male, 20 were female; they were aged 41 to 82 years ; disease duration (Parkinson's disease) was 1 to 152 months (mean disease duration 49 months) . 21 patiLong-term blood pressure measurements were performed with “Mobilograph,” an oscillometric recorder . Patients were asked to maintain their normal physical activity. Nondipper patients were defined as having nocturnal decrease of mean systolic and diastolic blood pressure less than 10%. Presence or absence of orthostatic hypotension was determined by Schellong's test ; orthostatic hypotension was defined as a minimum decrease in systolic and/or diastolic blood pressure of 20 mmHg or 10 mmHg, respectively, on standing at the third minute .Results are presented as mean values (± standard deviation (SD)). A correlation analysis was performed between the presence of loss of nocturnal blood pressure fall and disease duration, age and presence of arterial hypertension/use of antihypertensive medication.Ambulatory blood pressure was recorded without interruption in all patients with a mean measurement period of 23 hours . The average number of measurements obtained during the 24-hour recording was 88% (± 11). Results of the mean diurnal arterial pressure showed 18 patients with hypertensive blood pressure values and 22 patients with normal blood pressure. Beside the 21 patients who were already diagnosed with arterial hypertension 6 patients showed hypertensive blood pressure values in ambulatory blood pressure recording; thus a total of 27 patients (68%) had arterial hypertension .We identified 35 non dipper patients (88%) among 40 examined patients with Parkinson's disease; 25 patients (63%) showed nocturnal increase of blood pressure . CorrelaAutonomic test results showed 21 patients with orthostatic hypotension. Loss of nocturnal blood pressure fall was detected in 20 patients with orthostatic hypotension (95%); this was also detected in 15 patients without orthostatic hypotension (79%). We found 11 patients with supine hypertension who were non dipper (100%) and 24 patients without supine hypertension who were non dipper (83%) .We investigated ambulatory blood pressure monitorings in patients with Parkinson's disease to evaluate the circadian blood pressure profile, especially the presence or absence of loss of nocturnal blood pressure fall (non dipper). Results of earlier studies show a variable percentage of non dipper patients , mainly Our results show that most of our examined patients with Parkinson's disease were non dipper (88%); this was even found in 79% of the patients without orthostatic hypotension. In conclusion, ambulatory blood pressure monitoring could be useful to detect autonomic dysfunction even in the early stages of Parkinson's disease when orthostatic hypotension is not present. α-adrenergic receptors due to sympathetic denervation in patients with Parkinson's disease may contribute to supine hypertension during the night due to exaggerated vasoconstriction [The alterations in the circadian blood pressure profile may be caused by impairment of the autonomic regulatory mechanisms, possibly through postural dysregulation of blood pressure as mentioned in earlier reports . This dytriction , 23, 24.Presence or absence of arterial hypertension was additionally investigated in our patients  to evaluate a possible association with loss of nocturnal blood pressure fall; there was no correlation between the presence of non-dipping and the presence of arterial hypertension or use of antihypertensive medication. 27 patients were additionally diagnosed with arterial hypertension or had hypertensive blood pressure values in ambulatory blood pressure recording (68%). 24-hour blood pressure monitoring should be performed in patients with Parkinson's disease and coexisting arterial hypertension, who are treated with antihypertensive medication. For prevention of an increased prevalence of end-organ damage we propose treatment of nocturnal hypertension in patients with Parkinson's disease. Treatment with antihypertensive drugs in patients with orthostatic hypotension may be complicated by worsening of the postural blood pressure fall in the upright position in these patients and thus may cause syncopes or cerebral ischemia. Consequently, vasoactive drugs should be used carefully in patients with Parkinson's disease and autonomic dysfunction. Alternatively sleeping in a 12° head-up tilt position may be helpful . Concerning an increased cardio—and cerebrovascular risk in patients with nondipping, Cornélissen et al. showed that—compared with dipping—abnormal chronobiological end points such as reduced circadian standard deviation of heart rate or elevated pulse pressure are more useful to discriminate between patients who are at a higher risk and who are not . HoweverIn conclusion 24-hour ambulatory blood pressure monitoring showed a high prevalence of non-dipping in 40 patients with Parkinson's disease with and without orthostatic hypotension independent of the coexisting arterial hypertension, and antihypertensive treatment. Our data strengthens the role of ambulatory blood pressure recording for detection of non-dipping not only in patients with orthostatic hypotension, but also in patients without orthostatic hypotension. Beside other autonomic tests  24-hour blood pressure monitoring may be helpful to support the detection of autonomic dysfunction in patients with Parkinson's disease even in the early stages of the disease.


There is an error in the legend for Figure 3. The legend refers to figures 3a, 3b and 3c, which is not correct. Figure legend 3 should read:Figure 3: The figures display the mean % BOLD signal change for each of the 3 tasks  for each delay  in the ROI. The graphs show: the medial temporal cortex (V5), the supramarginal gyrus (SMG), and superior parietal lobe (SPL), the cerebellum (CBM), primary visual cortex (V1), the frontal eye fields (FEF), supplementary eye fields (mid SEF), and dorsolateral prefrontal cortex (DLPFC). The centre of mass for the regions of interest used in the analysis are highlighted within a template brain on either side of the % signal change graph for all figures and brain coordinates are presented using the Montreal Neurological Institute (MNI). T values and significance corrected cluster level P values are also displayed for each ROI.


Pancoast syndrome (Pancoast’s syndrome) is characterized by a malignant neoplasm of the superior sulcus of the lung (lung cancer) with destructive lesions of the thoracic inlet and involvement of the brachial plexus and cervical sympathetic nerves (stellate ganglion) which causes Horner syndrome .Following trimodality treatment for (SSTs), the 5-year survival rate has significantly improved. The objective of this study was mortality and morbitity and quality of life after the resection of superior sulcus tumours following neoadjuvant chemoradiation.Between Januar 2002 and December 2012 ; 55 consecutive patients were treated with primary radiotherapy followed by surgery or with primary excision and subsequent radiotherapy in the absence of an initial histological diagnosis.In total, 55 patients participated in this study (45 men and 10 women). The median age was 60 years (range 40–73), median radiation dose 45 Gy (range 40–60) and median follow-up 50 months (range 4–101). The mortality of patients given the combined treatment was 8.6% (one death due to pulmonary embolism and one death due to myocard ischemia), and the five year survival rate was 20.6% for all patients and 37% for those who underwent complete resection without N2 disease.Stage III lung cancer, classified as Pancoast tumour according to strict, consistent criteria, is best treated by primary radiotherapy; combined treatment should be used only for patients with potentially resectable cancer without N2 disease and/or malignant invasion of the first rib.


Despite the increasing public health burden associated with chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD), the molecular mechanisms responsible for the pathogenesis of these diseases remain unclear. The goal of this study was to comprehensively profile the lung small RNA transcriptome via next-generation sequencing, and elucidate microRNAs that might contribute to COPD and ILD pathogenesis.n = 150) or ILD (n = 149), and from normal lung tissue (n = 65). 319 lung tissue samples were sequenced via multiplexing on the Illumina HiSeq 2000 (10 samples/lane) and 45 samples were sequenced on the Illumina GAIIx (one sample per lane). Reads were trimmed using the FASTX toolkit and aligned to hg19 using Bowtie. Reads mapping to a microRNA locus were grouped into those with the canonical seed and those with an alternative seed due to variation in the 5' start position (isomiRs). Negative binomial generalized linear models were used to identify microRNAs and isomiRs differentially expressed between phenotypes. The large RNA from a subset of these samples was also sequenced using the Illumina GAIIx (one sample per lane). A network was generated by identifying all microRNA-mRNA pairs that were significantly anti-correlated  <0.05) and had a predicted microRNA-binding site in the mRNA 3'UTR (TargetScan 6.0).As part of the Lung Genomics Research Consortium, we sequenced the small RNA in lung tissue samples from subjects with COPD (n = 72). 2,133 genes in the network (65%) were predicted to be regulated by at least one differentially expressed isomiR. Both the canonical form and three isomiRs of miR-338-3p were significantly downregulated in ILD. Predicted targets of the canonical form of miR-338-3p were enriched in extracellular matrix genes while the predicted targets of a miR-338-3p isomiR were enriched in the Wnt signaling pathway (P<0.001), suggesting different roles for multiple forms of this microRNA in ILD.An average of 26.3 million and 7.1 million reads were sequenced per sample using the singleplex and multiplex protocols, respectively. An average of 73% of reads per sample aligned to the human genome with one or fewer mismatches at 10 or fewer locations. 287 novel microRNA precursors were predicted using the miRDeep algorithm. One of these candidates was validated by quantitative RT-PCR and found to be expressed across a range of human tissues. The expression of 309 canonical microRNAs was significantly different between patients with disease and controls (FDR <0.05). In addition, we found that 242 isomiRs from 159 different microRNA loci were also differentially expressed (FDR <0.05). We developed a network of microRNA-mRNA interactions by integrating small RNA and large RNA sequencing data generated on the same samples (Our results demonstrate the power of deep sequencing to reveal additional complexity in the microRNA transcriptome, such as novel microRNAs and isomiRs. The disease-related patterns of microRNA expression can provide insights into the molecular pathogenesis of chronic lung diseases and novel targets for therapy.