Among the many factors determining protein evolutionary rate, protein-protein interaction degree (PPID) has been intensively investigated in recent years, but its precise effect on protein evolutionary rate is still heavily debated.We first confirmed that the correlation between protein evolutionary rate and PPID varies considerably across different protein interaction datasets. Specifically, because of the maximal inconsistency between yeast two-hybrid and other datasets, we reasoned that the difference in experimental methods contributes to our inability to clearly define how PPID affects protein evolutionary rate. To address this, we integrated protein interaction and gene co-expression data to derive a co-expressed protein-protein interaction degree (ePPID) measure, which reflects the number of partners with which a protein can permanently interact. Thus, irrespective of the experimental method employed, we found that (1) ePPID is a better predictor of protein evolutionary rate than PPID, (2) ePPID is a more robust predictor of protein evolutionary rate than PPID, and (3) the contribution of ePPID to protein evolutionary rate is statistically independent of expression level. Analysis of hub proteins in the Structural Interaction Network further supported ePPID as a better predictor of protein evolutionary rate than the number of distinct binding interfaces and clarified the slower evolution of co-expressed multi-interface hub proteins over that of other hub proteins.Our study firmly established ePPID as a robust predictor of protein evolutionary rate, irrespective of experimental method, and underscored the importance of permanent interactions in shaping the evolutionary outcome. Among the many factors determining protein evolutionary rate -5, proteThe relationship between protein evolutionary rate and PPID is mostly studied through hub proteins, i.e., proteins with a large number of interaction partners, from many different aspects -23. For Therefore, in this paper, we first re-investigated the relationship between protein evolutionary rate and protein-protein interaction degree (PPID) and confirmed that the correlation between protein evolutionary rate and PPID varies considerably across different protein interaction datasets. We then integrated protein interaction and gene co-expression data to derive a co-expressed protein-protein interaction degree (ePPID) measure, which reflects the number of partners with which a protein can permanently interact. Our results demonstrated that ePPID is a more robust predictor of protein evolutionary rate than PPID. It was further found that the contribution of ePPID to protein evolutionary rate is statistically independent of expression level. Finally, we established that ePPID could predict protein evolutionary rate better than the number of distinct binding interfaces for hub proteins in the Structural Interaction Network and clarified the slower evolution of co-expressed multi-interface hub proteins over that of other hub proteins.Researchers have found very different correlations between PPID and protein evolutionary rate ,8-17. ToWe next studied if the protein abundance effect may account for the above significant difference between the Y2 H and other datasets. We computed Spearman rank correlations between PPID and protein abundance  and founProteins with higher PPID are assumed to have a greater proportion of residues involved in interactions and thus evolve more slowly than proteins with lower PPID ,9. This Several studies have addressed the difference in selective constraints between permanent and transient interactions on protein evolution ,20,22,39Therefore, we proposed a co-expressed protein-protein interaction degree (ePPID), defined as the maximal number of co-expressed interaction partners of a given protein in all gene expression datasets we used . Despite this effect, permanent interactions do place higher selective constrains on protein evolution than transient interactions do, further illustrating why our ePPID measure could better predict protein evolutionary rate.However, the variance of protein evolutionary rate explained by ePPID is still the lowest in the "Y2H-union" dataset, which may be explained in three ways. First, ePPID cannot filter out all transient protein interactions, partly because of noise in the gene expression datasets we used. Second, Y2 H datasets may contain co-expressed protein pairs which are localized to different cellular compartments and seldom interact in nature. Third, ePPID may be underestimated based on incompleteness of Y2 H datasets ,32, whicA number of genomic variables, such as expression level ,25,50-52For this purpose, we collected two expression-related variables, mRNA abundance and protein abundance; two function-related variables, gene dispensability and gene pleiotropy, which were measured by the associated number of GO biological process terms of each gene; and two network-related variables, ePPID and betweenness (see Methods). We then carried out a principal component regression ,56 of prResults show that the first principal component explains much more variance of protein evolutionary rate than the other components in all the six datasets. Thus, in the following, we focus on the first principal component to study the percentage contribution of ePPID. In the "Combined-AP/MS", "LC-multiple", "Updated-HC", "DIP-CORE" and "DIP-FULL" datasets, the contribution of ePPID to the first principal component is more than that of all other variables. In the "Y2H-union" dataset, the ePPID contribution is more than betweenness, but less than the other four variables. Consistently, the independent contribution of ePPID to the total variance of protein evolutionary rate dN explained by all the six principal components in most datasets is comparable to that of the expression-related variables of mRNA abundance and protein abundance  in the "SIN" dataset  and studIt is reported that protein evolutionary rate is actually more reflective of the number of distinct binding interfaces . Yet we It is also reported that multi-interface hubs have lower evolutionary rate than singlish-interface hubs  . TherefoTo see whether our result for yeast can be obtained in other species, we obtained the relevant data for human and computed ePPID for each protein  and protein evolutionary rate.Proteins with higher PPID are assumed to have a greater proportion of residues involved in interactions and thus evolve more slowly than proteins with lower PPID ,9. This In this work, we performed extensive studies to identify how protein interactions, as measured by PPID, affect protein evolutionary rate. By carefully comparing experimental setups, we observed that Y2 H assays may have introduced a considerable amount of transient protein interactions. On this basis, we hypothesized that the difference in experimental methods contributes to our inability to clearly define how PPID affects protein evolutionary rate. This hypothesis was confirmed by introducing a new protein interaction degree measure, the co-expressed protein-protein interaction degree (ePPID). Since ePPID is a measure that integrates protein interactions with gene co-expression information, it can filter out many transient protein interactions. As a result, ePPID gives a better prediction of protein evolutionary rate than PPID in the various protein interaction datasets tested. The relationship between ePPID and protein evolutionary rate is also robustly significant in all protein interaction datasets, which was not possible when using PPID in previous studies. We also investigated the redundancy between several variables that may affect protein evolutionary rate against the contribution of ePPID and found that ePPID makes an independent contribution to protein evolutionary rate. This result suggests the novelty of ePPID as an important determinant of protein evolutionary rate. Moreover, the application on hub proteins in the Structural Interaction Network provides further support that ePPID also gives a better prediction of protein evolutionary rate than the number of distinct binding interfaces and clarified the slower evolution of co-expressed multi-interface hub proteins over that of other hub proteins.In summary, our work provides a new protein interaction degree measure by integrating protein interaction datasets with gene expression datasets. This new measure has, at least in part, resolved the longstanding debates on the role of protein interactions in affecting protein evolutionary rate. Finally, we have found that this result also holds in human. We will study if this can be observed in more species in the future.To study the effect of experimental bias, reliability and completeness, we collected nine different yeast protein interaction datasets. We used the "Y2H-union", "Combined-AP/MS" and "LC-multiple" datasets to represent typical protein interaction datasets obtained from Y2 H assays, the AP/MS method and literature curation, respectively. To account for data quality (confidence), we also used the filtered yeast interactome ("FYI"), the Structural Interaction Network ("SIN"), "DIP-CORE" and the updated high-confidence dataset ("Updated-HC") as high-confidence datasets. In addition, the "SIN" dataset was also used to study the evolutionary rate of hub proteins through a mechanistic perspective. In contrast, we used the "DIP-FULL" and "Eight-union" datasets to account for completeness. The nine datasets are listed below.1) Y2H-union: the union of three high-throughput Y2 H datasets: Uetz-screen , Ito-cor2) Combined-AP/MS: an integrated dataset  of two h3) LC-multiple: a protein interaction dataset based on the literature. Each protein interaction must have been curated from ≥2 different publications .4) FYI: the filtered yeast interactome obtained from .5) SIN: the Structural Interaction Network dataset obtained from .6) Updated-HC: the updated high-confidence dataset obtained from .7) DIP-CORE: the core dataset derived from the database of interacting proteins (DIP) ,43.8) DIP-FULL: the full dataset derived from the database of interacting proteins (DIP) .9) Eight-union: the union of the above eight datasets.The network properties of the nine datasets are summarized in Additional file Saccharomyces cerevisiae genes, were obtained from [S.cer vs S.par, S.cer vs S.mik and S.cer vs S.bay) were obtained from [Saccharomyces Genome Database [Evolutionary rate data (non-synonymous substitutions per site dN), which is based on the four-way yeast species alignments for 3,036 ned from . Specifined from  and dN wned from . Three oned from . mRNA abned from . Proteinned from . Codon aned from , was obtned from . The assned from , used asDatabase . ProteinDatabase ,47, was Database  with theDatabase .Partial correlation analysis is frequently used to determine the confounding effect of variables such as protein abundance on the relationship between PPID and protein evolutionary rate ,25,51,71Saccharomyces Genome Database [We collected ten gene expression datasets -83, eachDatabase . Genes wDatabase , and tecr is used as a similarity measure between the expression profiles of two genes. The PCC r was then converted into z-score using Fisher transformation:Pearson correlation coefficient (PCC) n is the sample size. We only considered positive correlations since they are reported to be more reflective of functional similarity than negative correlations [which approximately follows a standard normal distribution under the hypothesis of independence, where elations . Next, Pelations , and theelations ,88 and wg denote the set of interaction partners of a given protein g. By filtering out the potential transient interaction partners in PPIg, we want to integrate gene co-expression in a way that allows us to identify the partners with which protein g can permanently interact. To explain, we can calculate the z-scores of co-expression between protein g and all genes in PPIg for each gene expression dataset. Let ePPIg(i) denote the number of genes that are found to be significantly co-expressed with gene g in gene expression dataset i . The co-expressed protein-protein interaction degree (ePPID) of protein g is then defined as ePPIDg = max(ePPIg(i); i = 1,2,...,10). In addition, we tried other co-expressed protein-protein interaction degree measures to demonstrate the robustness of our results  in each protein interaction dataset. Similar to the concept behind the date and party hub definition in , we furtTo study the contribution of permanent and transient interfaces to protein evolutionary rate in the "SIN" dataset, the ePPID of hub proteins in the "SIN" dataset was calculated similarly, and the hub proteins were further grouped into four classes: non-co-expressed singlish-interface hubs, non-co-expressed multi-interface hubs, co-expressed singlish-interface hubs and co-expressed multi-interface hubs by taking intersections.APCC: average Pearson correlation coefficient; AP/MS: affinity purifications followed by mass spectrometry; CAI: codon adaptation index; dN: non-synonymous substitution rate; ePPID: co-expressed protein-protein interaction degree; FDR: false discovery rate; GO: Gene Ontology; PCC: Pearson correlation coefficient; PER: percent of all possible correlations; PPID: protein-protein interaction degree; Y2H: yeast two-hybrid.KP and XM conceived and designed the experiment; HS and XM supervised the experiment; KP, CC, ZX, HS and XM conducted the experiment and wrote the manuscript. All authors have read and approved the final manuscript.Supplementary texts, figures and tables. This file contains Supplementary Texts S1-S11, Figures S1-S4 and Tables S1-S38.Click here for file


These studies suggest that the hepatic flares are frequently asymptomatic, but life-threatening liver failure occurs in nearly 10% of cases. Rituximab, a chimeric mouse-human monoclonal antibody directed to the CD20 antigen expressed on pre-B lymphocytes and mature lymphocytes, causes a profound B-cell depletion. Due to its peculiar characteristics, this drug has been used to treat oncohaematological diseases, B cell-related autoimmune diseases, rheumatoid arthritis, and, more recently, HCV-associated mixed cryoglobulinaemic vasculitis. Rituximab-based treatment, however, may induce an increased replication of several viruses such as hepatitis B virus, cytomegalovirus, varicella-zoster virus, echovirus, and parvovirus B19. Recent data suggest that rituximab-based chemotherapy induces an increase in HCV expression in hepatic cells, which may become a target for a cell-mediated immune reaction after the withdrawal of treatment and the restoration of the immune control. Only a  Hepatitis C virus (HCV) is responsible worldwide for chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma . BesidesIn the last decade, rituximab, a chimeric mouse-human monoclonal antibody directed to the CD20 antigen expressed on pre-B lymphocytes and mature lymphocytes , has beeIt is well known that rituximab-based chemotherapy is frequently followed by a reactivation of viral infections and correlated diseases. A frequent increase in viral replication under R-CHOP  was demonstrated by Aksoy et al. for several viruses including hepatitis B virus (HBV), cytomegalovirus, varicella-zoster virus, echovirus, and parvovirus B19 . Worthy According to the fragmentar data available, R-CHOP can induce an increase in HCV replication in oncohaematological patients , 35, a bThe uncertainty dominating this topic arises from the inconsistent data available and from the marked differences in the studies as regards the design, age of the patients, type and stage of the oncohaematological diseases, type of chemotherapy used, and stage of liver disease. In addition, the criteria used to define HCV reactivation and a hepatic flare differ from study to study.Some of these papers are case reports. Akosy et al. described HCV reactivation not associated to a hepatic flare in a patient with HCV-related cirrhosis and NHL treated only with rituximab . Nooka eOnly a few studies evaluated both HCV reactivation and the development of a hepatic flare in small series of patients with oncohaematological diseases receiving R-CHOP . In a prIn a retrospective study on 131 patients with HCV infection and NHL treated with rituximab and prednisone-based chemotherapy, Ennishi et al.  describeIn a retrospective study, Marignani et al.  describePitini et al. recently described HCV reactivation and a hepatic flare in 10 patients with HCV infection and NHL treated with R-CHOP . The hepSimilar results were reported by Tsutsumi et al.  in a proIn a retrospective study on 160 patients with Hodgkin's lymphoma and HCV infection, Arcaini et al. described hepatotoxicity in 5 (17.9%) of 28 patients with B-cell lymphoma treated with R-CHOP . For 25 Petrarca et al. treated HCV-associated mixed cryoglobulinaemia with rituximab and found this treatment useful and safe even in patients with a severe liver disease ; the patWorthy of mention is the literature showing a frequent occurrence of hepatic flares in patients receiving high-dose corticosteroids plus rituximab –40, probVisco et al. reported an association between R-CHOP treatment for DLBCL and a mild increase in the liver enzyme level not requiring discontinuation of treatment in 5 (14%) of 35 patients ; this stCoppola et al. described 28 anti-HCV/HCV RNA-negative patients receiving chemotherapy (rituximab based in 61% of cases) for oncohaematological diseases. These patients remained HCV RNA-negative both in plasma and PBMC and no hepatic flare was observed over an observation period of 6–24 months , most pr Fragmentary information from case reports, small studies, and retrospective investigations suggest, on the whole, that rituximab-based chemotherapy favours viral replication in patients with HCV infection and oncohaematological diseases. Only a few small studies evaluated both variations in the HCV viral load and the development of a hepatic flare during or after R-CHOP treatment, but the results are concordant and strongly suggest a close association between these two events. A possible interpretation of this association is that R-CHOP induces an enhancement of HCV replication followed by a spontaneous subsequent decrease once treatment is reduced or discontinued. The consequent restoration of the immune control may induce a hepatic flare of varying clinical impact, that is, asymptomatic, symptomatic or life threatening, most probably reflecting the extent of cell-mediated hepatocellular necrosis. The clinical effects seem more evident in patients treated with a combination of rituximab and high-dose corticosteroids. The high prevalence worldwide of HCV infection in oncohaematological patients and the progressive increase in the use of R-CHOP for treating these diseases may create a heavy burden in the future for the health care authorities in several countries.


The regulatory information encoded in the DNA of promoter regions usually enforces a minimal, non-zero distance between the coding regions of neighboring genes. However, the size of this minimal regulatory space is not generally known. In particular, it is unclear if minimal promoter size differs between species and between uni- and bi-directionally acting regulatory regions.S. cerevisiae and K. waltii) are shorter than the lower limit observed in older regions.Analyzing the genomes of 11 yeasts, we show that the lower size limit on promoter-containing regions is species-specific within a relatively narrow range (80-255 bp). This size limit applies equally to regions that initiate transcription on one or both strands, indicating that bi-directional promoters and uni-directional promoters are constrained similarly. We further find that young, species-specific regions are on average much longer than older regions, suggesting either a bias towards deletions or selection for genome compactness in yeasts. While the length evolution of promoter-less intergenic regions is well described by a simplistic, purely neutral model, regions containing promoters typically show an excess of unusually long regions. Regions flanked by divergently transcribed genes have a bi-modal length distribution, with short lengths found preferentially among older regions. These old, short regions likely harbor evolutionarily conserved bi-directionally active promoters. Surprisingly, some of the evolutionarily youngest regions in two of the eleven species (The minimal chromosomal space required for transcriptional regulation appears to be relatively similar across yeast species, and is the same for uni-directional and bi-directional promoters. New intergenic regions created by genome rearrangements tend to evolve towards the more narrow size distribution found among older regions. It is likely that this regulatory information - including spacers and the 5' untranslated region (UTR) - needs a minimum length of DNA sequence. In most cases, promoter regions will not overlap with the coding or UTR regions of neighbouring genes, both because of functional constraints and because transcription of the neighbour gene would interfere with transcription initiation at the promoter [Expression of a gene requires a functional promoter region 5' of the coding sequence. Promoter regions encompass the binding sites for DNA polymerase, usually accompanied by promoter ,2. A lowWe can classify inter-CDS regions according to the relative orientation of the two genes flanking the region. First, the two genes can be located on the same strand, and will thus be transcribed uni-directionally . It is conceivable that sometimes this impairment is outweighed by accompanying expression changes that are advantageous , and sucBy far the strongest predictor of gene neighbourhood conservation is intergenic or inter-CDS distance . This isIn this paper, we thus analyse the length distributions of inter-CDS regions across yeast species to examine the following questions: (i) are promoter-containing regions longer than promoterless regions, indicating a minimal promoter length?; (ii) does minimal promoter size differ between uni-directional and divergent regions?; (iii) does minimal promoter size differ between species?; and (iv) can the size distribution of inter-CDS regions be explained by a simplistic neutral model of rearrangements?We identified inter-CDS regions flanked by protein coding genes in 11 fully sequenced and well-annotated yeast species ,10. RegiInter-CDS regions that were recently formed by genomic rearrangements may not have had time to evolve towards their 'optimal' length . For eaccis-regulatory sequences are organized into promoter regions located directly 5' of their regulated genes. Accordingly, few cis-regulatory sequences should be found in inter-CDS regions flanked by the 3' ends of convergently transcribed genes. We thus expect that such 'convergent' inter-CDS regions have no hard limits on their size distribution. Furthermore, if DNA insertions and deletions were selectively neutral and unbiased in these regions, then length changes per unit time via indels should be proportional to the region length (as each nucleotide would have the same chance of being an insertion or deletion point). On log-scale, such random length changes over time would be additive, and we hence expect a log-normal distribution of lengths under these conditions. In agreement with this expectation, Figure It is generally assumed that most Kluyveromyces waltii to 316 bp in Saccharomyces bayanus . This relatively narrow range indicates that each yeast has its species-specific equilibrium length of convergent regions. A 'soft' lower limit >0 bp is likely due to the 3' UTRs, and may further be enforced by the avoidance of transcriptional interference between the flanking genes .The age of inter-CDS regions can be approximated by the divergence time of the last common ancestor of the query genome and the most distal genome with an orthologous inter-CDS region. We reconstructed the phylogenetic relationships of the 11 yeast genomes using As shown in Figure R = 0.87, p = 0.00058) and by genome size . In contrast, we find no significant correlation between minimal promoter length and the number  or total length  of protein-coding genes.In the presence of either a bias towards deletions or selection for genome compactness, we expect a strong correlation between total intergenic space and minimal promoter size. Furthermore, as genome size is the sum of gene-coding and intergenic space, we also expect minimal promoter size to be correlated with genome size. Indeed, minimal promoter length across the 11 yeast species is well predicted by total intergenic space  inter-CDS regions. The length distributions of promoter-containing young regions also respect the minimal length derived from old inter-CDS regions in all species - with the exception of i Figure . Both ofS. cerevisiae and K. waltii  independent promoter regions; conversely, old regions often contain one bi-directional promoter Figure , and hene.g., neighbouring genes may be co-regulated through changes in chromatin state [In a careful study, Poyatos and Hurst  found thin state ,15, or min state . Howeverin state , and it old regions now . In each time step, we randomly chose an inter-CDS region and removed it from the distribution with a probability proportional to its length; this simulates rearrangements, where ancestral regions are destroyed to create new regions. This random removal was repeated until the number of rearrangements was identical to the lower bound on the number of rearrangements in the real data, i.e., until we removed Nyoung regions. It has to be pointed out that our model is very simplistic, not only in its assumption that rearrangements are strictly proportional to the length of the inter-CDS region. Importantly, length changes also occur through insertions and deletions; these are ignored here.Thus, we employed a simulation in which the probability that an inter-CDS region is broken up depends only on its length. We start from the total observed length distribution (combining old and young regions) of a given species, using this as an approximation to the total length distribution in the common ancestor of the species and its closest relative in our dataset. We then test if random rearrangements can reduce this total 'ancestral' distribution to the distribution seen for S. cerevisiae and K. waltii show a dearth of very short regions in the real data compared to the simulations. This suggests that either the real ancestor did not have a similar number of very short young regions as the extant species, or that these regions were preferentially removed or extended by natural selection.If the model approximately reflects the underlying biology, then the simulated length distribution should closely match the length distribution of old inter-CDS regions. As evident from Figure e.g., TY elements) in inter-CDS regions may serve as hot-spots for non-homologous recombination. Their presence may therefore modify gene neighbourhood in a non-random, non-selective manner. However, all results presented above remain essentially unchanged after excluding repeat-containing inter-CDS regions identified using RepeatMasker [Repeat-sequences  may vary according to the complexity of expression patterns among genes. However, this will depend on how expression signals are integrated along the hierarchy of transcriptional regulators. For example, a transcription factor may be induced according to complicated combinatorial cues by a variety of environmental signals. This single transcription factor may then simply transfer these integrated signals to all its regulatory targets - all the cis-regulatory complexity is then accounted for by the promoter region of the transcription factor, allowing for complex expression patterns with simple cis-regulatory sites for the majority of genes.Promoter-containing inter-CDS regions harbour cis-regulatory sites. That this is indeed the case is evidenced by the length distributions of inter-CDS regions in Figure i.e., those regions containing promoters.We therefore expect that many genes require little more than a minimal promoter configuration . However, it appears highly likely that successful (and orderly) initiation of transcription still requires a certain minimum of Many eukaryotic promoters initiate transcription on both strands, even if transcription often stalls in one direction ,4. ConsiS. cerevisiae, a canonical promoter of ~140 bp upstream to transcription start sites (TSS) was previously described [S. cerevisiae, which represents the lower limit of a functional promoter space.In escribed . This diescribed ) coincidescribed  , or they may even be polymorphic in the respective populations.What is puzzling is that in two species, s Figure . These aeighbour ,21); in In this study we show that the minimal chromosomal space is required for transcriptional regulation in yeast species; while the minimal length varies between yeast species, this variation is relatively small. The position of the left edge of the distribution varies between 78 bp and 255 bp Figure . SubtracKluyveromyces lactis , Ashbya gossypii, Saccharomyces kluyveri, Kluyveromyces waltii, Kluyveromyces thermotolerans, Zygosaccharomyces rouxii, Kluyveromyces polysporus, Candida glabrata, Saccharomyces castellii, Saccharomyces cerevisiae, Saccharomyces bayanus) from the Yeast Gene Order Browser [We downloaded protein sequences and genome annotations for eleven yeast species  in April 2010. The resulting tree is shown in Additional file To reconstruct the phylogenic relationships among downloaded yeast species, we selected ~40 universal single-copy marker genes previously suggested by Ref. , and alies pombe  as an ouS. cerevisiae into four age groups, according to the most distal genomes in which conserved neighbourhood was found. 'young' regions are those without conservation in any other species; 'plain old' (PO) are regions with orthologs in S. bayanus or S. castellii; 'very old' (VO) are regions with orthologs in any of C. glabrata, K. polysporus and Z. rouxii; and 'extra old' (XO) are those regions with orthologs in any of K. thermotolerans, K. waltii, S. kluyveri, A. gossypii and K. lactis  pairs. We assume that the inter-CDS length distribution is in equilibrium, i.e., the length distribution at the ancestral node was identical to the total length distribution observed now. We then set the probability that each inter-CDS nucleotide becomes the point of a rearrangement to p := Nyoung divided by the sum of all inter-CDS lengths .In a given genome, we have Ntotal inter-CDS regions (assumed to describe the length distribution in the ancestor that divides old from young regions). For each pair, we calculate the probability that it will be broken up in the given time interval as P = p × length (Model B: effective length); then we draw a random number r between 0 and 1 and put the pair into a list of 'surviving' pairs if r <P. We repeat this simulation until the final list contains Nold regions. The complete simulation is repeated 10 times, and the simulated length distributions shown are averaged over the 10 simulation runs.We start with the complete set of all WHC assembled the dataset, carried out the analyses and drafted the manuscript. MJL revised the manuscript. WHC, WW and MJL conceived of and designed the study together through iterative discussions, and read and approved the final manuscript.Size distribution of old and young inter-CDS regions for the remaining eight species; this figure is complementary to Figure Click here for fileStatistics of the underrepresentation of divergent and uni-directional old pairs in the 11 yeast species. The NULL expectation here is that if all pairs are distributed randomly, at given inter-CDS range, we would expect 25% of the total pairs to be divergent, 25% to be convergent, and 50% to be co-oriented .Click here for fileSchizosaccharomyces pombe as an outgroupReconstruction of phylogenetic relationships of the 11 analysed yeast species, using . The red star indicates the location of the recent whole genome duplication (WGD). Full species names for the abbreviations are: kla - Kluyveromyces lactis, ago -Ashbya gossypii, skl - Saccharomyces kluyveri, kwa - Kluyveromyces waltii, kth - Kluyveromyces thermotolerans, zro - Zygosaccharomyces rouxii, kpo - Kluyveromyces polysporus, cgl - Candida glabrata, sca - Saccharomyces castellii, sce - Saccharomyces cerevisiae, sba - Saccharomyces bayanus, spo - Schizosaccharomyces pombe.Click here for fileFitting one and two Gaussians to the distributions of inter-CDS distances (log10-transformed) of old, divergently transcribed regions in the 11 analyzed yeast species.Click here for fileModelling-B of size distribution of inter-CDS regions for the remaining eight species; this figure is complementary to Figure Click here for fileSame as Figure 3& Additional file 5combined, but showing only results from simulations with 'uncorrected' distances.Click here for file


Pulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images.The elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining.The present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages. Pulmonary alveolar proteinosis is a rare disease characterized by dense accumulation of surfactant and phospholipids in the alveoli and distal airways . ProgresThe aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis for particles and intracellular iron in lung tissue specimens combined with Berlin blue staining for iron.PAP lung tissue was obtained from a 64-year-old woman at video-assisted thoracoscopic surgery (VATS). She was a hairdresser, and a current smoker (10 pack-years). Serum anti-GM-CSF antibody was negative analysis. Pathological examination revealed interstitial pneumonia with interstitial fibrosis and periodic acid-Schiff (PAS)-positive material in the alveolar spaces. The pathological diagnosis was pulmonary alveolar proteinosis. As a control, normal lung tissue was obtained from a 72-years-old woman with lung cancer of adenocarcinoma. She was a housewife, and a never smoker without history of occupational exposure of particles. She received a lobectomy at surgical resection, and the normal lung of the margin of tumor was used for the analysis. Tissues were subjected to in-air micro-PIXE analysis and Berlin blue staining for iron.For in-air micro-PIXE analysis, paraffin-embedded lung tissue specimens were cut into sections 5 μm thick. Each section was dried, placed onto 5 μm polycarbonate film, and fixed in the sample holder as described previously . After iBerlin blue staining revealed that basically, two morphologic characteristics of present PAP case needed to study, i.e. in lung tissue cells with dense particles and alveolar macrophages in the alveoli digesting deposits of surfactant. Elemental analysis of the PAP lung tissue was performed on an area containing dense particles phagocytosed by macrophages (54 μm × 61 μm) with the focused beam. High Kα peaks of magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganese (Mn), iron (Fe), and zinc (Zn) were obtained. The Kβ peak of Fe appeared separately from Kα peak, and near the peak of cobalt (Co) (data not shown). The elemental map showed a high Fe contents strongly associated with Si, as well as metals in the particles. Serial sections of lung tissue with Berlin blue staining showed dense black particles that had been phagocytosed and accumulated in iron-rich alveolar macrophages Figure .Elemental analysis of the alveolar macrophages from a surfactant-rich area (54 μm × 61 μm) with the focused beam area showed high S and Fe peaks Figure , howeverThe Fe/P ratios calculated by in-air PIXE analysis were 0.28, 0.36 and 0.0036 for a dense particles phagocytosed by macrophages in PAP, an alveolar macrophage in surfactant-rich area of PAP and an alveolar macrophage of control, respectively.Disturbance of iron homeostasis has been reported in PAP , and alvCases of PAP had been reported in association with occupational and environmental exposure to substances such as indium oxide, indium-tin oxide, silica, titanium, aluminum, cotton, and fibrous material -8. A recAs a factor in the onset of PAP, iron-induced oxidative stress and lysosomal rupture following the disturbance of iron homeostasis may play a role ,11. In tApplication of in-air micro-PIXE is possibly useful for evaluation of iron as a disease marker of PAP, assessing the distribution of iron in particles and alveolar macrophages, and for determining the intracellular iron content in alveolar macrophages. Secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.The authors declare that they have no competing interests.YS designed this study, prepared the sample, immunostained the lung tissues, analysed the datas, and wrote this manuscript. SM prepared the sample, analysed the datas and irradiated to the sample. NY prepared the sample, TS analysed the datas, irradiated the sample and gave useful suggestion on this study. MK, AY, TO, YI, TK irradiated to the sammple. KD irradiated the sample and gave useful suggestions on this study. MM gave useful suggestion on this study.


Although the soleus (Sol), medial gastrocnemius (MG), and lateral gastrocnemius (LG) muscles differ in function, composition, and innervations, it is a common practice is to investigate them as single H-reflex recording. The purpose of this study was to compare H-reflex recordings between these three sections of the triceps surae muscle group of healthy participants while lying and standing during three different ankle positions.The Sol, MG and LG muscles' H-reflexes were recorded from ten participants during prone lying and standing with the ankle in neutral, maximum dorsiflexion, and maximum plantarflexion positions. Four traces were averaged for each combination of conditions. Three-way ANOVAs (posture X ankle position X muscle) with planned comparisons were used for statistical comparisons.p ≤ .05).Although the H-reflex in the three muscle sections differed in latency and amplitude, its dependency on posture and ankle position was similar. The H-reflex amplitudes and maximum H-reflex to M-response (H/M) ratios were significantly 1) lower during standing compared to lying with the ankle in neutral, 2) greater during standing with the ankle in plantarflexion compared to neutral, and 3) less with the ankle in dorsiflexion compared to neutral during lying and standing for all muscles (Varying demands are required for muscles activated during distinctly different postures and ankle movement tasks. Hoffmann reflex (H-reflex) recordings from muscles around the ankle joint are useful in examining the activation and inhibition during normal function. It is also useful for examining dysfunctional pathologies. For example, H-reflex recordings from the soleus (Sol) muscle are useful electrophysiological procedures for the evaluation of patients with S1 radiculopathy .Existing H-reflex procedures do not allow valid examination of the L5 nerve root. Clinicians either measure the L4 and L5 nerve roots by recording the vastus medialis H-reflex or measure the L5 and S1 nerve roots by recording the Sol H-reflex. Then inference is used to diagnosis L5 root impingement with a corroborating physical exam and/or radiographic imaging.H-reflex recordings from the lateral gastrocnemius (LG) would provide L5 diagnosis in patients with radiculopathy, given that the LG is innervated by L5 more than S1 nerve roots and the Sol is innervated mostly by the S1 nerve root . Thus, iNo previous study has compared H-reflex recordings between the LG, medial gastrocnemius (MG), and Sol during varied body postures, foot positions, and muscle function. Yet, it is commonly accepted that the Sol, LG and MG which function as members of the triceps surae muscle group have similar activation patterns, although they perform different functions , differ Ten healthy males  participated in this study. None of the participants reported any history of musculoskeletal, metabolic, systemic, or neurologic disorders within the past two years. All participants read and signed an informed consent approved by the Institutional Ethical Review Board.The MG, LG, and Sol H-reflexes were stimulated and recorded according to the method of Sabaahi and Khalil  and AlroIn brief, an EMG  unit  electrically stimulated and recorded the MG, LG, and Sol H-reflexes. To stimulate the H-reflexes, one Ag/AgCl surface stimulating bar electrode was applied over the tibial nerve longitudinally at the midline of the popliteal fossa with the active electrode positioned proximal to the reference electrode to avoid anodal block  , maximum active dorsiflexion, or maximum active plantarflexion position over the end of a treatment table. In the standing position, each participant was asked to perform each of three standing conditions: standing upright relaxed ; standing on heels (maximum active dorsiflexion); or standing on metatarsals (maximum active plantarflexion) while maintaining equal weight on both lower extremities. Maximum muscle activation during plantarflexion and dorsiflexion while lying and standing was used to simulate strenuous activities of the feet during daily living activities. The orders of recordings from the muscles, postures and ankle positions were randomly alternated to compensate for the potential inherent changes of the H-max recording during the course of the experiment . Five trThe maximum peak-to-peak amplitude and latency of the largest four traces from the Sol, MG, and LG H-reflexes were measured and averaged for each testing position. Also, the peak-to-peak amplitude of the maximum M-wave was measured. Then, the H/M (maximum H-reflex to maximum M-response) ratio was calculated for each muscle/posture/ankle-position condition.Means and standard deviations for the Sol, MG, and LG H-reflex maximum amplitudes, latencies, and H/M ratios for each condition were calculated over subjects. Statistical analysis included two dependent variables (H-reflex maximum amplitude and H/M ratio) and three independent variables: 1) muscles ; 2) ankle positions ; and 3) body postures (prone lying and standing). A 3 × 3 × 2 (muscles X ankle positions X body postures) repeated measures ANOVA with planned comparisons was performed with a global alpha level of .05. Planned comparisons included the mean differences of H-reflex maximum amplitudes and H/M ratios recorded from each muscle during prone lying and standing in dorsiflexion and plantarflexion positions to the mean values when the ankle was in the neutral position. Planned comparisons of H-reflex means were also made among muscles during prone lying and standing with the ankle in the neutral position. Statistical analyses were conducted using SPSS .p = .006, .009, and .029; Figure p = .0005 and .007 for Sol, Figure p = .001 and .04 for MG, Figure p = .0005 and .0005 for LG, Figure p = .2, .19, and .47), and the Sol, MG, and LG maximum amplitude was significantly greater during ankle plantarflexion compared to the neutral ankle position during the standing posture . The results for the Sol, MG, and LG H-reflex H/M ratio were comparable to the H-reflex maximum amplitude results during prone lying or standing for all ankle positions , Sol and LG (p = .0005), but not between the MG and LG (p = .49) across all ankle positions and both postures.Table The two primary results of this study showed: 1) The Sol, MG, and LG H-reflexes during prone lying and standing for three active ankle positions were similar in some respects. The Sol, MG, and LG H-reflex maximum amplitudes were inhibited during standing compared to lying with the ankle in the neutral position, inhibited during dorsiflexion compared to the neutral ankle position during lying and standing, and facilitated during plantarflexion compared to the neutral ankle position while standing; and 2) The Sol, MG, and LG muscle activities differed in terms of reflex recruitment (peak-to-peak amplitudes and thresholds of stimulation).The similarities between the muscles  H-reflex average maximum amplitudes, particularly during standing, may be explained by their role as antigravity postural muscles in which the suppression of these muscles would prevent body instability (swaying) and retain balance necessary for upright posture. Similarities may also be explained by how these muscles perform during locomotion .The suppression of the Sol, MG, and LG H-reflex maximum amplitudes during standing upright may be due to presynaptic inhibition, in which somatosensory afferents from the foot sole and/or stretch receptors from the soleus muscle may have been the source of presynaptic inputs. This was previously reported as a possible mechanism for this unintuitive result of greater H-reflex amplitudes during prone lying compared with active standing ,8,10-14.The facilitation of the Sol, MG, and LG H-reflexes during standing on metatarsal (maximum plantarflexion) may be explained by the increase in the excitability drive to the alpha motoneurons of the Sol, MG, and LG muscles as subjects contracted these muscles against gravity on the body's mass . By compThe inhibition of the Sol and MG H-reflexes during ankle dorsiflexion and facilitation during ankle plantarflexion reported in this study were in agreement with a previously reported study . PinnigeAlthough the major goal of this study was to establish the normal activation patterns of the muscles around the ankle joint under varied recordings condition, the study findings may be of clinical relevance. It provides a reference standard for comparison of patients with L5 nerve root impingement, as existing H-reflex procedures do not allow valid examination of the L5 nerve root. However, the validation of the clinical diagnosis of L5 and S1 radiculopathies requires patients with proven radiculopathies at different levels and it is the focus of ongoing research. Nevertheless, testing patients with proven radiculopathies using the H-reflex has been shown to be more useful during standing as compared to lying. It increases the sensitivity of the H-reflex for detecting subtle changes . Thus, fThe differences between the Sol, MG, and LG H-reflex maximum peak-to-peak amplitudes and H/M ratios reported in this study were in agreement with the work of Tucker and Türker . The difIn this study, tibialis anterior muscle activity was not monitored before or during active ankle movements while prone lying and standing. Previous research showed co-contraction of agonist and antagonist muscles depressed the H-reflex amplitudes . The preThe results of this study suggested the varying demands for static/tonic standing requires multiple physiologic responses around the ankle which are performed by the multi-task triceps surae muscle group. The results also suggested the sensitivity of H-reflex procedures to detect subtle changes in physiologic activity of functionally closely related muscles  bundled in one compartment group (triceps surae) but perform contractions across the multiaxial ankle joint. The results of this study provide a reference standard for comparison of patients with nerve root impingement at the L5 and S1 levels. Future studies of patients with L5/S1 radiculopathies may find it beneficial to use this method of examining Sol, MG, and LG H-reflexes during plantarflexion, neutral and dorsiflexion standing for differentiating L5 and S1 impingements.The authors declare that they have no competing interests.HNM contributed to the following activities: concept and research design, writing, project management, and data collection and analysis. MAS contributed to the following activities: concept and research design, and writing. BE contributed to writing and consultation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/65/prepub


Disk diffusion test is the usual applicable method for assessing the antimicrobial susceptibility pattern in most institutions and hospitals. The aim of this study was to determine the reliability of resistant-reported results of disk diffusion test for 6 routinely used antibiotics against Gram-positive microorganisms of nosocomial origin, using E-test method.e.g. blood, tracheal secretions, wound secretions, urine, etc.) were obtained from hospitalized patients with defined nosocomial infection and were cultured. Isolated Gram-positive bacteria underwent disk diffusion test for cephalothin, oxacillin, clindamycin, ciprofloxacin, vancomycin, teicoplanin (only for Enterococci), and meropenem antibiotics. E-test method was performed for all isolates resistant or intermediately sensitive to the disks of any mentioned antibiotics.Over a 1-year period, clinical specimens  and Enterococcus spp .Data showed compatible results of disk diffusion test with the results of E-test method for cephalothin, oxacillin, ciprofloxacin, vancomycin, and teicoplanin. None of ciprofloxacin- and vancomycin-resistant isolates in disk diffusion test showed sensitivity in E-test method. Significant differences between the results of disk diffusion and E-test methods were observed for clindamycin and meropenem against In order to increase the reliability of antimicrobial susceptibility results, it is recommended to perform E-test for nosocomial Gram-positive microorganisms that show antibiotic resistance by disk diffusion test and it is more important for clindamycin and meropenem. Staphylococcus aureus and Enterococcus spp. are extremely important pathogens in the hospital environment  were obtained from hospitalized patients with nosocomial infection diagnosed using the Centers for Disease Control and Prevention/National Healthcare Safety Network (CDC/NHSN) definition of health care-associated infections , clinical specimens (fections  and sentfections . After tfections . AntibioTo determine the reliability of disk diffusion test and the MIC (minimum inhibitory concentration) of applied antibiotics for bacterial isolates, E-test method was performed for all resistant isolated or intermediately sensitive to disks of any above-mentioned antibiotics using the antibiotic E-test strips  according to the manufacturer’s guidelines. After the inoculation of Mueller-Hinton agar culture medium (blood agar for Streptococci) with a direct saline suspension of isolated colonies adjusted to 0.5 McFarland turbidity standard, E-test strips were placed on the agar surface. After 16-20 h of incubation (24 h for vancomycin and teicoplanin) at 35°C, the MIC values, where the edge of the inhibition ellipse intersects the side of the strip, were read. The results were interpreted as either sensitive, intermediate or resistant according to the MIC values using manufacturer’s breakpoints.Statistical analysisSPSS software (version 17) was used for statistical analysis. For comparing the susceptibility results between the resistant microorganisms in disk diffusion and E-test methods, chi-square and fisher exact test were used. P-value less than 0.05 was considered significant.Staphylococcus aureus was the most frequently isolated microorganism  followed by Enterococcus spp. , Staphylococcus epidermidis , Staphylococcus hemolyticus , Streptococcus group D , Streptococcus pneumoniae , and Streptococcus viridans . All of the Staphylococci were sensitive to vancomycin; therefore, vancomycin E-test was not performed for these strains.During the study period, a total of 137 isolates of nosocomial Gram-positive bacteria were obtained. S. aureus  and Enterococcus spp . There were not any significant differences between the disk diffusion and E-test results for other microorganisms.Periodical assessment of common microorganisms’ resistance pattern in the hospitals is essential for the selection of appropriate antibiotic regimen in patients with manifestation of an infection. Proper patient evaluation, collection of suitable patient’s biological sample and coordination with an expert clinical microbiology department help the health care workers accordingly. There are some controversies about the results of methods that were used for the assessment of microorganisms’ antimicrobial susceptibility. Disk diffusion method is the routine laboratory test for microorganisms’ antibiotic susceptibility test in our hospitals. The results of this test are reported as susceptible, intermediate resistant, or resistant. With the development of microbial resistance and change of bacterial sensitivity to antibiotics, it is important to evaluate the MIC for each pathogenic microorganism. Serial dilution or E-test stripes are used for determining MIC. Serial dilution is a precise but time consuming and personnel-dependent method.et al., E-test method was carried out for E.coli strains resistant to five antibiotics in disk diffusion test. By E-test method, 47.7% of strains were sensitive to nitrofurantoin, 21.1% sensitive to gentamicin and 10.5% sensitive to cotrimoxazole, ciprofloxacin and ceftazidime  susceptibility to these antibiotics may show resistance to disks. Moreover, due to some reports of reduced susceptibility of clinically significant Staphylococci to glycopeptide antimicrobials such as vancomycin (Staphylococcus strain show resistance to its disk. The same conclusion was made by a study that compared these two methods of vancomycin against the coagulase-negative Staphylococci isolates (CoNS)  ; in thisIn order to increase the reliability of antimicrobial susceptibility results, it is recommended to perform E-test for nosocomial Gram-positive microorganisms that show antibiotic resistance via disk diffusion test and it is more important for clindamycin and meropenem.


The autophagic vacuolar myopathies (AVM) are a group of inherited myopathies defined by the presence of autophagic vacuoles in pathological muscle specimens. AVM can be categorized into three groups: acid maltase deficiency, myopathies characterized by autophagic vacuoles with unique sarcolemmal features, and rimmed vacuolar myopathies (RVM). While the pathogeneses of these conditions are still being elucidated, some drugs  can also cause AVM. Minocycline is a disease-modifying anti-rheumatic drug that may be used in the treatment of rheumatoid arthritis (RA). Here, we describe the first case of minocycline-associated AVM with rimmed vacuole formation.A 75-year-old woman suffering from RA has been continuously treated with minocycline (200 mg/day) for the past 7 years. During this time, she developed a myopathy that predominantly affected her lower limbs. Histological studies of biopsied muscle revealed scattered atrophic myofibers with rimmed vacuoles that contained pigment granules. Histochemical staining revealed that the pigment comprised both iron and melanin, which is consistent with type II minocycline-induced cutaneous pigmentation. Under electron microscopy, autophagic vacuoles were consistently observed in association with numerous collections of pigment granules.This is the first report of minocycline-induced pigmentation in skeletal muscle. The strong association between autophagic vacuoles and the accumulation of minocycline-induced pigments suggest that long-term minocycline treatment induced pigment accumulation, leading to elevation of autophagic activity and RVM. It might also be possible that minocycline directly activated autophagy, as the observed pigments are known to form complexes containing minocycline and/or its metabolites. As long-term minocycline treatment is expected to be used more widely in the future, we must draw attention to this adverse effect. Autophagy, which is a lysosomal degradation pathway that is essential for cell survival , plays aMinocycline is a semi-synthetic derivative of tetracycline that functions as a broad-spectrum antimicrobial agent and has proven to be a moderately effective disease-modifying anti-rheumatic drug in the treatment of rheumatoid arthritis (RA) . The druA 75-year-old woman was admitted to our hospital due to a 2-year history of gradually progressive gait disturbance. She had suffered from RA since the age of 25. Because of an exacerbation of this disease, she started taking prednisolone (5 mg/day) and sulfasalazine at 67 years, and had been continuously treated with minocycline (200 mg/day) since the age of 68. She noticed a gradual increase in blue-black skin pigmentation on both legs at 72 years. Thereafter, she began to have difficulty walking, and would frequently catch the tip of her foot on the ground. No exposure to other drugs known to cause pigmentary changes was recorded.Examination of the patient revealed blue-black pigmentation related to minocycline therapy on the distal parts of the legs Figure . The musThe biopsied specimens were snap-frozen in isopentane-liquid nitrogen, and cryosections were stained with a standard battery of histological and histochemical reactions. Paraffin-embedded tissue sections were also stained with Prussian blue stain (for iron), Masson Fontana preparation (for melanin) and hydrogen peroxide melanin bleach. Histopathological studies of the biopsied muscle revealed modest variability in myofiber diameter with scattered angular atrophic fibers Figure A. There Electron microscopy was performed on glutaraldehyde-fixed tissue using a standard electron microscopic processing protocol and a transmission electron microscope. Electron microscopy revealed that the pigment granules were localized in the cytoplasm of histiocytes, in the spaces between myofibers, and adjacent to small blood vessels Figure A. In theIn addition to the standard battery of histological stains, the sural nerve biopsy specimen was embedded in epoxy resin and examined by light and electron microscopy. Light microscopy of the sural nerve showed a reduction in the number of large diameter myelinated fibers, but no other specific features Figure F. HoweveThe incidences of minocycline-induced cutaneous pigmentation have been reported in up to 41% of patients with RA who take minocycline . HistochThe sural nerve biopsy demonstrated the features of a chronic axonal neuropathy, with minimal pigment observed in the cytoplasm of Schwann cells. Only a few cases of minocycline-associated sensory neuropathy have been reported, and axonal-type sensory neuropathy is a common complication in RA . Thus, iTissue pigmentation due to minocycline treatment is not usually harmful . HoweverOne year after the cessation of minocycline therapy, our patient exhibited no worsening of gait disturbance. There was little improvement in muscle weakness, however, perhaps because she had taken minocycline for an extremely long duration even after the appearance of skin pigmentation. This is an unusual condition. However, long-term minocycline treatment may be used more widely in the future, as on-going clinical trials are assessing the therapeutic use of minocycline as an anti-inflammatory agent in a broad range of disorders. Thus, we must draw attention to this adverse effect because its insidious onset, slowly progressive course, and lack of creatine kinase elevation could complicate its prompt identification in patients.Chloroquine and hydroxychloroquine are anti-malarial drugs that are also used to treat RA and systemic lupus erythematosus. Hydroxychloroquine is used much more frequently than chloroquine, as the latter is more likely to cause irreversible retinal damage. Long-term hydroxychloroquine treatment rarely causes myopathy and sensory neuropathy; the former typically manifests with an insidious onset and normal-to-mildly-elevated creatine kinase levels, and muscle biopsy consistently reveals curvilinear bodies and muscle fiber atrophy with vacuolar changes ,22. AfteMinocycline has proven to be an effective disease-modifying anti-rheumatic drug. However, we herein report for the first time that long-term minocycline treatment may cause RVM via the accumulation of minocycline-induced pigmentation in skeletal muscle. The clinical features of minocycline-associated RVM are very similar to those observed in hydroxychloroquine myopathy, suggesting that there may be a common pathogenic mechanism underlying these conditions. As long-term minocycline treatment may be used more widely in the future, we must draw attention to this adverse effect.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-In-Chief of this journal.AVM: Autophagic vacuolar myopathy; RVM: Rimmed vacuolar myopathy; RA: Rheumatoid arthritis; p62: p62/SQSTM1; TDP-43: TAR-DNA binding protein-43; DMRV: Distal myopathy with rimmed vacuoles.The authors declare that they have no competing interests.. All authors read and approved the final manuscript.Study concept and design: KS. Acquisition of the data: KB, ST, KS. Analysis and interpretation: KB, KS, KM, SM, TK. Wrote the paper: KB, KS, SMThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/12/140/prepub


Community-acquired pneumonia (CAP) is one of the most common infections presenting to the emergency department (ED). Increasingly, antibiotic resistant bacteria have been identified as causative pathogens in patients treated for CAP, especially in patients with healthcare exposure risk factors.in vitro activity against the isolated pathogen, served as the primary end point. Information regarding demographics, severity of illness, comorbidities, and antibiotic treatment was recorded. Logistic regression was used to determine factors independently associated with inappropriate treatment.We retrospectively identified adult subjects treated for CAP in the ED requiring hospital admission (January 2003-December 2011). Inappropriate antibiotic treatment, defined as an antibiotic regimen that lacked The initial cohort included 259 patients, 72 (27.8%) receiving inappropriate antibiotic treatment. There was no difference in hospital mortality between patients receiving inappropriate and appropriate treatment . Hospital length of stay  and 30-day readmission  were greater among patients receiving inappropriate treatment. Three variables were independently associated with inappropriate treatment: admission from long-term care , antibiotic exposure in the previous 30 days , and chronic obstructive pulmonary disease .Inappropriate antibiotic treatment of presumed CAP in the ED negatively impacts patient outcome and readmission rate. Knowledge of risk factors associated with inappropriate antibiotic treatment of presumed CAP could advance the management of patients with pneumonia presenting to the ED and potentially improve patient outcomes. Community-acquired pneumonia (CAP) is an important infection globally, and accounts for significant morbidity, mortality, and economic burden -5. MortaPatients with HCAP risk factors are at increased risk of infection with MDR pathogens -16. PhysLegionella pneumophilia serogroup 1 was used to document infection with this pathogen. The Washington University School of Medicine Human Studies Committee approved the study.We retrospectively identified all hospitalized adult (age >18 years) patients diagnosed and treated for culture-positive CAP in the ED between January 1, 2003 and December 31, 2011. Presumed CAP was defined as a new or progressive radiographic infiltrate plus one of the following: temperature greater than 38.3°C, WBC greater than 10,000/μL, or purulent tracheal secretions. One investigator (MHK), blinded to the clinical and microbiologic information adjudicated the chest imaging. To be included, patients had to be treated in the ED with a CAP antimicrobial regimen that included at our institution ceftriaxone plus azithromycin or moxifloxacin. Identification of pathogens was based on the results of cultures obtained in the ED from blood, pleural fluid, sputum, or the lower airways. A positive urinary antigen for in vitro activity against the isolated pathogen, were compared to those receiving appropriate antibiotic therapy. Covariates of interest included patient demographics, severity of illness, the Charlson score, and antibiotic treatment. Demographic factors included age, gender, and race. Comorbidities potentially influencing the isolated pathogen included residence in a long-term care facility, hospitalization in the last 90 days, receipt of antimicrobials in the last 30 days, end-stage renal disease requiring hemodialysis, and immunosuppression. Immunosuppression was defined as the presence of the human immunodeficiency virus (HIV), active malignancy undergoing chemotherapy, or treatment with immunosuppressants . Disease severity was assessed with the CURB-65 score, along with the need for either intensive care unit (ICU) admission or mechanical ventilation [in vitro activity against the pathogen(s) isolated and was administered within 6 hours of presentation.Patients receiving inappropriate therapy, defined as an antibiotic regimen that lacked tilation . InfectiWe completed univariate analyses with either the Fisher’s exact test or Student’s t-test as appropriate. Continuous, non-parametrically distributed data was compared via the Mann–Whitney U test. All analyses were two tailed, and a p value of < 0.05 was statistically significant. Logistic regression was used to determine independent factors associated with inappropriate treatment. Variables significant at p < 0.10 in univariate analyses were entered into the model. To arrive at the most parsimonious model we utilized a step-wise backward elimination approach. Co-linearity was explored with correlation matrices. Adjusted odds ratios (AORs) and ninety-five percent confidence intervals (CIs) are reported where appropriate. The model’s goodness-of-fit was assessed via calculation of the R2 value and the Hosmer-Lemeshow c-statistic. All analyses were performed with SPSS 11.0 .During the study period there were 1367 patients with culture-positive bacterial pneumonia admitted from the ED. Among these 1108 (81.5%) were diagnosed and treated for presumed HCAP, received initial treatment with broad-spectrum antibiotics and excluded from analysis. Of the 259 patients diagnosed and treated for CAP, 187 (72.2%) received appropriate antibiotic treatment and 72 (27.8%) received inappropriate treatment  followed by Pseudomonas aeruginosa (27.8%). Tracheal aspirates and bronchoalveolar lavage specimens were more often used to establish the microbiologic diagnosis among patients receiving inappropriate treatment. There was no difference in the presence of polymicrobial infection or secondary bacteremia between groups.With respect to infection-related characteristics, patients receiving appropriate treatment were more likely to be infected with ae Table . MRSA, PWe observed an association between comorbidities and inappropriate treatment. The median Charlson score was higher in patients receiving inappropriate treatment . Neurologic conditions such as dementia and stroke occurred equally between groups while other chronic diseases, such as chronic obstructive pulmonary disease (COPD), chronic kidney disease, and peripheral vascular disease occurred more often in patients receiving inappropriate treatment. Of the comorbidities evaluated, those linked to the definition of HCAP correlated with inappropriate treatment Table . For exaOverall, 129 (49.8%) patients were retrospectively found to have at least one HCAP risk factor. Patients having any of the HCAP risk factors were significantly less likely to receive appropriate antibiotic treatment compared to patients with no HCAP risk factors . Inappropriate antibiotic treatment was not associated with excess mortality . We found that almost one half of the patients treated for CAP in the ED had at least one risk factor for healthcare-associated infection. Healthcare-associated risk factors, including prior antibiotic exposure and long-term care residency, were found to be predictors for inappropriate antibiotic treatment. Our study is unique in showing that prior antibiotic exposure, presumably by predisposing to inappropriate antibiotic treatment, may also be associated with subsequent 30-day hospital readmission.These data add to the growing evidence elucidating the importance of discriminating between patients with pneumonia having healthcare-associated risk factors, including prior antibiotic exposure, from those without such risk factors. In one of the largest studies examining the epidemiology of pneumonia, approximately one-third of all patients with pneumonia admitted to the hospital were due to HCAP . SubsequThe importance of initial antibiotic therapy in patients admitted to the hospital with pneumonia is suggested by a recent medico-economic analysis conducted by Oster et al. . These iStreptococcus pneumoniae[The findings from our study also confirm the recent observation made by Shorr et al. that patients with pneumonia and healthcare-associated risk factors are more likely to require 30-day hospital readmission compared to pneumonia patients without healthcare-associated risk factors . Shorr aneumoniae, ventilaneumoniae and HCAPneumoniae. The comThere are several important limitations of our study that should be noted. First, the study was performed at an academic medical center and the results may not be generalizable to other institutions, especially those with a lower incidence of MDR pathogens. The large proportion of pneumonia patients with HCAP risk factors receiving broad-spectrum antibiotic therapy at our institution underscores this point. However, our findings are in agreement with those from other investigators supporting their overall validity -16,20. SAnother important limitation of our study is that not all healthcare-associated risk factors carry the same importance. A US study found recent hospitalization, residency in a long-term care facility, chronic hemodialysis, and admission to the ICU to be the best predictors of pneumonia due to an antibiotic-resistant pathogen . These fIn conclusion we found that inappropriate therapy for patients diagnosed and treated for CAP in the ED setting is relatively common. Patients receiving inappropriate therapy were significantly more likely to have HCAP risk factors compared to patients receiving appropriate therapy, suggesting that the identification of these risk factors, or recognition of their clinical importance, was underappreciated in these individuals. The association of inappropriate therapy with longer hospital lengths of stay and greater 30-day hospital readmission suggests that efforts to reduce inappropriate therapy in the ED would be beneficial. Several investigations have applied multi-disciplinary approaches to the management of such patients demonstrating improvements in antibiotic prescription to include more targeted use of antimicrobial agents ,36. FutuStaphylococcus aureus; MSSA: Methicillin-susceptible Staphylococcus aureus; NLFGNB: Non-lactose fermenting Gram-negative bacteria.AOR: Adjusted odds ratio; ATS/IDSA: American Thoracic Society/Infectious Disease Society of America; CAP: Community-acquired pneumonia; CI: Confidence interval; COPD: Chronic obstructive pulmonary disease; ED: Emergency department; HAP: Hospital-acquired pneumonia; HCAP: Healthcare-associated pneumonia; HIV: Human immunodeficiency virus; ICU: Intensive care unit; MDR: Multidrug-resistant; MRSA: Methicillin-resistant Kollef has served as a consultant, speaker for, or received grant support from: Cubist, Hospria, Merck, and Cardeas.Dr. Micek has received grant support from Cubist, Forest, Optimer, Merck, and Pfizer.The remaining authors have no potential conflicts.MHK, STM, NBH: had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis; contributed to the study conception and design, statistical analysis, drafting of the manuscript, and have given approval to the final version. AL: had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis; contributed to the study conception and data base construction, and drafting of the manuscript and has given approval to the final version. BMF: had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis; contributed to the study conception and design and drafting of the manuscript and has given approval to the final version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/61/prepub


MicroRNA-372 (miR-372) acts as either an oncogenic miRNA or an anti-oncomiR in various human malignancies. However, its roles in gliomas have not been elucidated. To address this problem, we here detected miR-372 expression in human gliomas and non-neoplastic brain tissues by real-time quantitative RT-PCR assay. The association of miR-372 expression with clinicopathological factors or prognosis of glioma patients was also statistically analyzed. As the results, miR-372 expression levels were significantly upregulated in glioma tissues compared to the corresponding non-neoplastic brain tissues (P<0.001). In addition, the high miR-372 expression was significantly associated with the advanced pathological grade (P=0.008) and the low Karnofsky performance score (KPS) of glioma patients (P=0.01). Moreover, the overall survival of patients with high miR-372 expression was dramatically shorter than those with low miR-372 expression (P<0.001). Furthermore, multivariate Cox regression analysis indicated that miR-372 expression was an independent prognostic factor for glioma patients (P=0.008). More importantly, subgroup analyses according to tumor pathological grade revealed that the cumulative overall survival of glioma patients with advanced pathological grades was significantly worse for high miR-372 expression group than for low miR-372 expression group (P<0.001), but no significant difference was found for patients with low pathological grades (P=0.08). Taken together, these data offer the convincing evidence for the first time that miR-372 may act as an oncogenic miRNA in gliomas and represent a potential regulator of aggressive development and a candidate prognostic marker for this malignancy, especially for advanced tumors with high pathological grades.http://www.diagnosticpathology.diagnomx.eu/vs/1707761328850011The virtual slide(s) for this article can be found here:  Human gliomas are a heterogeneous group of primary intracranial tumors for both children and adults . The ent′UTR regions . As determined by the log-rank test, the survival rate of patients with high miR-372 expression was significantly lower than those with low miR-372 expression  was significantly worse for high miR-372 expression group than for low miR-372 expression group (P<0.001, Figure Biomarker screening is an emerging field for neurooncology. Especially for gliomas, considerable progresses have been made in identifying, characterizing, and applying molecular markers. In the present study, we initially found that miR-372 was upregulated in human glioma tissues compared with non-neoplastic brain tissues. Then, the increased expression of miR-372 in glioma tissues was significantly correlated with advanced tumor progression and aggressive clinicopathological features. Next, the Kaplan-Meier analysis revealed that glioma patients with high miR-372 expression tend to have poorer overall survival. In addition, the multivariate analysis clearly demonstrated that high miR-372 expression was a statistically significant risk factor affecting overall survival in glioma patients, suggesting that miR-372 upregulation in gliomas is not only in a grade-dependent fashion, it is also a predictor of overall survival. Finally, subgroup analyses showed the significant prognostic value of miR-372 upregulation for glioma patients especially for those with advanced pathological grade.MiR-372, together with miR-371a, miR-371b and miR-373, belongs to miR-371~373 cluster which has been demonstrated to play important roles in tumorigenesis and tumor progression . Among tOur findings that miR-372 upregulation was associated with aggressive tumor progression mentioned above prompt us to investigate its possible prognostic value in glioma patients. According to the univariate and multivariate analyses, we identified miR-372 upregulation as an independent predictor for short overall survival of glioma patients, which was consistent with the findings of Yamashita et al.  in colonIn conclusion, our data offer the convincing evidence for the first time that miR-372 may act as an oncogenic miRNA in gliomas and represent a potential regulator of aggressive development and a candidate prognostic marker for this malignancy, especially for advanced tumors with high pathological grades. Further elucidation of the mechanism by which the oncogenic roles of miR-372 in gliomas are thwarted is worth to be done.The authors declare that they have no competing interests.SH and GG designed the study. GL and ZZ participated in the design and coordination, performed the molecular genetic evaluation, and drafted the manuscript. All the patients were followed up by YT and HL. And TJ and GC performed the statistical analysis, and joined into drafting the manuscript. GL, SH and GG all contributed to improving the draft of the manuscript. All authors have read and approved the final manuscript.