Streptococcus pneumoniae emerged in the U.S. and globally during the early 1990's. The RNA methylase encoded by erm(B) and the macrolide efflux genes mef(E) and mel were identified as the resistance determining factors. These genes are disseminated in the pneumococcus on mobile, often chimeric elements consisting of multiple smaller elements. To better understand the variety of elements encoding macrolide resistance and how they have evolved in the pre- and post-conjugate vaccine eras, the genomes of 121 invasive and ten carriage isolates from Atlanta from 1994 to 2011 were analyzed for mobile elements involved in the dissemination of macrolide resistance. The isolates were selected to provide broad coverage of the genetic variability of antibiotic resistant pneumococci and included 100 invasive isolates resistant to macrolides. Tn916-like elements carrying mef(E) and mel on the Macrolide Genetic Assembly (Mega) and erm(B) on the erm(B) element and Tn917 were integrated into the pneumococcal chromosome backbone and into larger Tn5253-like composite elements. The results reported here include identification of novel insertion sites for Mega and characterization of the insertion sites of Tn916-like elements in the pneumococcal chromosome and in larger composite elements. The data indicate that integration of elements by conjugation was infrequent compared to recombination. Thus, it appears that conjugative mobile elements allow the pneumococcus to acquire DNA from distantly related bacteria, but once integrated into a pneumococcal genome, transformation and recombination is the primary mechanism for transmission of novel DNA throughout the pneumococcal population.Macrolide resistance in  Streptococcus pneumoniae, the pneumococcus, remains a significant risk to human health. Treatment of pneumococcal disease has been hindered by emergence of resistance to the key antibiotics  and mel or the target-modifying RNA methylase gene erm(B). The mef(E)/mel operon confers the M-phenotype, that is, resistance to 14- and 15-membered macrolides but not lincosamides or streptogramin B  which is the MLSB-phenotype  and mel genes are located on the Macrolide Genetic Assembly, Mega /mel intergenic region. Expression of the mef(E)/mel operon is controlled by transcriptional attenuation and is inducible by 15- and 16-membered macrolides /mel operon are four co-transcribed open reading frames, orfs 3-6. This operon has organization and sequence similarity to a SOS-responsive operon on the pneumococcal conjugative transposons Tn5252 and Tn5253 . It is one of two ICE demonstrated to carry macrolide resistance determinants in pneumococci  located in the capsule biosynthesis locus. Class IV is in the RNA-methyltransferase rumA (SP_1029) located at the left flank of the pneumococcal pathogenicity island (PPI-1)  , named “erm(B) element,” inserted into Tn916 orf20 is Tn6002  is Tn2010  , mel and erm(B) by PCR as previously described .The capsule serotype of each isolate was determined by the institution from which it was acquired and verified by analyses of the capsule locus in the genome sequence. Minimal inhibitory concentrations (MIC) of erythromycin, clindamycin, tetracycline, and chloramphenicol were determined by microdilution or Etest . The Multi Locus Sequence Type (MLST) of selected isolates was determined by Sanger nucleotide sequencing of PCR products with MLST allele-specific primers as previously described  (Table http://www.beiresources.org).For each isolate sequenced, two shotgun libraries were constructed for 454 sequencing: one rapid non-paired-end library for coverage and one 3 kb paired-end library for assembly scaffolding. All isolates were sequenced using the 454 GS FLX Titanium to a depth of sequence coverage ranging from 16× to 57×. Shotgun reads were assembled into 4–32 contigs per isolate using Celera Assembler v6.1  Table  and the /) Table . Isolatehttps://www.perl.org) scripts and were implemented within the Ergatis framework (https://github.com/jorvis/ergatis)    to eliminate false positive SNP calls, only core SNPs in short sequences found in all 166 genomes were selected. Putative recombinogenic regions were not excluded. The set of 10,120 core SNP bases were concatenated from each of the input genomes to create a multi-FASTA file. This file was used for phylogenetic tree construction using the Geneious Basic software version 5.6.4 with neighbor-joining and the Hasegawa, Kishino and Yano (HKY) genetic distance model. The HKY model assumes that every base has a different equilibrium base frequency, and also assumes that transitions evolve at a different rate to transversions. A consensus tree was built based on 100 iterations to provide an estimate for the level of support for each clade in the final tree. We used a 100% consensus support threshold that resulted in a strict consensus tree where the clades included were those that were present in all the trees of the original set.Two high throughput reference-based pipelines were used to perform single nucleotide polymorphism (SNP) discovery and validation in 166 genomes using the TIGR4 genome as a reference , Tn5253   and others. Elements were characterized by the int genes they carried. Integrase genes were identified by BLASTN search with query sequences from described ICE including those mentioned above. All query sequences used are supplied as supporting information (Data File mef(E), mef(A), mel(msr(D)), erm(B), erm(A), tet(M) and cat  in the Atlanta metropolitan area . 74 displayed the M-phenotype as indicated by susceptibility to the lincosamide clindamycin and 24 were also resistant to clindamycin (MLSB-phenotype) (Table Isolates of e) Table . In addie) Table . In totae) Table .The 147 isolates from this study and 15 publically available genomes were compared using whole genome single nucleotide polymorphism (SNP) analyses. The resulting phylogenetic tree is shown in Figure mef(E)/mel intergenic region and 38 carried the 5,412 kb Mega type 2 (Mega-2) with a 99 bp deletion in the intergenic region . Analyses of the 454 raw reads revealed that both copies of the repeated sequence were contained on single reads and the tandem duplication region had greater than 40× sequence coverage. This confirmed that the duplication was not an assembly error. The authentic duplication resulted in an intergenic region that contained the 119 bp that immediately follows mef(E) in Mega-1, followed by a 38 codon ORF that includes the first 30 amino acids of Mel. The ORF had the mel RBS and appeared suitable for protein expression. Theoretically this could have a polar effect of mel translation, however, GA07643 had an erythromycin MIC of >16 μg/ml so it does not appear to have had a negative impact on resistance (Table mel overlapped the 3′ end of the duplicated ORF by 10 bp and maintained the native mel RBS. There was no correlation between the size of the mef(E)/mel intergenic region and the level of efflux-mediated erythromycin expressed by that strain (data not shown).Of the Atlanta invasive isolates, 86 contained Mega integrated into the pneumococcal chromosome backbone or nested within larger mobile elements. Mega was found in 17 serotypes, 16 clonal complexes and two singletons (Table ce Table . The ful916-like elements (class V), and in one novel site (Table mef(E) and mel , Mega-2 (n = 35) and the novel Mega-3 in strain GA07643   that encoded a tRNA methyltransferase and was located at the left end of the Pneumococcal Pathogenicity Island-1 (PPI-1)     contained the only class IVc Mega insertion identified (Tables S. suis 05HAS68 (Accession no. CP002007). Twenty-seven genomes contained Mega-1 inserted into orf6 of a Tn916-like element (class V)  or Mega-1.III (n = 1)  and seven serotypes  Table . Four dies Table . Tn6002 es Table . Tn2010 es Table .Tn916-like elements were integrated into four unique loci in the pneumococcal chromosome backbone and into several loci within Tn5253-like elements  encoding a hypothetical secreted protein  were independently acquired by a Tn916 residing in the SP_1947 locus. In addition, elements in this locus were identified in two clonal complexes  indicating transformation and recombination of the element in this locus between clonal complexes.To better understand the evolutionary relationships between these elements, the insertion site of each was determined. 2009 was also identified integrated into SP_1638 (TIGR4), an iron-dependent transcriptional regulator, in a single serotype 9V, CC156 isolate from 2007. The fact that no element was identified in this locus from an earlier collected isolate may indicate that this integration event occurred more recently than the SP_1947 integration. Likewise, a single isolate  from 2010 contained Tn6002 integrated into the ABC transporter gene SP_1438. Five serotype 6B, CC384 isolates contained Tn916 integrated into lytA (SP_1937) encoding an autolysin  involved in assembly of 23S ribosomes. Downstream of Tn5253-like elements in this locus is the immunoglobulin A1 protease gene iga as has been described previously  which encodes ribosomal protein L7/L12  and 19F (n = 2) and represent capsule switching from a PCV7 vaccine serotype to a non-vaccine serotype and a different vaccine serotype, respectively. This conclusion is supported by the close relationship between the isolates, all but one of which was also serotype 23F and CC81 . In both isolates with Tn3872, the element was integrated into orf20/orf18 in the same orientation as Tn2009 in this location  element between orf43 and orf20. These data suggest that de novo integration Tn916-like elements into pneumococcal chromosome backbones or ICE was infrequent relative to the rate of horizontal transfer by transformation and recombination.In the other composites identified, the TnS. pneumoniae is extremely plastic due to its natural competency and possession of numerous MGE. While transformation and conjugation are both involved in shaping the pneumococcal chromosome, the diversity of macrolide resistance elements, both independent and integrated into larger conjugative elements, raises the question of the relative role of each mechanism in the dissemination of macrolide resistance in pneumococci. Recombination events involving various macrolide resistance determinants were evidenced by an apparent interchangeability of Mega, the erm(B) element and Tn917 in Tn916-like elements. The conversion of Tn6002 and Tn6003 (containing aminoglycoside resistance gene aphA-3) has been reported  and was associated with Mega. However, the possibility of S. suis and S. equi having acquired these elements from the pneumococcus cannot be disregarded. In fact, there is no data to suggest that these elements cannot flow equally in both directions.We identified Mega inserted in the four previously described locations in the pneumococcal chromosome backbone  often integrate in Tn916-like elements  element, and Tn917 also carrying erm(B). All of these elements were integrated into the same location in the pneumococcal chromosome backbone: the ribobiogenesis GTPase, rbgA. The second group was similar to ICESp23FST81 and were all inserted into the ribosome protein encoding gene rplL. The conservation of insertion sites reflects the specificity of different integrases encoded on each element. Because of the specificity of these integrases, it is to be expected that they would integrate into the exact location repeatedly. Therefore, we can make no conclusions on the frequency of transfer of the element into or out of pneumococci.ICE appear to be well-established in the pneumococcal population. They were present during a rapid emergence of macrolide resistant invasive pneumococcal disease during the early to mid-1990's  genes carried on composite mobile elements.Comparison of the sequence types and serotypes of the pneumococcal isolates carrying macrolide resistance determinants showed closely related isolates with variable macrolide resistance elements indicating horizontal transfer. For example, in Figure 2010, which was not detected in the pre-vaccine era. Interestingly, Tn2010 was detected in only one chromosome backbone site and was not found in any ICE (Table The 7-valent Pneumococcal Conjugate Vaccine PCV7 was introduced into Atlanta in late 2000. We analyzed the genomic data in respect to the pre-vaccine and post-vaccine years. There appeared to be little effect on the types of elements harboring macrolide resistance determinants, however, the sequence types and serotypes harboring these elements were changed significantly. Some examples of this can be seen in the whole genome phylogenetic analyses where blue text indicated isolation in the pre-vaccine era and red indicated post-vaccine isolation (Figure 916-like element integration events appear infrequent, transformation and homologous recombination appear to be the primary means of horizontal transfer into the pneumococcus. With limited sites for insertion, the element has multiplied its capacity to disseminate throughout the pneumococcal population by co-opting the insertion sites of other elements. Thus, conjugation allows acquisition of DNA from more distantly related bacteria. Once in a pneumococcal genome, if beneficial and selected, the conjugated transposon is subsequently disseminated by transformation throughout the pneumococcal population.In conclusion, macrolide resistance dissemination in pneumococci has been greatly influenced by conjugation and transformation. Because Mega and TnThe 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.


Chronic Obstructive Pulmonary Disease (COPD) is characterized by an excessive activation of the adaptive immune system and, in particular, uncontrolled expansion of the B-cell pool. One of the key promoters of B cell expansion is A PRoliferation-Inducing Ligand (APRIL). APRIL has been strongly linked to non small cell lung cancer (NSCLC) onset and progression previously. However, little is known about the expression of APRIL in the lungs of COPD patients.1 33 ± 20 % predicted), 4 subjects with primary diagnosis of NSCLC, 4 subjects diagnosed with both COPD and NSCLC, smokers without COPD or NSCLC and 3 healthy never-smokers. The percentage of B cells, alveolar macrophages (AMs) and polymorphonuclear neutrophils (PMNs) in the lung and alveolar epithelial cells (AECs) that stained positively for APRIL was quantified using epi-fluorescence microscopy and image analysis software.Using immuno-fluorescence staining, the expression of APRIL was assessed in sections of lungs from 4 subjects with primary diagnosis of COPD . The percentage of APRIL-expressing AMs and AECs (but not in B cells) was higher in patients with NSCLC alone than in patients with COPD alone. The percentage of APRIL-expressing AECs (but not B cells or AMs) was higher in COPD patients than in SC and NSC . The percentage of APRIL-expressing B cells, AMs and AECs cells was similar in NSC and SC.The percentage of APRIL-expressing B cells, AMs, PMNs and alveolar epithelial cells (AECs) was higher in patients having both COPD and NSCLC than in patients with either COPD or NSCLC alone, SC or NSC (The percentage of APRIL-expressing B cells, AMs and AECs is higher in the lungs of patients with both COPD and NSCLC than in patients with COPD or NSCLC alone or control subjects. These findings suggest that APRIL may contribute to the pathogenesis of both COPD and NSCLC, and possibly to the development of NSCLC in patients with established COPD. Chronic obstructive pulmonary disease (COPD) is characterized by an enhanced pulmonary and systemic inflammatory response to inhaled particles and gases, particularly those found in tobacco smoke. COPD patients have an increased risk of developing non-small cell lung cancer (NSCLC) that is independent of smoking pack-year history , 2. HoweBoth innate immune cells  and adaptive immune cells (T and B lymphocytes) participate in the chronic inflammatory responses occurring in COPD lungs –10. ThisTNF-related death ligand-1), which share two receptors (B-cell maturation antigen (BCMA) and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). A third receptor, the BAFF receptor, only binds to BAFF [Regulation of B-cell homeostasis involves two members of the TNF-alpha family: B-cell Activating Factor (BAFF) and A proliferation-inducing ligand , or the Department of Pathology at Brigham and Women’s Hospital, Boston. None of the subjects studied had evidence of respiratory tract infection at the time of lung tissue sampling. Among the subjects studied, 8 subjects had a primary diagnosis of COPD , 4 subjects had a primary diagnosis of NSCLC, 3 were healthy ever-smokers (SC), and 3 were healthy never-smokers (NSC). Among the COPD patients, 4 had a secondary diagnosis of NSCLC.All studies conducted on human subjects were approved by the Partners Institutional Review Board. The characteristics of the lung tissue cohort studied are shown in Fig. 2 conjugated to Alexa-488 to identify APRIL-positive cells. PMNs were identified by their characteristic multi-lobed nuclei . AMs were defined as mononuclear cells (having ample cytoplasm) present in the alveolar spaces. Bronchial and alveolar epithelial cells were identified in bright-field images. To identify B cells, we immunostained the sections with a murine anti-CD20 IgG followed by a goat anti-murine IgG conjugated to Alexa-546. All of the antibodies (Abs) listed were purchased from Abcam  and secondary antibodies were obtained from ThermoFisher Scientific . Lung sections were also immuno-stained with appropriate isotype-matched non-immune control antibodies. Images of the immuno-stained lung sections were captured and analyzed using an epi-fluorescence microscope .Formalin-fixed and paraffin-embedded sections (5 μm thick) of peripheral lung sections from the subjects were studied. Lung sections were deparaffinized, and antigen (Ag) retrieval was performed by boiling the slides immersed in 0.01 M sodium citrate and 2 mM citrate buffer (pH 6.0) in a microwave. We used a rabbit anti-APRIL IgG followed by goat anti-rabbit F(ab’)In order to quantify APRIL expression in PMNs and B cells, PMNs and B cells in at least 50 non-consecutive high-magnification fields were analyzed. At least 20 AMs within the alveolar spaces were evaluated for each patient. Results were expressed as percentage of all AMs, PMNs and B-cells that were counted that stained positively for APRIL. For evaluation of APRIL in AECs, cells in the alveolar walls in 10 non-consecutive high magnification fields were evaluated for each subject. The area of the alveolar wall was measured using MetaMorph software . APRIL-positive cells within the alveolar walls were counted and the results were expressed as the number of positive cells/pixel of alveolar wall.1 % predicted and pack/years) followed by pair-wise comparisons using student’s t-tests or Mann-Whitney U tests. The Chi-square test was used to analyze categorical variables. P < 0.05 was considered statistically significant.Data were analyzed using one-way ANOVA tests for continuous variables  was not available for the NSC and was only available for one of the four patients with both COPD and NSCLC. In the latter group, COPD was diagnosed on the basis of the presence of emphysema in high resolution computed tomography (HRCT) scans of the thorax and an FEV1/FVC ratio < 0.7, but the FEV1% predicted was not available from the electronic medical records. There was a significant difference in FEV1 % pred between COPD patients and both SC and NSC by design.The demographic characteristics of the patients are shown in Table APRIL expression was detected in some B cells, AECs and AMs in lung sections from all subjects studied albeit at low levels in SC and NSC. No staining was present in bronchial epithelial cells in any of the subject groups, or in lung sections stained with non-immune isotype-matched non-immune antibodies.p < 0.005 for all comparisons). The percentage of APRIL-positive B cells was higher in lung sections from patients with NSCLC alone than in the lungs of SC or NSC . The percentage of APRIL-positive AMs was higher in patients with NSCLC alone than in patients with COPD alone or NSC . Although low-level positive staining for APRIL was detected in PMNs in lung sections from SC and NSC, we could not quantify the expression of APRIL in cells from these subjects due to the very small number of PMNs present in peripheral lung sections from these subject groups.The percentage of APRIL-positive PMNs was higher in lung sections from patients with COPD and NSCLC than in patients with either COPD alone or NSCLC alone . Thus, we were not able to correlate APRIL expression with COPD severity, pack-year smoking history, current versus former smoker status or NSCLC cell type. Another limitation of our study is that we did not measure the APRIL expression levels per cell (only the percentage of APRIL-expressing cells). We also did not correlate APRIL expression with readouts of cellular functions germane to COPD or NSCLC. These studies will be the focus of future studies in our laboratory.Our study has several limitations. First, we studied a small number of subjects. Patients with both COPD and lung cancer were significantly older than those with COPD alone. Second, in the group of patients with both COPD and NSCLC, only one out of four patients had PFT values available, and the pack-year smoking histories were not available for this group. In the latter group COPD was diagnosed using HRCT scans and FEVHerein, we report an increased APRIL expression in lung leukocytes and AECs in patients with COPD alone and NSCLC alone, and even greater increases in these cells in patients with both diseases. Our results suggest that exaggerated APRIL expression in lung leukocytes and/or AECs could create an environment conducive to the development and/or progression of NSCLC in patients with COPD.


It has been well-established that type I interferons (IFN-Is) have pleiotropic effects and play an early central role in the control of many acute viral infections. However, their pleiotropic effects are not always beneficial to the host and in fact several reports suggest that the induction of IFN-Is exacerbate disease outcomes against some bacterial and chronic viral infections. In this brief review, we probe into this mystery and try to develop answers based on past and recent studies evaluating the roles of IFN-Is in infection and immunity as this is vital for developing effective IFN-Is based therapeutics and vaccines. We also discuss the biological roles of an emerging IFN-I, namely IFN-ε, and discuss its potential use as a mucosal therapeutic and/or vaccine adjuvant. Overall, we anticipate the discussions generated in this review will provide new insights for better exploiting the biological functions of IFN-Is in developing efficacious therapeutics and vaccines in the future. Since the initial discovery of type I interferons (IFN-Is) as anti-viral agents , these cThe importance of IFN-Is in protecting hosts against pathogens has been demonstrated in several contexts. Firstly, IFN-AR deficient mice tend to be more susceptible to infection with viruses  compared to wild-type mice. Some examples include Henipavirus , acute Fin vitro where De la Maza and colleagues  of HIV-1 life cycle –53. HoweApart from their most celebrated role as direct anti-viral agents, IFN-Is have also been increasingly recognized as potent regulators of cellular immune responses. Of particular interest to vaccine development has been the ability of these cytokines to regulate adaptive immune responses and this aspect is discussed here.Dendritic cells are often crucial for initiating adaptive immune responses and serve as important targets for IFN-Is to regulate adaptive immunity. Exposure of IFN-Is facilitates maturation of DCs via increasing the expression of DC-associated chemokine receptors, co-stimulatory molecules, and major histocompatibility complex class I and class II antigen presentation –60. Consin vitro  could dictate the success of IFN-I treatment or IFN-I based vaccine formulations. Secondly, members of the IFN-I family have different binding affinities and kinetics to the IFN-AR subunits with current comparative studies suggesting that IFN-β has the highest affinity to IFN-AR and anti-viral capacity –113. A cCollectively, IFN-Is have shown considerable promise for the treatment of cancers and pathogen infections  in some clinical settings. IFN-Is are also promising for use as vaccine adjuvants, but the species of IFN-Is used for this purpose could have a significant bearing on adaptive immunity generated at certain immune compartments. For instance, IFN-β could be used to effectively enhance systemic T cell immune responses, whereas IFN-ε is more promising as an adjuvant to enhance mucosal T cell immunity in the lung and the gut mucosae.+CD107a+IFN-γ+ cell population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) highly functional CD8+CD4+ double positive T cell subset [CD3highC–C chemokine receptor (CCR)7highCD62Llow] in lung lymph nodes . Specifically, IFN-ε’s ability to enhance CD8epletion ] and alsThe dual roles of IFN-Is in providing beneficial and detrimental effects to the host in pathogen control is intriguing for developing IFN-I based vaccines and therapies. Lessons learned from acute viral infection models and studies comparing acute versus chronic infection states suggest that transient, but not sustained and/or excessive induction of IFN-Is is likely to confer protective outcomes. IFN-Is have also proven to be promising therapeutic agents against various pathogens and cancers and could also be used as vaccine adjuvants. The caveat here is that the vaccine vector used should ideally not chronically stimulate the production of IFN-Is, which is expected to be detrimental for the generation of robust adaptive immune responses. Our laboratory and others have demonstrated that IFN-ε has great potential to provide protective outcomes against not only mucosal viral infections, but also certain mucosal bacterial infections. Keeping this in mind, more studies need to evaluate the contribution of the different species of IFN-Is not just IFN-α and -β in immunity against infections. These studies are expected to pave way for the development of novel and effective IFN-I based vaccines/therapies against chronic pathogens and cancers.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.


The coupling of aortic wall thickening and stiffening has been recognized in hypertension. However, specific relationship between aortic wall thickness (AWT) and stiffness has not yet been documented in community-based studies. The purpose of this study is to evaluate associations between AWT and arterial stiffness measured by aortic distensibility and PWV in the MESA cohort.423 studies by 1.5-T whole-body MRI were analyzed. CMRI protocol included cardiac function and late gadolinium enhancement. Only participants with no findings of myocardial scar were included. AWT images were obtained using a double inversion recovery black-blood fast spin-echo sequence and phase contrast cine gradient echo sequence was used to evaluate aortic stiffness. Aortic sagittal oblique plane with black blood sequence was acquired to position the aortic imaging and allowed for the measurement of the distance between the ascending and descending aorta. Images of the ascending and descending aorta were obtained in the transverse plane at the level of the right pulmonary artery perpendicular to the vessel lumen. The thickness of the midthoracic descending aortic wall was measured using electronic calipers at 4 standard positions: 12, 3, 6, and 9 o'clock (QMASS 7.2). The average value of these 4 measurements was calculated. Distensibiliy of the ascending aorta and PWV were performed using validated automated software (ARTFUN. INSERM U678).Table We have demonstrated aortic functional alterations with hypertension (lower distensibility and higher PWV) and the coupling between vessel wall thickness and function. Without hypertension, morphological changes is associated more strongly with distensibility, however, PWV are more affected by aortic wall thickening in hypertension.This research was supported by contracts N01-HC-95159 through N01-HC-95168 from the National Heart, Lung, and Blood Institute.


Glioblastoma (GB) is a highly invasive primary brain tumor that almost systematically recurs despite aggressive therapies. One of the most challenging problems in therapy of GB is its extremely complex and heterogeneous molecular biology. To explore this heterogeneity, we performed a genome-wide integrative screening of three molecular levels: genome, transcriptome, and methylome. We analyzed tumor biopsies obtained by neuro-navigation in four distinct areas for 10 GB patients . We classified samples and deciphered a key genes signature of intratumor heterogeneity by Principal Component Analysis and Weighted Gene Co-expression Network Analysis. At the genome level, we identified common GB copy number alterations and but a strong interindividual molecular heterogeneity. Transcriptome analysis highlighted a pronounced intratumor architecture reflecting the surgical sampling plan of the study and identified gene modules associated with hallmarks of cancer. We provide a signature of key cancer-heterogeneity genes highly associated with the intratumor spatial gradient and show that it is enriched in genes with correlation between methylation and expression levels. Our study confirms that GBs are molecularly highly diverse and that a single tumor can harbor different transcriptional GB subtypes depending on its spatial architecture. Glioblastoma (GB) is the most frequent and most aggressive malignant primary brain tumor , 2. The The clinical hallmarks of GB are its aggressive growth and inexorable recurrence despite multimodal therapy. Complete surgical resection of these infiltrative tumors is virtually impossible and GB almost systematically recurs after therapy, mainly at the margin of the resection cavity in the peritumoral brain zone , 13. DecGrand Ouest Glioma Project: ‘from the core to beyond the margin’ (GOGP) is a translational program based on a multimodal analysis of GB areas  (6 cases), but also the [ and Classical] combination (1 case). FT08 presented strong tumor heterogeneity with two Mesenchymal and two Classical biopsies (p = 9.10-9)  Figure . This hiMesenchymal genes  and that the turquoise module was enriched with Proneural (p = 0.002) and Neural (p = 0.02) genes.We performed weighted gene co-expression network analysis to map genes and pathways onto the intra-tumor heterogeneity highlighted by PCA. We restricted this analysis to the 8000 most informative probes (see methods). It identified seven co-expression modules hierarchically organized into two distinct groups Figure . The fisr < −0.6) in the whole dataset. To assess the importance of epigenetics mechanisms in the definition of intratumor heterogeneity, we analyzed the proportion of anti-correlated genes with respect to their contribution to the transcriptome PCA data structure. This proportion was compared to random samplings (n = 1000) of the genes. The percentage of anti-correlated genes significantly increased with the gene contribution to the PCA data structure. The highest percentage of anti-correlated genes  was reached for the top 370 genes  showed the presence of particular intratumor architecture as observed at the transcriptome level . There ws Figure .VEGF, CD44, GFAP, EZH2, CHI3L1, NES, and IGFBP2. Hierarchical clustering based on this selection conserved the inner structure of the data identified by PCA and the biological meaning of the coexpression modules  and NES (blue module).We focused on the difference between infiltrated brain zone and macroscopically normal brain . We selected the 100 most informative genes in terms of (i) importance in the PCA data structure, (ii) differential expression, and (iii) module membership (25 genes per significant module). Among these genes we found well-known GB genes such as: s Figure . As for s Figure . SixteenProneural subtypes and the other in Classical/Mesenchymal subtypes. Most of the time, different tumor samples from the same patient were classified into these different subgroups. In both studies, our key cancer-heterogeneity signature showed that the Neural subtype was clearly associated with biopsies performed in the margin/non-enhancing tumor region.We performed external validations of the key cancer-heterogeneity signature with two independent studies of GB intratumor heterogeneity , 31: theThe genomic profiling of the GB samples included in our study confirmed the presence of common glioblastoma alterations reported in previous studies : loss/paAt the transcriptome level, we observed that the molecular heterogeneity was much more important within tumors than between patients. PCA-based analysis highlighted zone-specific profiles with pronounced intratumor architecture reflecting the surgical sampling plan of the study: ‘from the core to beyond the margin’. Our findings also support the implicit notion of coexpression network analysis that genes with similar expression behavior are related biologically. Indeed, the gene modules identified and their functional enrichments clearly evidenced modularity in the GB transcriptome, a feature already evidenced by Bredel and colleagues . These mCHI3L1/YKL-40, brown module) is a mesenchymal marker that promotes tumor angiogenesis and malignancy . This β-value provides a continuous and quantitative measurement of DNA methylation, ranging from 0 (completely unmethylated) to 1 (completely methylated). Missing values were imputed by nearest neighbors averaging (impute R package). All samples were G-CIMP negative [MGMT unmethylated [DNA methylation profiling was performed with the Infinium HumanMethylation27 beadchip (Illumina Inc.). DNA from GBM samples and control brains were bisulfite-modified, using the EZ DNA methylation kit (Zymo Research) and hybridized according to the manufacturer's instructions. For each interrogated CpG site, methylation status is calculated by dividing the signal from the methylated probe (M) by the sum of signals for both methylated and unmethylated (U) probes (negative . All samthylated  except Fn = 13612). Anti-correlated genes were defined as genes presenting a negative correlation between expression and methylation levels (Pearson correlation coefficient less than –0.6).Correlations between transcriptome and methylome data were computed on a gene basis for all the genes with available data on the two molecular levels  and Hierarchical Clustering on Principal Components (HCPC) to classify samples and select genes. PCA is a linear dimensionality reduction technique  that seeks to identify a small number of components that capture most of the relevant structure in a dataset. HCPC is an agglomerative hierarchical classification method based on the principal components of the factorial analysis. PCA and HCPC were performed on transcriptome data with the u et al.  in the fp-values were calculated by controlling for the FDR with the Benjamini & Hochberg (BH) procedure. Absolute fold-changes (FC) between two conditions were also computed. Genes were considered significantly differentially expressed between 2 conditions for an adjusted p-value lower than 0.001 and an absolute FC greater than 2.A one-way Welch's anova was performed to compare the mean gene expression levels between HCPC clusters and to identify genes up- or downregulated in at least one cluster. Adjusted p-values from the analysis of variance performed between HCPC clusters. All network analyses were performed using the WGCNA R package with default parameters except for the minModuleSize that was set to 175. Functional analyses were generated through the use of IPA  and GOMiner [We constructed a signed weighted gene co-expression network on the basis of the expression data of the most informative genes. Gene co-expression networks are increasingly used to explore the system-level functionality of genes. The network construction is conceptually straightforward: nodes represent genes that are connected if the corresponding genes are significantly co-expressed across appropriately chosen tissue samples. In a weighted gene co-expression network, a connection weight is assigned to each gene pair. This weight reflects the co-expression measure between the two genes (correlation) and is based on the network spatial relationship between these genes (adjacency) . Pearson GOMiner . For eacGAPDH  and TBP (TATA box binding protein) RNAs were chosen as internal controls. Calibration was performed with FirstChoice Human Brain Reference Total RNA (Ambion). The relative amounts of the gene transcripts were determined using the delta-delta-Ct method. Primers are provided in Q-PCR reactions were done with the 7900HT Fast Real-Time PCR System using the SYBR TMGreen PCR Master Mix (Applied Biosystems).


N = 46) and healthy controls (N = 57) using magnetoencephalography (MEG). Time-frequency decomposition was performed to determine regions of significant changes in gamma band power by group in response to unisensory and multisensory stimuli relative to baseline levels. Results showed significant behavioral differences between groups in response to unisensory and multisensory stimuli. In addition, time-frequency analysis revealed significant decreases and increases in gamma-band power in schizophrenia patients relative to healthy controls, which emerged both early and late over both sensory and frontal regions in response to unisensory and multisensory stimuli. Unisensory gamma-band power predicted multisensory gamma-band power differently by group. Furthermore, gamma-band power in these regions predicted performance in select measures of the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) test battery differently by group. These results reveal a unique pattern of task-related gamma-band power in schizophrenia patients relative to controls that may indicate reduced inhibition in combination with impaired oscillatory mechanisms in patients with schizophrenia.Deficits in auditory and visual unisensory responses are well documented in patients with schizophrenia; however, potential abnormalities elicited from multisensory audio-visual stimuli are less understood. Further, schizophrenia patients have shown abnormal patterns in task-related and task-independent oscillatory brain activity, particularly in the gamma frequency band. We examined oscillatory responses to basic unisensory and multisensory stimuli in schizophrenia patients ( On a moment-by-moment basis, behaviorally salient information reaches us from multiple senses and seamlessly shapes our perceptions and actions in the world. The capacity to organize the many stimuli arriving from different sensory modalities into a coherent and operable percept remains one of the marvels of the central nervous system. Despite a rich history exploring unisensory processes in the brain, understanding the processes involved in multisensory integration remains a central challenge in systems neuroscience.Empirical and computational approaches have revealed some of the neural mechanisms subserving multisensory integration at the cellular and systems level. Based on both human and animal studies, it is clear that a broad network of cortical . Though studies examining these multisensory cells have played an important role in advancing research in this area, research at the cellular level does not provide a complete view of multisensory integration. Gamma-band oscillations (>30 Hz), in particular, are implicated in aspects of feature binding  by establishing temporal synchrony both within and across cortical regions  and healthy controls (HC) to perform a multisensory integration task while brain activity was recorded using magnetoencephalography (MEG). A simple multisensory paradigm was employed by presenting both unisensory  stimuli as well as multisensory (AV) stimuli within a forced choice reaction time task. Based on the previously reported deficits in gamma-band oscillations, we hypothesized that gamma-band power would be reduced in SP relative to HC. The task also required that the participants identify the location of the stimulus within a perspective drawing, thereby presenting participants with near and far stimuli which corresponded to both peripherally- and centrally-presented visual stimuli, respectively. Peripherally-presented visual stimuli preferentially activate the dorsal visual stream . All participants provided written informed consent prior to study procedures. All procedures were performed in accordance with the Declaration of Helsinki and with prior approval from the University of New Mexico Health Sciences Center Human Research Review Committee. All SP met DSM-IV criteria for a diagnosis of schizophrenia or schizoaffective disorder, were stable on their medications for at least 1 month prior to study participation, and were periodically assessed throughout study enrollment to confirm clinical and pharmacological stability. HC and their first-degree relatives possessed no prior history of any psychiatric disorder based on the SCID-NP. None of the participants suffered from substance abuse, prior head trauma, or other neurological disorders, based on a standard neurological exam. Participant demographics are provided in Table To assess responses to unisensory and multisensory stimuli, participants performed a simple stimulus discrimination task. During the task, participants were presented with ecologically relevant audio-visual stimuli designed to mimic the image of a soccer ball and the sound of a soccer ball “bounce.” As control conditions, visual-only  and auditory-only  stimuli were also presented. During the presentation of all stimuli, participants viewed a static background on a projection screen positioned at a distance of 1 m from nasion. A simplified soccer field with a goalie and net provided a perspective-drawing framework, and participants were asked to fixate upon the goalie during all stimulus presentations Figure . There wThe visual-only near stimulus condition (VN): During the VN condition, the image of a black and white soccer ball appeared on the static soccer field background for 200 ms. The ball was centered at 8° below fixation and subtended 2.7° of visual angle, giving the appearance that the ball was “downfield” and near to the participant : During the VF condition, the soccer ball image appeared on the soccer field at 1.8° below fixation and subtended 1° of visual angle, giving the impression that the ball was “upfield” and farther away from the participant. The ball remained on the screen for 200 ms : During the AN condition, a 550 Hz tone was presented binaurally for 200 ms through a set of ear plugs at a volume of 63 dB above hearing threshold. Threshold was determined uniquely for each participant prior to task performance. No other visual stimuli were presented during the AN condition, other than the static soccer field background : The AF condition was identical to the AN condition except that the tone was presented at a lower volume of 45 dB above hearing threshold in order to mimic a more distant sound.The audio-visual near stimulus condition (AVSN): The AVSN condition consisted of the presentation of the VN stimulus followed by the AN stimulus with a 5 ms delay.The audio-visual far stimulus condition (AVSF): During the AVSF condition, the VF stimulus was presented and followed by the AF stimulus after a 5 ms delay.The audio-visual asynchronous near stimulus condition (AVAN) and The audio-visual asynchronous far stimulus condition (AVAF): The final two audio-visual conditions were identical to the AVN and AVF conditions except that the delay between the visual and auditory stimuli was increased from 5 to 50 ms to emulate the natural delay between the two stimulus types which occur at a distance from the participant due to the differential speed of light and sound.During each trial, one of the eight stimulus conditions was randomly presented. Participants were asked to indicate whether the stimulus was near to them or farther away by pressing one of two buttons on a response device with either their right index finger or right middle finger, respectively. During 20% of the trials, feedback regarding response accuracy was given. If the response was correct on an audio-visual trial, the image of the ball rolled into the soccer goal along with the sound of a cheering crowd. If an incorrect response or no response was given the ball rolled away from the goal accompanied by a “groaning crowd” sound. Unisensory trial feedback was matched to the sensory modality . Trial duration was between 1500 and 1900 ms and varied randomly from trial to trial, rendering an inter-stimulus interval (ISI) between 1300 and 1700 ms. Trials were organized into six blocks of 200 trials, separated by short breaks, where each condition was randomly presented with equal probability such that there were approximately 150 trials of each condition. Participants received pre-recorded instructions and were given a brief practice run to ensure that they understood the task prior to MEG data collection. The entire task, including auditory threshold determination, instructions, the practice run and the six trial blocks, was performed in a magnetically shielded room while participants were seated in the MEG chair. MEG and behavioral data used in the analyses were collected during the six trial blocks.martinos.org/mne).MEG data were collected with a whole head, 306-channel Elekta Neuromag system located at the Mind Research Network in Albuquerque, NM and data were acquired at a sampling rate of 1000 Hz with a 0.1 Hz high-pass and 330 Hz low-pass anti-aliasing filter. To permit comparisons between participants and groups, MEG sensor data for each participant were interpolated to the same reference head position using Neuromag Maxfilter software . Baseline-corrected spectral maps were computed by frequency for each trial in decibels (dB), with average spectral power from −100 to 0 ms relative to stimulus onset as the measure of baseline noise. Spectral power at each MEG gradiometer, time point, and frequency was then averaged across trials for each condition and participant. Based on the spatial specificity of planar gradiometers , three-way multivariate analysis-of-variance tests (MANOVAs) were performed on the reaction times and accuracy (% correct) for the unisensory and multisensory stimuli. In these tests, stimulus type  and stimulus location (near or far) were treated as within-subject factors, while group identification (SP or HC) was treated as a between-subjects factor. Reaction times were only assessed for correct responses. Separate three-way MANOVAs were performed comparing reaction times and accuracy in which synchrony (synchronous or asynchronous) and stimulus location were treated as within-subjects factors and group identification was treated as a between-subjects factor. When significant main or interaction effects were detected, Bonferroni-corrected t-tests using FDR correction with q = 0.05. Group differences in spectral power were confirmed to overlap with the time/frequency windows of significant increases or decreases in power relative to baseline in at least one group, and those which did not overlap were excluded from further analysis. No group differences were found which did not overlap with event-related oscillations showing either significant increases or decreases in power relative to baseline.Event-related oscillations were determined by identifying significant increases or decreases in baseline-corrected spectral power within subject group in the 0–480 ms time window. Significance was determined by one-sample t-tests comparing SP to HC were applied at each sensor, time, and frequency point. The results of these t-tests were then used to identify candidate clusters of significant group differences in gamma-band power. Candidate clusters were identified when at least 3 adjacent channels, 3 adjacent frequency points, and 20 adjacent time points were significantly different at the p < 0.05 threshold. This criterion alone limits spurious results as described by Guthrie and Buchwald (p < 0.05). For these tests, participants' data were randomly re-assigned group identification (shuffled) while keeping the number of SP and HC constant. T-tests were applied to the data points in the identified clusters using these new group identifications. Group permutations were performed 5253 times (the number of unique permutations of 103 binary numbers) for each cluster and t-statistics were calculated and summed for each cluster for each permutation. When this summed t-statistic after permutation exceeded the summed t-statistic from the candidate cluster in more than 5% of the permutations, the cluster was rejected as not significantly different by group and was excluded from further analysis.Spectral power was compared between HC and SP across the 30–50 Hz (gamma-band) frequency range from 0 to 480 ms post-stimulus for each condition. We limited our time window to 0–480 ms to focus on event-related oscillations following stimulus presentation. We focused our analysis on the 30–50 Hz gamma-band range based on unisensory and multisensory studies reporting results in this frequency range. Independent sample Buchwald  when datBuchwald , furthert-scores for subtests of interest . Regression models were considered significant at p < 0.025 (0.05 divided by the number of multisensory clusters) to correct for multiple comparisons.Stepwise multiple regressions were performed separately for HC and SP to assess the extent to which mean spectral power of unisensory clusters predicted mean spectral power of multisensory clusters while limiting comparisons to the same stimulus location (near or far). Regression analyses were only performed for the clusters, which differed significantly from baseline values within group. Additionally, regressions were performed by group to determine if gamma-band power in each of the clusters predicted multisensory reaction time facilitation and MATRICS composite Finally, Pearson correlation was used to assess the relationships among mean spectral power of unisensory clusters, medication dosage , and positive/negative symptoms .F = 410.26, p < 0.001] in which reaction times to visual-only and audio-visual stimuli were significantly faster when compared to auditory-only stimuli . Audio-visual stimuli also evoked faster reaction times than visual-only stimuli . By comparing the AV RTs to the fastest unisensory RTs (V for both HC and SP), this result indicates significant multisensory facilitation based on average RT. A significant stimulus type by group interaction was also detected , in which differences between visual-only and audio-visual responses were significantly greater for SP than HC , where RTs to near stimuli were significantly faster. Additionally, there was a significant stimulus type by stimulus location interaction , in which RTs to the VN condition were significantly faster than RTs to the VF condition . Likewise AVN RTs were faster than AVF RTs .A significant effect of stimulus location (near vs. far) was also detected . No significant group effects were detected based on the synchrony of the AV stimuli .A significant main effect of audio-visual synchrony was detected when comparing AVN/AVF RTs to AVAN/AVAF RTs. Responses to synchronous presentations were faster than asynchronous presentations , where more correct responses occurred in the visual-only and audio-visual conditions compared to the auditory-only condition . There were also more correct responses to audio-visual presentations when directly compared to visual-only presentations . A significant stimulus type by group interaction was found  such that HC had significantly more correct responses to all stimulus types compared to SP , where more correct responses were made for near stimuli. A significant stimulus location by group interaction was also detected , in which HC had significantly more correct responses to both near stimuli  and far stimuli , where more correct responses were found for VN than VF stimuli and AVN than AVF stimuli . This provides evidence that the peripheral visual stimuli and higher volume auditory stimuli lead to improved accuracy in this task and the combined AV condition improved accuracy, especially for the near condition.There was also a significant main effect of stimulus location [t-test comparisons within one or both diagnostic groups. This provides evidence of task-related increases or decreases in gamma-band power in either one or both groups in each of the group difference clusters .Tables decreases and increases in gamma-band power in SP relative to HC. These group differences in multisensory gamma-band power were not directly accounted for by group differences in unisensory gamma-band power . Despite the lack of spatio-temporal overlap, certain unisensory clusters predicted gamma-band power in multisensory clusters indicating a cortical network of local gamma-band power that may influence gamma-band power in other regions. Finally, synchrony of the AV stimuli modulated group differences such that group differences were only obtained for the synchronous multisensory conditions. These results are discussed in more detail below.The aim of this study was to determine if differences in 30–50 Hz gamma-band power in response to unisensory and multisensory stimuli in a large cohort of schizophrenia patients relative to age-matched healthy controls explained the multisensory facilitation observed in our previous study. Similar to our previous study, this larger cohort of SP showed greater behavioral facilitation to multisensory stimuli than HC. However, gamma-band power was not directly associated with RT facilitation. Despite this lack of association with RT, gamma-band power predicted performance on MATRICS scores differently by group suggesting that gamma-band power may play a role in cognitive deficits in SP. Also, medication was positively correlated with gamma-band power for only one of the multisensory clusters suggesting that medication alone cannot account for the differences in gamma-band power. Furthermore, SP showed decreases in gamma-band power in the peripheral visual (VN) condition relative to HC, as hypothesized. In contrast to our hypothesis, we identified both Our results are consistent with the previous literature  this represented a failure to suppress gamma-band power following the stimulus in SP rather than a significant increase from baseline gamma-band power. Only 2 of the clusters (AN-RT2 and AVSN-LF) represented increased gamma-band power from baseline in SP as well as significantly greater gamma-band power than HC. Increased gamma-band power in the AVSN-LF cluster may in part be explained by the positive correlation between gamma-band power and medication level in SP. Significant differences in gamma-band power in schizophrenia have been observed in a number of paradigms, but reports of decreases in gamma-band power in SP are more common than increases . Our results only reveal group differences in gamma-band power during the peripheral—Near visual conditions. These results are consistent with previous studies indicating dorsal stream deficits in SP (Butler and Javitt, Finally, we investigated whether event-related power in the gamma-band clusters was predictive of cognitive outcome on five subtests of the MATRICS, and whether it was related to symptomology in SP. As shown in Tables B in schizophrenia (Farzan et al., B may explain increases in gamma-band power in SP as an improper inhibition of cortical oscillations. Another potential source of gamma-band differences is reduced or altered connectivity within and between brain areas in schizophrenia. Disruptions in anatomical and functional brain networks have been observed in individuals with schizophrenia (Burns et al., The underlying pathophysiology responsible for the gamma-band differences observed in this study and others could arise from a number of sources. GABAergic interneurons have been implicated in the generation and modulation of gamma-band oscillations in cortex and hippocampus (Traub et al., Our results extend previous work characterizing multisensory differences in task-related activity in schizophrenia (Stone et al., In summary, this study demonstrates that group differences between SP relative to HC in gamma-band activity are present in response to both unisensory and multisensory stimuli. Yet, the unisensory deficits do not directly map onto changes in gamma-band power in response to multisensory stimuli. Future work will tease apart the role of sensory parameters and attention in performing unisensory and multisensory tasks. Characterizing oscillations across the frequency spectrum will also likely provide broader insight into multisensory integration in schizophrenia and help provide a link between sensory and cognitive functions.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.


N-acetylcysteine, cysteine hydrochloride or glutathione) can form specific, self-assembled nanostructures when deposited on mica by dip coating. The mechanism of adsorption is based on molecule-to-substrate interactions between carboxylic groups and mica. Intermolecular cross-linking by hydrogen bonds was also observed due to the donor–acceptor character of the functional groups. The texture of supramolecular nanostructures formed by the studied materials on mica was analysed with atomic force microscopy and their specific surface energy was estimated by contact angle measurements. Significant differences in the surface roughness, thickness and the arrangement of macromolecules were noted depending on the kind of functional groups on the side chains. Specific changes in the morphology of the surface layer were observed when mica was primed with a monolayer of small organic compounds . The adsorption of both silsesquioxane oligomers and organic primers was confirmed with attenuated total reflectance infrared spectroscopy. The observed physiochemical and textural variations in the adsorbed materials correlate with the differences in the chemical structure of the applied oligomers and primers.Linear oligomeric silsesquioxanes with polar side moieties (e.g., carboxylic groups and derivatives of  The modification of surface properties can be used as a versatile tool in materials engineering for biological and medical purposes –4. The fWell-defined substrates made of small molecule self-assembled monolayers (SAMs) ,14,23–30332(Si3AlO10)(OH)2] that exhibits interesting surface properties and chemical specificity. Potassium ions electrostatically bind the alternating aluminosilicate sheets in the lamellar structure of mica. The mineral can be easily cleaved along the plane located in the K+ layer to expose a perfectly smooth surface [+ becomes accessible to acidic molecules and can be involved in the formation of surface salts. For example, potassium carboxylates generated on the surface of mica assist the process of adsorption of fatty acids [We have recently found that linear oligosilsesquioxanes functionalized with 2-(carboxymethylthio)ethyl side groups (LPSQ-COOH) can adsorb from their solutions and spontaneously form well-ordered and stable, PSAM-type, 2D nanolayers at the surface of muscovite mica, which renders the surface exceptionally hydrophilic –38. Musc surface  that can surface –41 and p surface –43. Uponty acids –47 and tty acids .N-acetylcysteine (NAC), cysteine hydrochloride (Cys-HCl) and glutathione (GSH). Such self-assembled PSAMs based on polysilsesquioxane materials are attractive for surface nanopatterning and bioengineering, including preparation of surfaces rich in organic groups typical of the extracellular matrix in living organisms .The character of the interactions between the oligomers and the substrate also defines the structure of the assemblies of LPSQ-COOH on mica . It was N-acetylcysteine, citric acid or thioglycolic acid. The primers are bound to mica by ionic bonds (carboxylates) and simultaneously provide the substrate with new organic functions capable of hydrogen bonding [We have investigated the effect of the kind of functional groups in side chains of LPSQ-COOH/X on the structure  of the prepared PSAMs. Native mica was used bare or primed, prior to the coating with LPSQ-COOH/X, with a monolayer of  bonding .Atomic force microscopy (AFM) and attenuated total reflectance infrared spectroscopy (ATR-FTIR) were used as analytic tools for the studies. The changes in the free surface energy of the prepared hydrophilic surfaces were also investigated for all LPSQ-COOH/X adsorbed on native and primed mica. The obtained results suggest that both the composition of side polymer chains and the kind of functional groups on the surface are key factors defining the structure and properties of PSAMs based on LPSQ-COOH/X.N-acetylcysteine (NAC), glutathione (GSH) and cysteine hydrochloride (Cys-HCl), to the side chains of vinyl-containing LPSQ precursors obtained by polycondensation of cyclic tetravinylsiloxanetetraols [Functionalized ladder-like silsesquioxanes (LPSQ-COOH/X) were prepared , Table 1tetraols . The thiThe structure and reactivity of LPSQ-COOH/X makes the polymers suitable for the formation of planar PSAMs on various reactive surfaces. Side carboxylic groups in LPSQ-COOH/X allow for a very efficient polymer anchoring on the surface due to both multipoint ionic substrate–adsorbate interactions and adsorbate–adsorbate hydrogen bonding . The forC=O band at ≈1700 cm−1 and emergence of diffuse bands in the formed PSAMs was observed).The results obtained for LPSQ-COOH/X using AFM (topographic images and height profiles) suggest a specific packing of the chains on the surface of mica. LPSQ-COOH can form fine nanolayers  of thickS) of each studied PSAM sample was determined by measuring the contact angle of water and glycerol as reference liquids (sessile drop technique and Owens–Wendt geometric mean Equation S1 described in −NH2+– and –COO−NH3+–)) and establish a network of hydrogen bonds. The slightly poorer wettability of P4 can be ascribed to the presence of the acetyl group, protecting the NH2 function of NAC.The surface free energy (γS (Rq = 0.03 nm for native mica and ≈0.1 ÷ 0.25 nm for PSAMs). No correlation could be found between Rq and γS. The increase of γS observed for all studied PSAMs can be tentatively linked to the presence of specific side groups in the adsorbed polymers, capable of effective hydrogen bonding.We have analysed the effect of surface roughness on changes in γS . All sur+ ions by adsorption of small molecules [N-acetylcysteine (NAC) were selected and used as primers to modify the surface properties of mica  and amide II at 1523 cm−1 (bending vibrations of the N–H bond)) disappeared after its adsorption on mica. This phenomenon can be ascribed to changes in geometry of NAC and possible interactions of C=O in the amide unit with K+ on the surface of mica.The formation of surface salts (potassium carboxylates) by the used primers was confirmed by ATR-FTIR analysis . Comparioxylates . The onlRq parameter estimated with AFM for these substrates is low . For both compounds patches of base layers can be observed that are more smooth (Rq = 0.077 and 0.047 nm). They do not exhibit clear phase contrast and it cannot be asserted whether they are areas of well-packed molecules or bare mica (Rq = 0.032 nm for bare mica). The surface of mica covered with TG bearing thiol functions (less effective in hydrogen bonding) is uniform and very smooth (Rq = 0.117 nm) except for visible drops of excess primer. Priming mica with NAC, CA and TG thus yields smooth, chemo-specific, hydrophilic supports  indicate a significant alteration in the nature of interactions between the studied macromolecules and the surface  structures resulting in a characteristic FTIR νC=O band at ≈1720 cm−1 [C=O band to 1600 cm−1 [−NH2+– and –COO−NH3+–).ATR-FTIR studies in the sensitive region . Their ability for the formation of hydrogen bonds with probe liquids (H2O and glycerol) defines the wettability and chemical specificity of the modified supports , the wettability of the coated samples is lower.The modification of mica-CA by adsorption of LPSQ-COOH/X results in a decrease of the surface free energy . The mosThe structure and properties of PSAMs made of ladder-like oligosilsesquioxanes LPSQ-COOH/X on chemo-reactive supports  have been analysed. The AFM studies showed that linear oligomers adsorb on the surface of mica and form various types of structures, depending both on the morphology of LPSQ-COOH/X and the chemical specificity of the support. The functional groups in side chains have a significant impact on the arrangement of macromolecules, surface pattern and hydrophilicity. The distribution of the studied macromolecules within the adsorbed PSAMs is a consequence of both polymer–substrate as well as inter- and intramolecular bonding. The homopolymer LPSQ-COOH can form the smoothest layers with macromolecules arranged horizontally in the monolayer due to the specific mechanism of their adsorption on mica. An alternative adsorption mechanism and the shifting of the orientation of the silsesquioxane chains towards more mushroom-like shapes allows for the possibly for other LPSQ-COOH/X schemes. It was also found that priming the substrate with small organic compounds can alter the structure of the adsorbed polymeric films due to the change of specific interactions between the polymer and the surface. The presented, simple approach for the preparation of hydrophilic, nanopatterned surfaces rich in organic polar groups can be especially useful in bioengineering.The supporting information features all experimental procedures, characterization methods, and NMR spectral data for the prepared LPSQ-COOH/X materials in addition to the ATR-FTIR spectra of P1, P2, P3 and P4, adsorbed on mica-CA, mica-TG and mica-NAC.File 1Experimental part.


Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limited success of the past clinical trials involving peptide vaccine therapy to treat colorectal cancer, it is necessary to revise our knowledge of the immune system and its potential use in tackling cancer. This review presents the efforts of the scientific community in the development of peptide vaccine therapy for colorectal cancer. We review recent clinical trials and the strategies for immunologic monitoring of responses to peptide vaccine therapy. We also discuss the mechanisms underlying the therapy and potential molecular targets in colon cancer. Colorectal cancer (CRC) is the third most common cause of cancer-related deaths among men and women in the western world ,2. EuropSince the discovery of tumour antigens in 1991, immunotherapy has become a potential alternative to conventional chemotherapy . In 2010Immunotherapy is based on the principle of aberrant expression of proteins by tumours, which either over-express ubiquitous proteins or express proteins that unusually occur in the original tissue of the cancer. Tumour cells that bear these antigens can be distinguished from the normal tissue by the immune system in the same way that bacteria or virus-infected cells are. Recognition of an unusual antigen starts an immune response aimed at elimination of the cell and generation of antigen-specific immune cells that provide long-lasting immunity. However, immune-mediated regression of cancer hardly ever takes place in real life due to the low effectiveness of wild-type antigens (generated by the tumour) in stimulating host immune responses. The aim of vaccine therapy is to provide a highly immunogenic antigen, such as peptides, which are capable of stimulating immune system to mount a cytotoxic attack against the tumour cells. The desired effect would be stabilization or even regression of the disease.Following an inoculation, the circulating peptides are degraded in the circulation and the antigenic products are endocytosed by antigen-presenting cells (APC), which migrate to lymph nodes where they present the antigen in a processed form to T cells. These are at the forefront of the battle against the tumour. There are two main types of T cells according to the surface markers and function. CD8+ T cells, also known as cytotoxic T lymphocytes (CTL), are responsible for direct killing of tumour cells. They induce apoptosis by release of granzymes and perforins or by presenting Fas ligand (FasL) to Fas death receptor on the target cells. Another consequence of granzyme release is target cell necrosis [The interaction between individual cells is mediated by two major histocompatibility complex (MHC) proteins, MHC classes I and II , present on the surface of cells. The antigenic protein from the circulation is processed by cellular machinery into a several-amino acid-peptide that can enter the MHC molecule for presentation to T cell receptor (TCR). ●MHC class I is responsible for presentation of the vaccine-derived peptide between APC and naïve CTLs. Primed CTLs are able to recognize the genuine tumour antigen presented by the MHC I on the surface of the tumour. Thus activated CTL sends out a death signal to the tumour.●MHC class II is responsible for “the talk” between APC and CD4+ T cells and subsequent generation of helper T cells. Cytokines secreted by helper T cells, such as IL-2, are essential for activation of strong cytotoxic pathways and potentiation of anti-tumour response [response . All in Various immune cells are normally found infiltrating the mass of a tumour, some of which are specialized to recognize the tumour-associated antigens. Although there is evidence of a positive correlation between the number of these cells and good prognosis, their effect on the tumour is considered insignificant ,11. It h●One such mechanism is expression of FasL, which is a ligand for Fas receptor present on CTL. Fas functions as a death receptor which, upon binding of its ligand expressed on the tumour surface, induces CTL to undergo apoptosis. Additionally, expression of FasL provides a degree of resistance to Fas-induced apoptosis in tumour cells themselves .●Another mechanism involves expression of altered peptide ligands for T cell receptors (TCR) by the tumour. “Altered Peptide Ligand” (APL) are analogs of immunogenic peptides in which the TCR contact sites have been manipulated . Recogni●Oncogenic signalling pathways within tumour cells and immunologic checkpoints in the tumour microenvironment also play a crucial role in promoting immunologic tolerance. For example, tumour releases factors that induce inhibition of both innate and adaptive antitumor immunity. Stat3 activation in tumours, as well as Braf activation, can induce release of factors such as IL10 that induce Stat3 signalling in NK cells, granulocytes, inhibiting their tumouricidal activity. Stat3 is also activated within conventional dendritic cells (CDC) in the tumour, converting them to toleragenic DC, which can induce T cell anergy and possibly regulatory T cells (Treg). Plasmacytoid DC (PDC) or PDC-related cells in the tumour microenvironment upregulate indoleamine 2,3-dioxygenase (IDO), an enzyme that metabolizes tryptophan. T cells are very sensitive to tryptophan depletion. Tumours can express co-inhibitory B7 family members, such as B7-H1 and B7-H4, which downregulate T cell activation and/or cytolytic activity. They can also induce B7-H1 and B7-H4 expression on tumour associated macrophages (TAM). Related immature myeloid cells or myeloid suppressor cells can further inhibit antitumor T cells via production of NO by the enzyme arginase. ,17.●Regulatory T cells are an important inhibitor of antitumor immunity. T cell activation in the absence of appropriate co-stimulatory signals leads to T cell anergy and generation of induced regulatory T cells (Treg). Treg, characterized by the FoxP3 transcription factor, upregulate a number of cell membrane molecules, including Lag3, CTLA4, GITR, and neuropilin. Treg can inhibit effector T cell activation and function via T-T inhibition or inhibition of antigen presenting cells .●Finally, cancers avoid recognition by the immune system by means of defective antigen presentation. This can be achieved by reduced expression of HLA type I (MHC I), which is a common event in colorectal cancer , as wellImmune-competence of cancer patients who receive chemotherapy is already partially compromised. The design of peptide vaccines is aimed at overcoming the described problems by delivering highly immunogenic antigens combined with adjuvants that stimulate the immune system.Special design of the amino acid sequence of a peptide can enhance the interaction with the TCR. This usually involves creation of a peptide epitope with single amino acid substitutions, which exhibits improved binding to MHC or affinity for the TCR. Such manipulations are capable of potentiating secretion of interleukin 2 , which is a potent immune-boosting cytokine. Other modifications, involving substitution at the peptide terminals, results in improved bio-stability and reduced degradation by seric proteases . EffectiThe use of synthetic peptides is a fairly recent endeavour. In the early days of immune therapy tumour antigens were delivered to the patient in a form of tumour lysates. Nowadays tumour lysates are being pulsed into dendritic cells (DC) as a way of facilitating peptide presentation. Another recent development is the use of a retrovirus carrying the antigen-encoding gene, which inserts itself in the host genome leading to production of large quantities of the antigen . AgainstThe main drawback is the human leukocyte antigen (HLA)-type restricted nature of the therapy. HLA molecules are expressed in a multitude of super-types and individual antigens are only capable of interacting with a particular HLA-super type. In the clinical context this means that only patients carrying the required HLA allele will be able to respond to the therapy. This substantially limits the application of the therapy. Moreover, use of self-antigens carries a risk of inducing autoimmunity. This kind of therapy is also contraindicated in patients with existing autoimmune diseases, where exposure of the over-reactive immune system to a highly immunogenic antigen can have unpredictable consequences. An example of that comes from trials on immune therapy in Parkinson’s disease where the patients’ condition was reported to be aggravated following vaccination against a neuroprotein .Two aspects of response to peptide therapy are commonly evaluated in clinical setting: clinical outcome as the primary endpoint and stimulation of cell-mediated toxicity as a secondary endpoint. Several techniques have been used to measure the frequency and activity of antigen-specific CTLs, including ELISPOT assay, flow cytometry, RT-PCR, HLA/epitope tetramer assay. This is often combined with evaluation by means of serum cytokines and chromium (υ¹Cr) release assays, and proliferation studies . The υ¹C●ELISPOT assays are used to assess secretion of proteins by CTLs which are indicative of an activated state, such as, INF-γ, granzyme B, IL-2 . The ass●15, EL4 and T2 lymphoma cells with a cell tracker dye DDAO-SE and staining permeabilized cells with antibody against cleaved (activated) caspase-3. This assay proved to be robust and reliable in evaluating antigen-specific CTL [Following T-cell receptor recognition of antigenic peptide–MHC class I complexes on the surface of target cells, CTLs induce target cell apoptosis through directed exocytosis of perforin and granzymes. The cytotoxic signalling leads to the activation of the caspase cascade that can be measured using flow cytometer. The assay involved labelling of P8ific CTL .●Another approach is to use flow cytometry for detection of apoptosis in CTL target cells . During apoptosis, phosphatidylserine (PS) is externalized on the surface of the cells and is available for binding of annexin V. Both granule marker and annexin V assays allow evaluation of the cytotoxic potential of tumour-specific CTLs .●By contrast, HLA/epitope tetramer assay is used for mere enumeration of the antigen-specific CTLs. However, when combined with intracellular cytokine staining it gives a complete picture of quantity and function of the TCLs.●Lastly, RT-PCR has been used to evaluate cytokine gene expression, including INF-gamma and granzyme B, by CTLs ,26. CellIdeally the immune response should correlate with the primary endpoints, which reflect the disease progression. Sadly this has been exceptionally rare in the clinical trials so far. In addition to patient survival, the levels of tumour markers are measured using fluorescence-based immunoassays for monitoring of tumour growth and response to therapy . Also, dTumour self-antigens are broadly categorized according to their specificity for the tumour. Differentiation tumour antigens present in both tumour and its original tissue, but are expressed at higher levels in the tumour. Tumour-specific antigens occur in tumours cells but are absent in normal adult tissues. Tumour/testis antigens are additionally expressed in the gonads. Unique tumour antigens constitute a separate group. These arise from nucleotide deletions or alternative splicing and characterize each single tumour. The choice of each one of these targets for vaccine therapy carries its specific risks as well as benefits. Targeting differentiation antigens, owing to their presence outside the tumour, can elicit a degree of adverse reaction in the healthy tissue. An example of that comes from clinical trials in melanoma where use of peptide vaccines directed against proteins involved in melanin biosynthesis leads to development of skin depigmentation (vitiligo). With regards to unique antigens, some evidence speaks in favour of their higher immunogenicity. The immune response against unique antigens has been found to persist for longer following tumour resection compared with shared tumour-specific antigens . Howeveri.e., melanoma. Nonetheless, the few antigen-based vaccines that have been developed show the ability to induce antigen-specific CTL and cause reduction in the tumour size in animal studies. Translation of these findings to a clinical setting was not completely successful. Tumour-specific antigens have been the most popular targets for vaccine therapy in general and in the context of colon cancer in particular. It must be noted that immunogenicity of colorectal cancers is considerably lower compared to the most immunogenic among cancers, in vivo studies and clinical trials. The remainder are summarized in the One particular pattern of response to peptide vaccine therapy prevails across all clinical studies. That is, strong induction of antigen-specific CTLs is not associated with improved clinical outcome. Frequently reported side effects of the therapy are: ulceration at the injection site, fever, fatigue, nausea, anorexia. However, serious adverse reactions are very rare. The protocols commonly require 3 or more injection courses at 2–3 weeks intervals and patients who show satisfactory response are offered continuation of the therapy. It has been argued that the lag between the first jab and the clinically relevant response is too long, especially in the case of advanced cancer patients who may not survive the necessary time. Below is a brief review of selected antigen targets in colon cancer including their biological role, results of Ephrin type-A receptor 2 (EphA2) is a member of a large tyrosine kinase receptor family. Eph receptors play an important role in oncogenesis  and tumoin vitro [Survivin is a protein which inhibits cancer cell apoptosis, is highly expressed during embryological development, and becomes undetectable in adult tissues . A studyin vitro . In 2003in vitro . The thein vitro ,48,49,50et al. used a vaccine formulation combining two SART3 antigenic epitopes recognized by HLA-A24-restricted CTLs with IFA in colorectal cancer patients. Immunization resulted in significant induction of tumour-specific CTL in 7 of 11 patients. Two patients presented with induction of INF-γ following the sixth vaccination time. However, augmentation of cellular immunity was not associated with improved clinical outcome. Likewise, no induction of IgG or IgE specific for the peptides was observed. An adverse reaction at the injection site manifested as itching and redness occurred in 6 patients. The median number of vaccination times was 8 and the median observation period was 5 months. Despite the somewhat promising result no further trials involving SART antigen has been published since 2001 [SART3 is a tumour-rejection antigen which is expressed in more than 70% colorectal cancers but is not present in normal non-malignant tissues . It was nce 2001 .Carcinoembryonic antigen (CEA) is a useful tumour marker which is correlated with tumour burden in the mouse metastasis model and has been used to monitor CRC metastasis development . A mouseOverall, immunization with CEA resulted in enhanced immune response and survival. Further improvement in both endpoints was seen with the combination therapy. This constitutes an interesting case for employment of both antibody-mediated and cell-mediated immunity to achieve greater anti-tumour response. It also consolidates the importance of T helper cells in stimulation of cytotoxic killing of the tumour cells . MUC-1 is an epithelial membrane antigen, which bound to the apical membrane of the secretory cells. It has been found that MUC-1 is expressed in more than 70% colorectal cancers and correlates with poor prognosis . An earlin vivo studies and clinical trials. The remainder are summarized in the We have discussed some of the selected antigen targets in colon cancer including their biological role, results of Development of a novel, less toxic therapeutic strategy to treat advanced and disseminated colorectal cancer has been a goal for oncologists and clinicians for many years. The discovery of tumour antigens in 1991 has opened a window for the use of immune therapy in the treatment of cancer. In the last two decades, a great deal of effort has been made to identify the molecular targets for immune therapy in colorectal cancer; some of those targets proved effective in a pre-clinical setting. Moreover, the immune therapy has been shown to cause minimal side effects in a clinical setting, which makes it a desirable alternative to the current systemic treatments. Its ability to induce strong and specific anti-tumour immune responses in patients has been well documented using the current immunologic monitoring techniques. However, these findings did not correlate with improved clinical outcome. This calls for more accurate strategies for immune monitoring and could become the next direction for research.


The objective was to re-evaluate the controversial reports of EMG-torque relation between impaired and non-impaired sides using linear electrode array EMG recordings. Ten subjects with chronic stroke performed a series of submaximal isometric elbow flexion tasks. A 20-channel linear array was used to record surface EMG of the biceps brachii muscles from both impaired and non-impaired sides. M-wave recordings for bilateral biceps brachii muscles were also made. Distribution of the slope of the EMG-torque relations for the individual channels showed a quasi-symmetrical “M” shaped pattern. The lowest value corresponded to the innervation zone (IZ) location. The highest value from the slope curve for each side was selected for comparison to minimize the effect of electrode placement and IZ asymmetry. The slope was greater on the impaired side in 4 of 10 subjects. There were a weak correlation between slope ratio and strength ratio and a moderate to high correlation between slope ratio and M-wave ratio between two sides. These findings suggest that the EMG-torque relations are likely mediated and influenced by multiple factors. Our findings emphasize the importance of electrode placement and suggest the primary role of peripheral adaptive changes in the EMG-torque relations in chronic stroke. Weakness for voluntary muscle contraction is a common sequela of a hemispheric stroke. Together with other clinical symptoms, weakness is a primary contributor to the overall impairment3457910These central and peripheral pathological changes profoundly affect the EMG-torque (force) relation in stroke survivors7The mechanism underlying the relation between isometric muscle force and surface EMG amplitude is very complex. In addition to the aforementioned intrinsic pathological neuromuscular changes, there are other technical factors that could affect the EMG-torque relations, such as electrode placement1415167To re-evaluate the EMG-torque relation in chronic stroke, we used a linear-electrode array to record surface EMG signals during isometric elbow flexion at different submaximal torque levels. The linear electrode array has dramatically expanded the applications of surface EMG in various areas21In this study, we aimed to utilize the advantages of the linear electrode array EMG recordings to re-examine the EMG-torque relations in stroke subjects. Specific aims were to 1) compare IZ locations between impaired and non-impaired biceps muscles; 2) to investigate the effect of IZ location on the EMG-torque relation by comparing slopes of EMG-torque from individual channel EMG recordings; 3) to examine correlations among slopes, strength and M-wave amplitudes to investigate possible underlying pathophysiological mechanisms of motor impairment.In this study, we recruited 10 hemiparetic stroke survivors . The subjects were seated on a height-adjustable chair. The arm to be tested was secured firmly on a customized apparatus . The elbA customized linear electrode array was used for EMG recordings. The array has 20 silver bars  arranged in a linear manner with 5 mm distance between each bar. Target skin area was cleaned thoroughly with alcohol wipes. After standard skin preparation the electrode array was placed from the proximal to distal portion of the biceps brachii muscle with the midline of the array electrode aligned with the midline of biceps longitudinally from the biciptal groove to the biceps tendon insertion. As such, the electrode array covered the main portion of the muscle . The samTorque signals were recorded with a torque sensor , digitized at 1,000Hz on a PC computer with a BNC-2090A data acquisition board  using custom LabView software . Both torque and surface EMG recordings were triggered simultaneously. Data was saved for offline analysis using a customized MATLAB  program.Subjects performed isometric elbow flexion tasks on each side. The order of side was randomized and balanced among subjects. Maximum voluntary contraction (MVC) of elbow flexion was first determined for each arm at approximately 90° of flexion after a few bouts of elbow flexion for warming up. Subjects were verbally encouraged to flex the elbow as hard as they can against the mechanical stops for 5 seconds . The higThe M-wave from both biceps muscles was recorded to estimate the peripheral neuromuscular capacity. We adopted a protocol for the M-wave measurement from our recent studyThe torque signal was digitally filtered using a zero lag low-pass, second-order Butterworth filter with a 10Hz cutoff. EMG signals were filtered using a fourth-order band-pass 20–450Hz) Butterworth filter and a 60Hz notch filter. Consistent flat response of the torque data was observed for at least 4 seconds across all trials and subjects  were then obtained. Correlation coefficient (r) was then calculated from R2. Similarly, slopes of the EMG-torque relation for each channel were calculated for each subject. r for all subjects ranged from 0.81 to 0.99. Since the average torque value over selected window remained the same for any given trial across EMG channels, the slope of EMG-torque relations can then be considered to correspond to EMG activities across different channels from linear array recordings. The slope for each channel was presented in the ‘Slope Curve’ in 7EMG-torque relations were first established using EMG values from individual channels. Linear regression on EMG-torque relations was performed  and ACTIVATION  to compare the IZ location. Highest and lowest points on the slope curve were compared between two sides by using a two-way ANOVA with factors of SIDE and SLOPE . Linear regression between strength ratio and slope ratio between two sides was performed. Linear regression between M-wave ratio and slope ratio between two sides was also performed. Our effect sizes were obtained from our preliminary trials, and were set to be 1.22 and 0.82 for slope and size (accounting for 60% and 40% of the variance respectively). Accordingly, statistical power was determined to be at least 80%. p < 0.05 was chosen to indicate statistically significant differences. = 24.43, p = 0.001) and SIDE  and a significant SLOPE×SIDE interaction (p < 0.005). Further one-way ANOVA analyses showed that the slopes were significantly greater on the non-impaired side than on the impaired side, and that the highest slope was significantly greater than the lowest slope . However, there was mixed observation of slopes from individual subjects. As shown in Previous studies7 = 0.59, p = 0.30) and also among contraction level  for the whole group. No significant interactions were found.In this study, the IZ location was detected using visual inspection. Even though we carefully placed the linear array symmetrically using anatomical landmarks, IZ location was not symmetrical between the impaired and non-impaired side . There wIn the present study, we re-examined the EMG-elbow flexion torque relation in hemiparetic stroke subjects using a linear electrode array. We confirmed the importance of IZ location on EMG-force relationset al.The innervation zone from the linear array recording was accurately localized by visual inspection. The IZ location did not show a clear change with the level of elbow voluntary flexion. It has been reported that IZ shifts with changes in joint angle and voluntary activation282930The novel finding of an “M” shaped pattern of the slopes of the individual channel EMG-torque relation further confirmed the relevance of IZ location. This pattern of slope curve indicated the importance of placement of bipolar surface electrodes with respect to the IZ location. Depending on placement of bipolar surface electrode with reference to the IZ location of the individual muscle , the slo7IZ asymmetry also has potential clinical applications. In stroke patients who need Botulinum toxin injection for spasticity management, best clinical outcome could be achieved if the toxins are directly injected to the innervation zonesOur observation of variable slope values in individual stroke subjects from linear array recordings confirmed findings from the previous studies7Our findings of a weak correlation between strength ratio and slope ratio suggest that strength is not likely correlated with the EMG-torque relation. As shown in Peripheral muscular changes – atrophy and reinnervation both occurred in the impaired muscles353456458133637In summary, re-evaluation of the EMG-elbow flexion torque relation using linear electrode array EMG recordings showed asymmetrical IZ locations and a difference in slope of the EMG-torque relations between impaired and non-impaired sides in stroke survivors. There was a weak correlation between slope ratio and strength ratio, but a stronger correlation between slope ratio and M-wave ratio between two sides of stroke survivors. These findings suggest that the EMG-torque relations are likely mediated and influenced by multiple factors. Possible mechanisms could include weakness and decreased descending activation, altered motor unit control and adaptive peripheral neuromuscular changes, asymmetries in IZ locations, and placement of bipolar electrodes. Our findings emphasize the importance of electrode placement and suggest the primary role of peripheral adaptive changes in the EMG-torque relations after stroke.How to cite this article: Bhadane, M. et al. Re-evaluation of EMG-torque relation in chronic stroke using linear electrode array EMG recordings. Sci. Rep.6, 28957; doi: 10.1038/srep28957 (2016).


We have now extended these studies to mammalian cell culture and mice. Depletion of pRB in non-tumorigenic hTERT-BJ cells leads to an increased cell population in S-phase and slight increases in the cell population doubling time. Acute loss of Rb1-/-in the mouse colon, but not the lung, leads to a robust increase in Ki67 staining within 96hrs. We therefore asked to what extent metabolic changes associated with proliferation increases are conserved between pRB-deficient cells in culture and acute loss of Rb1-/-in vivo. We performed isotopic tracer analyses with both glucose and glutamine and found that while loss of pRB in cell culture promotes increased isotopic enrichment from U13C-glutamine in TCA cycle intermediates, little to no change was seen in isotopic enrichment from U13C-glucose. In stark contrast, acute loss of Rb1 in the mouse colon leads to robust isotopic enrichment from U13C-glucose in TCA cycle intermediates and the pyrimidine precursor aspartate; while little to no change in isotopic enrichment from U13C-glutamine was found in all TCA cycle intermediates but alpha-ketoglutarate and no significant differences was seen in aspartate. Collectively these results reveal that loss of the tumor suppressor pRB has metabolic consequences in vivo.Inactivation of the p16/pRB axis is a rate-limiting step in tumorigenesis. Loss of these tumor suppressors is well known to disrupt control of cell proliferation but, additionally, these mutations cause changes in cell metabolism. To investigate the metabolic changes resulting from pRB inactivation we have exploited a Drosophila model. Metabolomics and